WO2020173473A1 - Polypeptides with chap domain and their use for treating sludge - Google Patents

Polypeptides with chap domain and their use for treating sludge Download PDF

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Publication number
WO2020173473A1
WO2020173473A1 PCT/CN2020/076877 CN2020076877W WO2020173473A1 WO 2020173473 A1 WO2020173473 A1 WO 2020173473A1 CN 2020076877 W CN2020076877 W CN 2020076877W WO 2020173473 A1 WO2020173473 A1 WO 2020173473A1
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seq
polypeptide
amino acids
nucleotides
sludge
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PCT/CN2020/076877
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French (fr)
Inventor
Mingjia ZHANG
Ming Li
Ye Liu
Bucong HAN
Ying Zhang
Shufang ZHAO
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Novozymes A/S
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Publication of WO2020173473A1 publication Critical patent/WO2020173473A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00—Treatment of sludge; Devices therefor
    • C02F11/12—Treatment of sludge; Devices therefor by de-watering, drying or thickening
    • C02F11/14—Treatment of sludge; Devices therefor by de-watering, drying or thickening with addition of chemical agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00—Treatment of sludge; Devices therefor
    • C02F11/02—Biological treatment
    • C02F11/04—Anaerobic treatment; Production of methane by such processes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00—Treatment of sludge; Devices therefor
    • C02F11/12—Treatment of sludge; Devices therefor by de-watering, drying or thickening
    • C02F11/14—Treatment of sludge; Devices therefor by de-watering, drying or thickening with addition of chemical agents
    • C02F11/147—Treatment of sludge; Devices therefor by de-watering, drying or thickening with addition of chemical agents using organic substances

Definitions

  • the present invention relates to polypeptides with amidase activity comprising a CHAP domain and polynucleotides encoding the polypeptides, and to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.
  • the invention also relates to the use of the polypeptides with amidase activity and with CHAP domain in sludge/wastewater treatment.
  • Sludge generated during the course of conventional wastewater treatment, is usually dewatered or concentrated prior to disposal by incineration, land application, land filling, composting, etc.
  • a basic dewatering scenario involves forming strong, shear-resistant sludge floes through the addition of a conditioning agent such as ferric sulphate and/or a flocculating agent (e.g. polyelectrolyte) followed by mechanical solid/liquid separation across gravity belt thickeners, belt filter presses, or centrifuges.
  • a conditioning agent such as ferric sulphate and/or a flocculating agent (e.g. polyelectrolyte) followed by mechanical solid/liquid separation across gravity belt thickeners, belt filter presses, or centrifuges.
  • WWTP wastewater treatment plant
  • the benefits of higher cake solids include: reduced dewatered sludge volume (less sludge to be“managed” by the plant); lower annual
  • transportation costs shipment the sludge to landfills or sites of land application
  • less water to be evaporated before sludge can be incinerated increasing the net energy value of the sludge when incineration is used for cogeneration purposes
  • a more concentrated feed to digesters and/or reduced volume of sludge to be landfilled or land applied.
  • WO 1999/027082 and WO 2003/006602 are of interest as they relate to proteases and variants thereof.
  • sludge remains problematic and dewatering difficult, there is a continuous need to improve methods for enhancing the dewaterability of residuals (i.e. sludge) generated by conventional wastewater treatment operations.
  • the CHAP domain is termed for the region of cysteine, histidine-dependent
  • amidohydrolase /peptidases The CHAP domain is associated with several families of amidases, which suggests that many of these proteins have multiple peptidoglycan hydrolytic activities. Experiments on glutathionylspermidine (GSP) amidase and other enzymes suggests that all the members of CHAP utilize a catalytic cysteine residue in a nucleophilic-attack mechanism.
  • GSP glutathionylspermidine
  • the use of the polypeptides of the present invention improve the flocculation performance of the sludge and thereby reduce the amount of polymer, and enhance the amount of solids per volumetric unit of sludge.
  • An aspect of the disclosure is directed to a method of treating sludge comprising the use of one or more amidase enzyme having a CHAP domain.
  • the invention provides for a method of treating sludge comprising the addition of one or more amidase enzyme having a CHAP domain, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids.
  • the invention provides for a method for improving sludge flocculation comprising i) adding one or more CHAP enzymes, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids.
  • the invention provides for a method for reducing polymer consumption, comprising i) adding or more CHAP enzymes, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids.
  • the present invention provides for a method of treating sludge comprising i) adding an effective amount of one or more amidase enzyme having a CHAP domain, typically further comprising adding one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids and separating the coagulated and flocculated solids from the water.
  • the present invention further provides isolated or purified polypeptides with CHAP domain and polynucleotides encoding the polypeptides.
  • the present invention relates to an isolated or purified polypeptide having amidase activity, selected from the group consisting of:
  • polypeptide having at least 60% sequence identity to mature polypeptide of any of SEQ ID NOs: 22-42 a polypeptide encoded by a polynucleotide that hybridizes under medium, medium-high, high or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA sequence thereof;
  • the purified polypeptide has CHAP domain.
  • the present invention still relates to the isolated polynucleotide encoding the above-mentioned polypeptides, the expression vectors or recombined host cells and their use in the method for producing the above-mentioned polypeptides.
  • the present invention relates to an enzyme composition comprising a protease and any one of the above-mentioned polypeptides. In another embodiment, the present invention also relates to the method for treating sludge with said enzyme composition.
  • the present invention relates to an enzyme composition comprising a protease and an enzyme with CHAP domain. In another embodiment, the present invention also relates to the method for treating sludge with said enzyme composition.
  • FIG. 1 shows the flocculation result of the polypeptide (SEQ ID NO: 30) in
  • the performance was conducted on two kinds of digested sludges. Wherein, the polypeptide was used in amount of 50 ug protein/t- DS, protease was used in amount of 0.5 kg/t-DS, and cellulase composition was used in amount of 0.5 kg/t-DS.
  • FIG. 2 shows the flocculation results of several polypeptides with CHAP domain in combination with the protease and cellulase composition. Each was tested in triplicated.
  • polypeptide was used in amount of 50 ug protein/t-DS
  • protease was used in amount of 0.5 kg/t-DS
  • cellulase composition was used in amount of 0.5 kg/t-DS.
  • Figure 3 shows the Flocculation result of the sludge with the same enzyme dosage.
  • the enzyme dose was maintained as 121 g protein/t-DS, wherein, the protease was fixed in amount of 21 g/t-DS. Shifting the amount of SEQ ID NO: 30 and cellulase composition from 0 to 100 g/t-DS respectively. Each condition was carried out in triplicated.
  • FIG. 4 shows the different flocculation status under score 1-5.
  • the flocculation status was classified into five groups according to the floes appearance and the clarity of bulk water.
  • groups 4 and 5 represented the desired flocculation status and thereby were scored as 4 and 5 respectively.
  • Reference to“about” a value or parameter herein includes aspects that are directed to that value or parameter per se.
  • description referring to“about X” includes the aspect“X”.
  • amidase means a kind of hydrolase which mainly acts on intramolecular C-N bond and catalyzes the hydrolysis of amide to produce corresponding carboxylic acid and ammonia.
  • amidase activity is determined according to the procedure described in the Examples.
  • cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
  • the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
  • cellulase composition means a blend of enzyme with cellulase or hemicelluase activity.
  • the cellulase composition comprises xylanase, cellulase, beta-glucosidase and a GH61A polypeptide.
  • xylanase means a 1 ,4-beta- D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans.
  • Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C.
  • One unit of xylanase activity is defined as 1.0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.
  • the term“cellulase” means one or more ( e.g ., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
  • Beta-glucosidase means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose.
  • Beta-glucosidase activity can be determined using p-nitrophenyl-beta-D- glucopyranoside as substrate according to the procedure of Venturi et ai, 2002, J. Basic Microbiol. 42: 55-66.
  • GH61 means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat B., 1991 , A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696.
  • the enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1 ,4-beta-D-glucanase activity in one family member.
  • CHAP domain is a term which defines the region of cysteine, histidine- dependent amidohydrolase /peptidases. This domain is between 110 and 140 amino acid in length and contains two invariant residues, a cysteine and a histidine. These residues form part of the active site of these proteins. A large family of proteins has been unified by the presence of CHAP domain. The CHAP domain is present in proteins containing at least three types of amidase domains.
  • the CHAP domain of the polypeptides of the present invention may be represented by amino acids 126 to 212 of SEQ ID NO: 22, amino acid 128 to 214 of SEQ ID NO:23, amino acids 129 to 215 of SEQ ID NO: 24, amino acid 125 to 211 of SEQ ID NO:25, amino acids 131 to 213 of SEQ ID NO: 26, amino acid 127 to 213 of SEQ ID NO:27, amino acids 125 to 211 of SEQ ID NO: 28, amino acid 126 to 212 of SEQ ID NO:29, amino acids 129 to 213 of SEQ ID NO: 30, amino acid 132 to 217 of SEQ ID NO:31 , amino acids 121 to 207 of SEQ ID NO: 32, amino acid 129 to 214 of SEQ ID NO:33, amino acids 127 to 213 of SEQ ID NO: 34, amino acid 128 to 211 of SEQ ID NO:35, amino acids 54 to 138 of SEQ ID NO: 36, amino acid 56 to 139 of SEQ ID NO:37, amino acids
  • coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA.
  • the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • control sequences means nucleic acid sequences necessary for expression of a polynucleotide encoding a polypeptide of the present invention.
  • Each control sequence may be native (i.e., from the same gene) or heterologous (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or heterologous to each other.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
  • expression means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
  • expression vector means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
  • fragment means a polypeptide, a catalytic domain, or a binding module having one or more ( e.g ., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has amidase activity.
  • the term "host cell” means any microbial or plant cell into which a nucleic acid construct or expression vector comprising a polynucleotide of the present invention has been introduced. Methods for introduction include but are not limited to protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. In some embodiments, the host cell is an isolated recombinant host cell that is partially or completely separated from at least one other component with, including but not limited to, proteins, nucleic acids, cells, etc.
  • hybrid polypeptide means a polypeptide comprising domains from two or more polypeptides, e.g., a binding module from one polypeptide and a catalytic domain from another polypeptide.
  • the domains may be fused at the N-terminus or the C-terminus.
  • hybridization means the pairing of substantially complementary strands of nucleic acids, using standard Southern blotting procedures. Hybridization may be performed under medium, medium-high, high or very high stringency conditions. Medium stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 55°C.
  • Medium-high stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 60°C.
  • High stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 65°C.
  • Very high stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 70°C.
  • isolated means a polypeptide, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component with which it is naturally associated as found in nature, including but not limited to, for example, other proteins, nucleic acids, cells, etc.
  • An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide.
  • mature polypeptide means a polypeptide in its mature form following N-terminal processing (e.g., removal of signal peptide).
  • the mature polypeptide is amino acids 21-236 of SEQ ID NO:22 based on the SignalP 3.0 program (Bendtsen et al., 2004, J.
  • the mature polypeptide is amino acids 21-238 of SEQ ID NO:23 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 23 are a signal peptide.
  • the mature polypeptide is amino acids 21-239 of SEQ ID NO:24 based on the SignalP 3.0 program
  • the mature polypeptide is amino acids 21-235 of SEQ ID NO:25 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783- 795) that predicts amino acids 1 to 20 of SEQ ID NO: 25 are a signal peptide.
  • the mature polypeptide is amino acids 21-237 of SEQ ID NO:26 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol.
  • the mature polypeptide is amino acids 21- 237 of SEQ ID NO:27 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 27 are a signal peptide.
  • the mature polypeptide is amino acids 21-235 of SEQ ID NO:28 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 28 are a signal peptide.
  • the mature polypeptide is amino acids
  • the mature polypeptide is amino acids 21-236 of SEQ ID NO:30 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 29 are a signal peptide.
  • the mature polypeptide is amino acids 21-236 of SEQ ID NO:30 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 30 are a signal peptide.
  • the mature polypeptide is amino acids
  • the mature polypeptide is amino acids 21-230 of SEQ ID NO:32 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 32 are a signal peptide.
  • the mature polypeptide is amino acids 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 33 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-239 of SEQ ID NO:34 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 34 are a signal peptide. In one aspect, the mature polypeptide is amino acids
  • the mature polypeptide is amino acid 17-161 of SEQ ID NO:36 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 16 of SEQ ID NO: 36 are a signal peptide.
  • the mature polypeptide is amino acids
  • the mature polypeptide is amino acids 21-242 of SEQ ID NO:38 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 38 are a signal peptide.
  • the mature polypeptide is amino acids
  • the mature polypeptide is amino acids 21-235 of SEQ ID NO:40 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 40 are a signal peptide.
  • the mature polypeptide is amino acids 21-235 of SEQ ID NO:40 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 40 are a signal peptide.
  • the mature polypeptide is amino acids
  • the mature polypeptide is amino acids 20-242 of SEQ ID NO:42 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 19 of SEQ ID NO: 42 are a signal peptide.
  • the mature polypeptide is amino acids 20-242 of SEQ ID NO:42 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 19 of SEQ ID NO: 42 are a signal peptide.
  • the mature polypeptide is amino acids
  • mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide having amidase activity.
  • the mature polypeptide coding sequence is nucleotides 61 to 711 of SEQ ID NO: 1. Nucleotides 1-60 of SEQ ID NO:1 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:2 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 403, 477-793 of SEQ ID NO: 3 or cDNA sequences thereof.
  • Nucleotides 1-60 of SEQ ID NO:3 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 391 , 456 to 772 of SEQ ID NO: 4 or cDNA sequences thereof.
  • Nucleotides 1-60 of SEQ ID NO:4 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 397, 470 to 786 of SEQ ID NO: 5 or cDNA sequences thereof.
  • Nucleotides 1-60 of SEQ ID NO:5 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 391 , 455 to 771 of SEQ ID NO: 6 or cDNA sequences thereof.
  • Nucleotides 1-60 of SEQ ID NO:6 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 391 , 452-768 of SEQ ID NO: 7 or cDNA sequences thereof.
  • Nucleotides 1-60 of SEQ ID NO:7 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 394, 455 to 771 of SEQ ID NO: 8 or cDNA sequences thereof.
  • Nucleotides 1-60 of SEQ ID NO:8 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 397, 475 to 788 of SEQ ID NO: 9 or cDNA sequences thereof.
  • Nucleotides 1-60 of SEQ ID NO:9 are signal.
  • the mature polypeptide coding sequence is nucleotides 64 to 411 , 492 to 800 of SEQ ID NO: 10 or cDNA sequences thereof.
  • Nucleotides 1-63 of SEQ ID NO:10 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 379, 482-795 of SEQ ID NO: 11 or cDNA sequences thereof.
  • Nucleotides 1-60 of SEQ ID NO:11 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 717 of SEQ ID NO: 12.
  • Nucleotides 1- 60 of SEQ ID NO: 12 are signal.
  • the mature polypeptide coding sequence is nucleotides 61 to 720 of SEQ ID NO: 13. Nucleotides 1-60 of SEQ ID NO:13 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 708 of SEQ ID NO: 14. Nucleotides 1-60 of SEQ ID NO:14 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 49 to 486 of SEQ ID NO: 15. Nucleotides 1-48 of SEQ ID NO: 15 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 55 to 492 of SEQ ID NO: 16. Nucleotides 1-54 of SEQ ID NO:16 are signal. In one aspect, the mature
  • polypeptide coding sequence is nucleotides 61 to 412, 463-779 of SEQ ID NO: 17 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:17 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 64 to 756 of SEQ ID NO: 18. Nucleotides 1-63 of SEQ ID NO:18 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 708 of SEQ ID NO: 19. Nucleotides 1-60 of SEQ ID NO:19 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 58 to 693 of SEQ ID NO: 20.
  • Nucleotides 1-57 of SEQ ID NO:20 are signal.
  • the mature polypeptide coding sequence is nucleotides 58 to 729 of SEQ ID NO: 21.
  • Nucleotides 1-57 of SEQ ID NO:21 are signal.
  • nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
  • operably linked means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
  • Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3).
  • Suitable protease in accordance with the present disclosure includes enzymes capable of cleaving the amide linkages in protein, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H.
  • purified means a nucleic acid or polypeptide that is substantially free from other components as determined by analytical techniques well known in the art (e.g ., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation).
  • a purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight on a molar basis).
  • a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique.
  • the term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.
  • recombinant when used in reference to a cell, nucleic acid, protein or vector, means that it has been modified from its native state.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.
  • Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g., a heterologous promoter in an expression vector.
  • Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences.
  • a vector comprising a nucleic acid encoding a polypeptide is a recombinant vector.
  • the term“recombinant” is synonymous with“genetically modified” and“transgenic”.
  • the sequence identity between two amino acid sequences is determined as the output of“longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later.
  • the parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the Needle program In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line.
  • the sequence identity between two polynucleotide sequences is determined as the output of“longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later.
  • the parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the Needle program In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line.
  • variant means a polypeptide having amidase activity comprising an alteration, i.e. , a substitution, insertion, and/or deletion, at one or more (e.g., several) positions.
  • a substitution means replacement of the amino acid occupying a position with a different amino acid;
  • a deletion means removal of the amino acid occupying a position; and
  • an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.
  • the invention relates, in one aspect to amidase enzymes having a CHAP domain.
  • CHAP is the amidase domain of bifunctional Escherichia coli glutathionylspermidine
  • the CHAP domain is a region between 110 and 140 amino acids that is found in proteins from bacteria, bacteriophages, archaea and eukaryotes of the family Trypanosomidae.
  • the domain is named after the acronym cysteine, histidine-dependent amidohydrolases/peptidases. Many of these proteins are uncharacterised, but it has been proposed that they may function mainly in peptidoglycan hydrolysis.
  • CHAP domain is found in a wide range of protein architectures; it is commonly associated with bacterial type SH3 domains and with several families of amidase domains. It has been suggested that CHAP domain containing proteins utilise a catalytic cysteine residue in a nucleophilic-attack mechanism.
  • the CHAP domain contains two invariant residues, a cysteine and a histidine. These residues form part of the putative active site of CHAP domain containing proteins. Secondary structure predictions show that the CHAP domain belongs to the alpha + beta structural class, with the N-terminal half largely containing predicted alpha helices and the C-terminal half principally composed of predicted beta strands.
  • Methanosarcina mazei protein MM2478 a putative chloride channel.
  • Cysteine peptidases belonging to MEROPS peptidase family C51 D-alanyl- glycyl endopeptidase, clan CA).
  • the polypeptides of SEQ ID Nos:22-42 all contain CHAP domain.
  • the present invention relates to a CHAP domain having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to amino acids 126 to 212 of SEQ ID NO: 22, amino acid 128 to 214 of SEQ ID NO:23, amino acids 129 to 215 of SEQ ID NO: 24, amino acid 125 to 211 of SEQ ID NO:25, amino acids 131 to 213 of SEQ ID NO: 26, amino acid 127 to 213 of SEQ ID NO:27, amino acids 125 to 211 of SEQ ID NO: 28, amino acid 126 to 212 of SEQ ID NO:29, amino acids 129 to 213 of SEQ ID NO: 30,
  • amidase enzyme having a CHAP domain is an isolated or purified polypeptide having amidase activity, selected from the group consisting of:
  • the fragment of the polypeptide of (a), (b), or (e) that has amidase activity typically as a chain length of at least 40% the chain length of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 80% of the amino acids of the CHAP domain, such as a chain length of at least 50% the chain length of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 80% of the amino acids of the CHAP domain, such as chain length of at least 60% of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 80% of the amino acids of the CHAP domain, such as chain length of at least 70% of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 80% of the amino acids of the CHAP domain
  • the invention is directed to the use of a polypeptide with amidase activity comprising a CHAP domain for the treatment of sludge.
  • the present invention relates to isolated or purified polypeptides having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the mature polypeptide of SEQ ID NOs: 22-42, which have amidase activity.
  • polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NOs: 22-42.
  • disclosure is directed to an isolated or purified polypeptide having amidase activity and a CHAP domain is selected from the group consisting of
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 22 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 23 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 24 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 25 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 125 to 21 1 of SEQ ID NO:25,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 29 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 31 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 132 to 217 of SEQ ID NO:31 ,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 32 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 121 to 207 of SEQ ID NO: 32,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 33 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 129 to 214 of SEQ ID NO:33,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 35 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid
  • the disclosure is directed to an isolated or purified polypeptide having amidase activity and a CHAP domain is selected from the group consisting of : i. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 236 of SEQ ID NO: 22 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 126 to 212 of SEQ ID NO: 22,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 238 of SEQ ID NO: 23 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 128 to 214 of SEQ ID NO:23,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 239 of SEQ ID NO: 24 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 129 to 215 of SEQ ID NO: 24,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 25 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 125 to 21 1 of SEQ ID NO:25,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 237 of SEQ ID NO: 26 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 131 to 213 of SEQ ID NO: 26,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 237 of SEQ ID NO: 27 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 127 to 213 of SEQ ID NO:27,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 236 of SEQ ID NO: 30 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 129 to 213 of SEQ ID NO: 30,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 22 to 239 of SEQ ID NO: 31 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 132 to 217 of SEQ ID NO:31 ,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 238 of SEQ ID NO: 33 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 129 to 214 of SEQ ID NO:33,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 239 of SEQ ID NO: 34 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 127 to 213 of SEQ ID NO: 34,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 35 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 128 to 21 1 of SEQ ID NO:35,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 17 to 161 of SEQ ID NO: 36 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 54 to 138 of SEQ ID NO: 36,
  • xvii a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 242 of SEQ ID NO: 38 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 136 to 218 of SEQ ID NO: 38, xviii.
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 22 to 251 of SEQ ID NO: 39 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 141 to 227 of SEQ ID NO:39,
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 20 to 230 of SEQ ID NO: 41 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 122 to 208 of SEQ ID NO:41 , and xxi.
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 20 to 242 of SEQ ID NO: 42 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 131 to 218 of SEQ ID NO: 42.
  • the isolated or purified polypeptide having amidase activity and a CHAP domain is selected from the group consisting of :
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 236 of SEQ ID NO: 22 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 126 to 212 of SEQ ID NO: 22, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.22, such as a chain length 95% to 105% of SEQ ID N0.22;
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 238 of SEQ ID NO: 23 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 128 to 214 of SEQ ID N0:23, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.23, such as a chain length 95% to 105% of SEQ ID N0.23;
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 239 of SEQ ID NO: 24 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 129 to 215 of SEQ ID NO: 24, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.24, such as a chain length 95% to 105% of SEQ ID N0.24;
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 25 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 125 to 21 1 of SEQ ID NO:25, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.25, such as a chain length 95% to 105% of SEQ ID N0.25;
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 237 of SEQ ID NO: 26 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 131 to 213 of SEQ ID NO: 26, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.26, such as a chain length 95% to 105% of SEQ ID N0.26;
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 237 of SEQ ID NO: 27 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 127 to 213 of SEQ ID NO:27, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.27, such as a chain length 95% to 105% of SEQ ID N0.27;
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 239 of SEQ ID NO: 34 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 127 to 213 of SEQ ID NO: 34, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.34, such as a chain length 95% to 105% of SEQ ID N0.34;
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 35 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 128 to 21 1 of SEQ ID NO:35, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.35, such as a chain length 95% to 105% of SEQ ID N0.35;
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 17 to 161 of SEQ ID NO: 36 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 54 to 138 of SEQ ID NO: 36, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.36, such as a chain length 95% to 105% of SEQ ID N0.36;
  • polypeptide having at least 70% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 242 of SEQ ID NO: 38 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 136 to 218 of SEQ ID NO: 38, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.38, such as a chain length 95% to 105% of SEQ ID N0.38;
  • the polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 22-42 or the mature polypeptide thereof; or is a fragment thereof having amidase activity.
  • the fragment comprises or contains amino acids 21- 236 of SEQ ID NO:22, amino acids 21-238 of SEQ ID NO:23, amino acids 21-239 of SEQ ID NO:24, amino acids 21-235 of SEQ ID NO:25, amino acids 21-237 of SEQ ID NO:26, amino acids 21-237 of SEQ ID NO:27, amino acids 21-235 of SEQ ID NO:28, amino acids 21-236 of SEQ ID NO:29, amino acids 21-236 of SEQ ID NO:30, amino acids 22-239 of SEQ ID NO:31 , amino acids 21-230 of SEQ ID NO:32, amino acids 21-238 of SEQ ID NO:33, amino acid 21- 239 of SEQ ID NO:34, amino acids 21-235 of SEQ ID NO:35, amino acid 17-161 of SEQ ID
  • the fragment of the polypeptide of the invention may comprise 1 to 30, typically 1 to 20, such as 1 to 10 amino acid substitutions, deletions and/or insertions within the sequence defined by any one of amino acids 21-236 of SEQ ID NO:22, amino acids 21-238 of SEQ ID NO:23, amino acids 21-239 of SEQ ID NO:24, amino acids 21-235 of SEQ ID NO:25, amino acids 21-237 of SEQ ID NO:26, amino acids 21-237 of SEQ ID NO:27, amino acids 21-235 of SEQ ID NO:28, amino acids 21-236 of SEQ ID NO:29, amino acids 21-236 of SEQ ID NO:30, amino acids 22-239 of SEQ ID NO:31 , amino acids 21-230 of SEQ ID NO:32, amino acids 21- 238 of SEQ ID NO:33, amino acid 21-239 of SEQ ID NO:34, amino acids 21-235 of SEQ ID NO:35, amino acid 17-161 of SEQ ID NO:36, amino acids 19-163 of SEQ ID NO:37
  • the fragment of the polypeptide of the invention may comprise 1 to 30 amino acid amino- or carboxyl-terminal extensions to the sequence defined by any one of amino acids 21-236 of SEQ ID NO:22, amino acids 21-238 of SEQ ID NO:23, amino acids 21-239 of SEQ ID NO:24, amino acids 21-235 of SEQ ID NO:25, amino acids 21-237 of SEQ ID NO:26, amino acids 21-237 of SEQ ID NO:27, amino acids 21- 235 of SEQ ID NO:28, amino acids 21-236 of SEQ ID NO:29, amino acids 21-236 of SEQ ID NO:30, amino acids 22-239 of SEQ ID NO:31 , amino acids 21-230 of SEQ ID NO:32, amino acids 21-238 of SEQ ID NO:33, amino acid 21-239 of SEQ ID NO:34, amino acids 21-235 of SEQ ID NO:35, amino acid 17-161 of SEQ ID NO:36, amino acids 19-163 of SEQ ID NO:37, amino acid 21-242 of SEQ
  • the present invention relates to isolated or purified polypeptides having amidase activity encoded by polynucleotides that hybridize under medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of SEQ ID NOs: 1-21 or the cDNA thereof (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).
  • the present invention relates to variants of any one of the mature polypeptides of SEQ ID NOs: 22-42 comprising a substitution, deletion, and/or insertion at one or more ( e.g ., several) positions.
  • the number of amino acid substitutions, deletions and/or insertions introduced into any of the mature polypeptides of SEQ ID NO: 22-42 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding module.
  • Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for amidase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708.
  • the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899- 904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64.
  • the identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
  • Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
  • Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No.
  • Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et ai., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
  • the polypeptide may be a hybrid polypeptide or a fusion polypeptide.
  • polypeptides of the present invention have CHAP domain.
  • a polypeptide having amidase activity of the present invention may be obtained from microorganisms of any genus.
  • the term“obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted.
  • the polypeptide obtained from a given source is secreted extracellularly.
  • the polypeptide of SEQ ID NO:22 is a Zopfiella polypeptide, e.g., a polypeptide obtained from Zopfiella sp. t180-6.
  • the polypeptide of SEQ ID NO:23 is a Subramaniula anamorphosa polypeptide.
  • the polypeptide of SEQ ID NO:24 is a Staphylotrichum boninense polypeptide.
  • the polypeptide of SEQ ID NO:25 is a Chaetomium megalocarpum polypeptide.
  • the polypeptide of SEQ ID NO:26 is a Chaetomium polypeptide, e.g., a polypeptide obtained from Chaetomium sp. ZY089.
  • the polypeptide of SEQ ID NO:27 is Thieiavia polypeptide, e.g., a polypeptide obtained from Thieiavia sp. ZY346.
  • the polypeptide of SEQ ID NO:28 is a Chaetomium jodhpurense polypeptide.
  • the polypeptide of SEQ ID NO:29 is a Taifanglania major polypeptide.
  • polypeptide of SEQ ID NO:30 is a Thermothelomyces hinnulea polypeptide.
  • polypeptide of SEQ ID NO:31 is a Humicola hyalothermophila polypeptide.
  • polypeptide of SEQ ID NO:32 is a
  • the polypeptide of SEQ ID NO:33 and 45 are Zopfiella latipes polypeptides.
  • the polypeptide of SEQ ID NO:34 is a Trichocladium asperum polypeptide.
  • the polypeptide of SEQ ID NO:35 is a Fusarium neocosmosporiellum polypeptide.
  • the polypeptide of SEQ ID NO:36 is a Sporormia fimetaria polypeptide.
  • the polypeptide of SEQ ID NO:37 is a Simplicillium obclavatum polypeptide.
  • the polypeptide of SEQ ID NO:38 is a Chaetomium polypeptide, e.g., a polypeptide obtained from Chaetomium sp. ZY474.
  • the polypeptide of SEQ ID NO:39 is a Geastrales polypeptide, e.g., a polypeptide obtained from Geastrales sp. LC1927.
  • the polypeptide of SEQ ID NO:40 is a
  • polypeptide of SEQ ID NO:41 is a
  • Gliomastix polypeptide e.g., a polypeptide obtained from Gliomastix sp-71728.
  • the polypeptide of SEQ ID NO:42 is a Sarocladium polypeptide, e.g., a polypeptide obtained from Sarocladium sp. XZ2014.
  • the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
  • ATCC American Type Culture Collection
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • the polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above- mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample.
  • the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
  • the present invention also relates to isolated polynucleotides encoding a polypeptide of the present invention, as described herein.
  • the present invention relates to isolated or purified polypeptides having amidase activity encoded by polynucleotides that hybridize under medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of SEQ ID NOs: 1-21 or the cDNA thereof (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).
  • polynucleotides of SEQ ID NOs: 1-21 or a subsequence thereof, as well as the mature polypeptides of SEQ ID NOs: 22-42 or a fragment thereof may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having amidase activity from strains of different genera or species according to methods well known in the art. Such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the
  • the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length.
  • Both DNA and RNA probes can be used.
  • the probes are typically labeled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
  • a genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having amidase activity.
  • Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
  • DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or another suitable carrier material.
  • the carrier material is used in a Southern blot.
  • hybridization indicates that the polynucleotides hybridize to a labeled nucleic acid probe corresponding to (i) SEQ ID NOs: 1-21 ; (ii) the mature polypeptide coding sequences of SEQ ID NOs: 1-21 ; (iii) the cDNA sequence thereof; (iv) the full-length complement thereof; or (v) a subsequence thereof; under medium to very high stringency conditions.
  • Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
  • the nucleic acid probe is a polynucleotide that encodes the mature polypeptide of any of SEQ ID NOs: 22-42; or a fragment thereof.
  • the nucleic acid probe is SEQ ID NO: 1 or nucleotides 61 to 711 of SEQ ID NO: 1 , SEQ ID NO: 2 or nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2, SEQ ID NO: 3 or nucleotides 61 to 403, 477-793 of SEQ ID NO: 3, SEQ ID NO: 4 or nucleotides 61 to 391 , 456 to 772 of SEQ ID NO:
  • SEQ ID NO: 9 or nucleotides 61 to 397, 475 to 788 of SEQ ID NO: 9, SEQ ID NO: 10 or nucleotides 64 to 41 1 , 492 to 800 of SEQ ID NO: 10, SEQ ID NO: 11 or nucleotides 61 to 379, 482-795 of SEQ ID NO: 11 , SEQ ID NO: 12 or nucleotides 61 to 717 of SEQ ID NO: 12, SEQ ID NO: 13 or nucleotides 61 to 720 of SEQ ID NO: 13, SEQ ID NO: 14 or nucleotides 61 to 708 of SEQ ID NO: 14, SEQ ID NO: 15 or nucleotides 49 to 486 of SEQ ID NO: 15, SEQ ID NO: 16 or nucleotides 55 to 492 of SEQ ID NO: 16, SEQ ID NO: 17 or nucleotides 61 to 412, 463-779 of SEQ ID NO: 17, SEQ ID NO: 18 or nucleotides 64 to 756 of
  • the present invention relates to isolated polypeptides having amidase activity encoded by polynucleotides having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the mature polypeptide coding sequence of SEQ ID
  • the techniques used to isolate or clone a polynucleotide include isolation from genomic DNA or cDNA, or a combination thereof.
  • the cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et ai, 1990, PCR: A Guide to Methods and Application, Academic Press, New York.
  • Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligation activated transcription
  • NASBA polynucleotide-based amplification
  • the polynucleotides may be cloned from a strain of Zopfiella sp. t180-6, Subramaniula anamorphosa, Staphylothchum boninense, Chaetomium megalocarpum, Chaetomium sp. ZY089, Thieiavia sp.
  • Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary for synthesizing polypeptides substantially similar to the polypeptide.
  • the term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide.
  • These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like.
  • the variants may be constructed on the basis of the polynucleotide presented as the mature polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA sequences thereof, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence.
  • nucleotide substitution see, e.g., Ford et ai, 1991 , Protein Expression and Purification 2: 95-107.
  • the present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • the polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
  • the control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention.
  • the promoter contains transcriptional control sequences that mediate the expression of the polypeptide.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene ( amyQ ), Bacillus licheniformis alpha-amylase gene ( amyL ), Bacillus licheniformis penicillinase gene ( penP ), Bacillus stearothermophilus
  • amyM maltogenic amylase gene
  • sacB Bacillus subtilis levansucrase gene
  • Bacillus subtilis xylA and xylB genes Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994,
  • E. col i lac operon E. coli trc promoter (Egon et ai, 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene ( dagA ), and prokaryotic beta- lactamase gene (Villa-Kamaroff et ai., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25).
  • promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase ( glaA ), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae those phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn
  • Rhizomucor miehei aspartic proteinase Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei x ylanase I, Trichoderma reesei xylanase II, Trichoderma reesei x ylanase III, Trichoderma reesei beta-xylosidase, and
  • Trichoderma reesei translation elongation factor as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha- amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof.
  • Other promoters are described in U.S. Patent No. 6,011 ,147.
  • useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1 , ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.
  • ENO-1 Saccharomyces cerevisiae enolase
  • GAL1 Saccharomyces cerevisiae galactokinase
  • ADH1 Alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
  • TPI Saccharomyces cerevisiae trios
  • the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
  • the terminator is operably linked to the 3’-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
  • Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease ( aprH ), Bacillus licheniformis alpha-amylase ( amyL ), and Escherichia coli ribosomal RNA ( rrnB ).
  • Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma ree
  • Trichoderma reesei translation elongation factor Trichoderma reesei translation elongation factor.
  • Preferred terminators for yeast host cells are obtained from the genes for
  • Saccharomyces cerevisiae enolase Saccharomyces cerevisiae cytochrome C (CYC1)
  • Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase Other useful terminators for yeast host cells are described by Romanos et ai, 1992, supra.
  • control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
  • mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et ai., 1995, J. Bacteriol. 177: 3465-3471).
  • the control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell.
  • the leader is operably linked to the 5’-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.
  • Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans those phosphate isomerase.
  • Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase,
  • Saccharomyces cerevisiae alpha-factor Saccharomyces cerevisiae alcohol
  • ADH2/GAP dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
  • the control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
  • Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
  • control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell’s secretory pathway.
  • the 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide.
  • the 5’-end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence.
  • a heterologous signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence.
  • a heterologous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance secretion of the polypeptide.
  • any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
  • Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus
  • stearothermophilus alpha-amylase Bacillus stearothermophilus neutral proteases ( nprT , nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiol. Rev. 57: 109-137.
  • Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
  • Useful signal peptides for yeast host cells are obtained from the genes for
  • Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase are described by Romanos et ai, 1992, supra.
  • the control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease ( aprE ), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
  • the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used.
  • the Aspergillus niger glucoamylase promoter In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used.
  • Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the
  • polynucleotide encoding the polypeptide would be operably linked to the regulatory sequence.
  • the present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals.
  • the various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites.
  • the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g ., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may be a linear or closed circular plasmid.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial
  • the vector may contain any means for assuring self-replication.
  • the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance.
  • Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.
  • Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosyl- aminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5’-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.
  • adeA phosphoribosylaminoimidazole-succinocarboxamide synthase
  • adeB phospho
  • Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.
  • the selectable marker may be a dual selectable marker system as described in WO 2010/039889.
  • the dual selectable marker is a hph-tk dual selectable marker system.
  • the vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination.
  • the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s).
  • the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.
  • the term“origin of replication” or“plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
  • bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and rAMb1 permitting replication in Bacillus.
  • origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1 , ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
  • AMA1 and ANSI examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et at., 1991 , Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163- 9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO
  • More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide.
  • An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the
  • polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
  • the present invention also relates to recombinant host cells, comprising a
  • polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.
  • a construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
  • the choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the polypeptide is heterologous to the recombinant host cell.
  • At least one of the one or more control sequences is
  • the recombinant host cell comprises at least two copies, e.g., three, four, or five, of the polynucleotide of the present invention.
  • the host cell may be any microbial or plant cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.
  • the prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.
  • Gram positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus,
  • Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas,
  • the bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
  • the bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.
  • the bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
  • the introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987,
  • the introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or
  • the introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et ai, 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g., Mazodier et ai., 1989, J. Bacteriol. 171 : 3583-3585), or transduction (see, e.g., Burke et ai, 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294).
  • the introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et ai, 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g., Mazodier et ai., 1989, J. Bacteriol. 171
  • Pseudomonas cell may be effected by electroporation (see, e.g., Choi et ai., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.
  • the host cell may be a fungal cell.“Fungi” as used herein includes the phyla
  • the fungal host cell may be a yeast cell.“Yeast” as used herein includes
  • yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
  • the yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,
  • Saccharomyces Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kiuyveri, Saccharomyces norbensis,
  • Saccharomyces oviformis Saccharomyces oviformis, or Yarrowia lipolytica cell.
  • the fungal host cell may be a filamentous fungal cell.“Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
  • the filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
  • the filamentous fungal host cell may be an Aspergillus awamori
  • Ceriporiopsis subrufa Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium iucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookweiiense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporo
  • Trichoderma harzianum Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et ai, 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
  • the present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.
  • the present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.
  • the host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art.
  • the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid-state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from
  • polypeptide If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
  • the polypeptide may be detected using methods known in the art that are specific for the polypeptides. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide
  • the polypeptide may be recovered using methods known in the art.
  • the polypeptide may be recovered from the fermentation medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • a whole fermentation broth comprising the polypeptide is recovered.
  • the polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • SDS-PAGE or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989)
  • the present invention also relates to a fermentation broth formulation or a cell composition comprising a polypeptide of the present invention.
  • the fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding the polypeptide of the present invention which are used to produce the polypeptide of interest), cell debris, biomass, fermentation media and/or fermentation products.
  • the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.
  • fermentation broth refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification.
  • fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium.
  • the fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation.
  • the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation.
  • the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.
  • the fermentation broth formulation and cell compositions comprise a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof.
  • the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid,
  • the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris.
  • the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these
  • the fermentation broth formulations or cell compositions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
  • a preservative and/or anti-microbial agent including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
  • the cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation.
  • the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon- limiting conditions to allow protein synthesis.
  • the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells.
  • the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.
  • a whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
  • the whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673.
  • Enzyme Compositions may be produced by a method described in WO 90/15861 or WO 2010/096673.
  • the present invention also relates to compositions comprising a polypeptide with amidase activity comprising a CHAP domain.
  • the enzyme composition of the present invention further comprises a protease.
  • the protease is a serine proteases.
  • a serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 "Principles of Biochemistry,” Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
  • the bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease.
  • the protease is a subtilase.
  • Subtilase refers to a sub-group of the serine proteases as proposed by Siezen et ai, Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases.
  • a subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et ai. now is a subgroup of the subtilases.
  • a wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al.( 1997).
  • subtilisin 168 subtilisin 168
  • subtilisin BPN' subtilisin BPN'
  • subtilisin Carlsberg ALCALASE ®
  • NOVOZYMES A/S subtilisin Carlsberg
  • subtilisin DY subtilisin DY
  • subtilases for use in accordance with the present disclosure includes the subtilases, I-S2 or high alkaline subtilisins, as recognized by Siezen et al. (supra).
  • Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprises enzymes such as subtilisin PB92 (BAALKP) (MAXACAL ® , Gist-Brocades NV), subtilisin 309
  • subtilisin 147 (BLS147) (ESPERASE ® , NOVOZYMES A/S), and alkaline elastase YaB (BSEYAB).
  • the suitable protease used in the present invention is subtilisin protease from B. Lentus as shown in WO 1999/027082 or WO 2003/006602, both of which are incorporated by reference in their entirety.
  • the enzyme composition comprises a protease at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to a protease as shown in SEQ ID NO:43.
  • Protease may be applied under conditions suitable to the sludge processing conditions, such as, for example, temperatures from 20 to 60°C, pH conditions from 4 to 10, and for a treatment time of 1 to 100 hours, 16 to 72 hours, or 1 , 2, 3, 4, 5, 6, 7 days.
  • conditions suitable to the sludge processing conditions such as, for example, temperatures from 20 to 60°C, pH conditions from 4 to 10, and for a treatment time of 1 to 100 hours, 16 to 72 hours, or 1 , 2, 3, 4, 5, 6, 7 days.
  • the enzyme composition further comprises a cellulase composition comprising xylanase and a cellulase preparation comprising beta-glucosidase and GH61A polypeptide.
  • the one or more (e.g., several) cellulase composition include a commercial cellulolytic enzyme preparation.
  • commercial cellulolytic enzyme preparations suitable for use in the present disclosure include, for example, CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S), CELLUCLASTTM (Novozymes A/S), NOVOZYMTM 188 (Novozymes A/S), CELLUZYMETM (Novozymes A/S), CEREFLOTM (Novozymes A/S), and ULTRAFLOTM (Novozymes A/S), ACCELERASETM (Genencor Int.), LAM IN EXTM (Genencor Int.), SPEZYMETM CP (Genencor Int.), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENTTM 7069 W (
  • the cellulase composition comprises an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a T choderma reesei cellulase preparation containing Aspergillus fumigatus beta- glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656).
  • the cellulase enzymes are added in amounts effective from about 0.005 wt % of solids, e.g., about 0.01 wt % of solids or about 0.1 wt % of solids.
  • the cellulase enzymes are added in amounts effective from about 0.005 to 0.1 wt % of solids.
  • the cellulase composition may be applied under conditions suitable to the sludge processing conditions, such as, for example, temperatures from 20 to 60°C, pH conditions from 4 to 10, and for a treatment time of 1 to 100 hours, 16 to 72 hours, or 1 , 2, 3, 4, 5, 6, 7 days.
  • the enzyme composition of the present invention may contain enzymatic component, e.g., a mono-component composition.
  • the compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or
  • transferase e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta- galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
  • transferase e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta- galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodext
  • deoxyribonuclease endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,
  • polyphenoloxidase polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
  • compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the compositions may be stabilized in accordance with methods known in the art.
  • the dosage of the composition and other conditions under which the composition is used may be determined on the basis of methods known in the art.
  • the present disclosure relates to an enzymatic method to facilitate and/or improve the process of dewatering sludge, such as sludge generated during conventional wastewater treatment.
  • An important aspect of the disclosure is directed to a method of treating sludge comprising the use of one or more amidase enzyme having a CHAP domain.
  • Sludges generated by the wastewater treatment industry are classified not only by the source of wastewater (e.g. municipal or industrial) but also by specific stages of the wastewater treatment process. In the broadest classification, sludge is considered primary, secondary or tertiary. Primary sludges are usually considered “raw” as they are often the result of settling of solids from raw wastewater influent passed across primary clarifiers. In most instances, the clarified water is then sent to activated sludge basins (ASBs) in which suspended floes of microorganisms remove soluble contaminants from the water. As the microorganisms replicate, they must be periodically removed from the ASB to avoid overgrowth.
  • ASBs activated sludge basins
  • This“secondary sludge” is considered“waste activated sludge” (WAS) and has a relatively universal presence at WWTPs employing biological nutrient removal (BNR) systems.
  • WAS biological nutrient removal
  • the sludge may be sent to aerobic (ambient aeration or pure oxygen) or anaerobic digesters which may be operated under either mesophilic or thermophilic conditions.
  • the resultant“tertiary” sludge is then known as “digested sludge” and may be further classified according to the specifics of digestion (e.g. thermophilic aerobically digested sludge). So, as can be seen, innumerable sludge types are produced during the treatment of wastewater. However, they can be loosely grouped as:
  • EPS extracellular polymeric substances
  • An aspect of the invention is directed to a method of treating sludge comprising i) adding an effective amount of one or more CHAP enzymes, or an enzyme composition comprising an effective amount of one or more CHAP enzymes, typically further comprising adding and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids and ii) separating the coagulated and flocculated solids from the water.
  • a suitable embodiment of the invention relates to method for treating sludge, comprising:
  • the method of the invention is suitable for all sludge(s) associated with conventional wastewater treatment specifically to improve
  • the enzyme compositions are applied to tertiary sludge(s) generated during treatment of industrial and municipal waste water.
  • the enzyme compositions of the present disclosure are applied to digested sludge form such as anaerobically or aerobically digested sludge.
  • a purpose of the present disclosure is to facilitate or improve the process of sludge dewatering including treating sludge with a combination of the polypeptides with CHAP domain and protease, prior to conventional sludge conditioning and dewatering operations.
  • steps further optional steps may be included, such as, for example, treating the sludge with enzymes post digestion and before dewatering stages.
  • enzyme composition of the present disclosure is contacted with sludge before mechanical dewatering of sludge in the waste water process stream.
  • the method of the invention and the enzyme composition in accordance with the present disclosure further comprise a protease together with polypeptides with amidase activity.
  • the enzyme composition in accordance with the present disclosure comprises a protease and polypeptides with CHAP domain.
  • the enzyme composition in accordance with the present disclosure further comprises a cellulase composition.
  • suitable amounts of the polypeptide with amidase activity of the present invention include 0.002 to 0.4 g protein per kg of total suspended solids, 0.01 to 0.1 g of protein per kg of total suspended solids, 0.02 to 0.08 g of protein per kg of total suspended solids.
  • said polypeptide is dosed at about 0.05g/kg TS (-50 ppm).
  • suitable amounts of the polypeptide with CHAP domain of the present invention include 0.002 to 0.4 g protein per kg of total suspended solids, 0.01 to 0.1 g of protein per kg of total suspended solids, 0.02 to 0.08 g of protein per kg of total suspended solids.
  • said polypeptide is dosed at about 0.05g/kg TS (-50 ppm).
  • suitable amounts of protease include 0.004 to 0.09 g protein per kg of total suspended solids, 0.005 to 0.03 g of protein per kg of total suspended solids, 0.010 to 0.025 g of protein per kg of total suspended solids. In embodiments, suitable protease is dosed at 0.011-0.025g/kg TS.
  • suitable amounts of cellulase composition used to combine with protease include 0.002 to 0.4 g protein per kg of total suspended solids, 0.01 to 0.1 g of protein per kg of total suspended solids, 0.04 to 0.09 g of protein per kg of total suspended solids. In embodiments, said cellulase composition is dosed at 0.045-0.09 g/kg TS.
  • the enzyme composition in accordance with this disclosure may be applied under conditions suitable to the sludge processing conditions, such as, for example, temperatures from 20 to 60°C, pH conditions from 4 to 10, and for a treatment time of 1 to 100 hours, 16 to 72 hours, or 1 , 2, 3, 4, 5, 6, 7 days.
  • conditions suitable to the sludge processing conditions such as, for example, temperatures from 20 to 60°C, pH conditions from 4 to 10, and for a treatment time of 1 to 100 hours, 16 to 72 hours, or 1 , 2, 3, 4, 5, 6, 7 days.
  • the present invention also relates to an isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 22, amino acids 1 to 20 of SEQ ID NO: 23, amino acids 1 to 20 of SEQ ID NO: 24, amino acids 1 to 20 of SEQ ID NO: 25, amino acids 1 to 20 of SEQ ID NO: 26, amino acids 1 to 20 of SEQ ID NO: 27, amino acids 1 to 20 of SEQ ID NO: 28, amino acids 1 to 20 of SEQ ID NO: 29, amino acids 1 to 20 of SEQ ID NO: 30, amino acids 1 to 21 of SEQ ID NO: 31 , amino acids 1 to 20 of SEQ ID NO: 32, amino acids 1 to 20 of SEQ ID NO: 33, amino acids 1 to 20 of SEQ ID NO: 34, amino acids 1 to 20 of SEQ ID NO: 35, amino acids 1 to 16 of SEQ ID NO: 36, amino acids 1 to 18 of SEQ ID NO: 37, amino acids 1 to 20 of SEQ ID NO: 38, amino acids 1 to 21 of SEQ ID NO
  • the present invention also relates to an isolated polynucleotide encoding a propeptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 22, amino acids 1 to 20 of SEQ ID NO: 23, amino acids 1 to 20 of SEQ ID NO: 24, amino acids 1 to 20 of SEQ ID NO: 25, amino acids 1 to 20 of SEQ ID NO: 26, amino acids 1 to 20 of SEQ ID NO: 27, amino acids 1 to 20 of SEQ ID NO: 28, amino acids 1 to 20 of SEQ ID NO: 29, amino acids 1 to 20 of SEQ ID NO: 30, amino acids 1 to 21 of SEQ ID NO: 31 , amino acids 1 to 20 of SEQ ID NO: 32, amino acids 1 to 20 of SEQ ID NO: 33, amino acids 1 to 20 of SEQ ID NO: 34, amino acids 1 to 20 of SEQ ID NO: 35, amino acids 1 to 16 of SEQ ID NO: 36, amino acids 1 to 18 of SEQ ID NO: 37, amino acids 1 to 20 of SEQ ID NO: 38, amino acids 1 to 21 of SEQ ID NO:
  • the polynucleotides may further comprise a gene encoding a protein, which is operably linked to the signal peptide and/or propeptide.
  • the protein is preferably heterologous to the signal peptide and/or propeptide.
  • the polynucleotide encoding the signal peptide is nucleotides 61 to 711 of SEQ ID NO: 1 , nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2, nucleotides 61 to 403, 477-793 of SEQ ID NO: 3, nucleotides 61 to 391 , 456 to 772 of SEQ ID NO: 4, nucleotides 61 to 397, 470 to 786 of SEQ ID NO: 5, nucleotides 61 to 391 , 455 to 771 of SEQ ID NO: 6, nucleotides 61 to 391 , 452-768 of SEQ ID NO: 7, nucleotides 61 to 394, 455 to 771 of
  • the polynucleotide encoding the propeptide is nucleotides 61 to 711 of SEQ ID NO: 1 , nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2, nucleotides 61 to 403, 477-793 of SEQ ID NO: 3, nucleotides 61 to 391 , 456 to 772 of SEQ ID NO: 4, nucleotides 61 to 397, 470 to 786 of SEQ ID NO: 5, nucleotides 61 to 391 , 455 to 771 of SEQ ID NO: 6, nucleotides 61 to 391 , 452-768 of SEQ ID NO: 7, nucleotides 61 to 394, 455 to 771 of SEQ ID NO: 8, nucleotides 61 to 397, 475 to 788 of SEQ ID NO: 9, nucleotides 64 to 411 , 492 to 800 of SEQ ID NO: 10, nucleotides 61 to 379,
  • the present invention also relates to nucleic acid constructs, expression vectors and recombinant host cells comprising such polynucleotides.
  • the present invention also relates to methods of producing a protein, comprising (a) cultivating a recombinant host cell comprising such polynucleotide; and optionally (b) recovering the protein.
  • the protein may be native or heterologous to a host cell.
  • the term“protein” is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and polypeptides.
  • the term“protein” also encompasses two or more polypeptides combined to form the encoded product.
  • the proteins also include hybrid polypeptides and fused polypeptides.
  • the protein is a hormone, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter.
  • the protein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase,
  • carbohydrase carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
  • the gene may be obtained from any prokaryotic, eukaryotic, or other source.
  • polypeptide having amidase activity selected from the group consisting of:
  • polypeptide having at least 60% sequence identity to mature polypeptide of any of SEQ ID NOs: 22-42 a polypeptide encoded by a polynucleotide that hybridizes under medium, medium-high, high or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA sequence thereof;
  • polypeptide of paragraph 1 having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to mature polypeptide of any of SEQ ID NOs: 22-42.
  • polypeptide of paragraphs 1 which is encoded by a polynucleotide that hybridizes under medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA thereof.
  • polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA thereof.
  • polypeptide of any one of paragraphs 1-4 which is a variant of the mature polypeptide of any of SEQ ID NOs: 22-42 comprising a substitution, deletion, and/or insertion at one or more positions.
  • polypeptide of any one of paragraphs 1-9 which is a fragment of any of SEQ ID NOs: 22-42 or the mature polypeptide thereof, wherein the fragment preferably contains amino acids 21-236 of SEQ ID NO:22, amino acids 21-238 of SEQ ID NO:23, amino acids 21-239 of SEQ ID NO:24, amino acids 21-235 of SEQ ID NO:25, amino acids 21-237 of SEQ ID NO:26, amino acids 21-237 of SEQ ID NO:27, amino acids 21-235 of SEQ ID NO:28, amino acids 21-236 of SEQ ID NO:29, amino acids 21-236 of SEQ ID NO:30, amino acids 22- 239 of SEQ ID NO:31 , amino acids 21-230 of SEQ ID NO:32, amino acids 21-238 of SEQ ID NO:33, amino acid 21-239 of SEQ ID NO:34, amino acids 21-235 of SEQ ID NO:35, amino acid 17-161 of SEQ ID NO:36, amino acids 19-163 of SEQ ID NO:37
  • polynucleotide of claim 8 which comprises SEQ ID NO: 1 or nucleotides 61 to 711 of SEQ ID NO: 1 , SEQ ID NO: 2 or nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2, SEQ ID NO: 3 or nucleotides 61 to 403, 477-793 of SEQ ID NO: 3, SEQ ID NO: 4 or nucleotides 61 to 391 , 456 to 772 of SEQ ID NO: 4, SEQ ID NO: 5 or nucleotides 61 to 397,
  • SEQ ID NO: 10 or nucleotides 64 to 411 , 492 to 800 of SEQ ID NO: 10, SEQ ID NO: 11 or nucleotides 61 to 379, 482-795 of SEQ ID NO: 11 , SEQ ID NO: 12 or nucleotides 61 to 717 of SEQ ID NO: 12, SEQ ID NO: 13 or nucleotides 61 to 720 of SEQ ID NO: 13, SEQ ID NO: 14 or nucleotides 61 to 708 of SEQ ID NO: 14, SEQ ID NO: 15 or nucleotides 49 to 486 of SEQ ID NO: 15, SEQ ID NO: 16 or nucleotides 55 to 492 of SEQ ID NO: 16, SEQ ID NO: 17 or nucleotides 61 to 412, 463-779 of SEQ ID NO: 17, SEQ ID NO: 18 or nucleotides 64 to 756 of SEQ ID NO: 18, SEQ ID NO: 19 or nucleotides
  • a nucleic acid construct or expression vector comprising the polynucleotide of paragraph 8 or 9, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.
  • a recombinant host cell comprising the polynucleotide of paragraph 8 or 9 operably linked to one or more control sequences that direct the production of the polypeptide.
  • the recombinant host cell of any one of paragraphs 11-14 which is a yeast recombinant host cell, e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. 16.
  • the recombinant host cell of any one of paragraphs 11-13 which is a yeast recombinant host cell, e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
  • filamentous fungal recombinant host cell e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, As
  • Trichoderma koningii Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
  • the recombinant host cell of any one of paragraphs 11-13 which is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,
  • Staphylococcus Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E. coli, Flavobacterium, Fusobacterium,
  • Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus
  • a method of producing the polypeptide of any one of paragraphs 1-7 comprising cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide.
  • a method of producing a polypeptide having amidase activity comprising cultivating the recombinant host cell of any one of paragraphs 1 1-17 under conditions conducive for production of the polypeptide.
  • amino acids 1 to 20 of SEQ ID NO: 22 An isolated or purified polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 22, amino acids 1 to 20 of SEQ ID NO: 23, amino acids 1 to 20 of SEQ ID NO: 24, amino acids 1 to 20 of SEQ ID NO: 25, amino acids 1 to 20 of SEQ ID NO: 26, amino acids 1 to 20 of SEQ ID NO: 27, amino acids 1 to 20 of SEQ ID NO: 28, amino acids 1 to 20 of SEQ ID NO: 29, amino acids 1 to 20 of SEQ ID NO: 30, amino acids 1 to 21 of SEQ ID NO: 31 , amino acids 1 to 20 of SEQ ID NO: 32, amino acids 1 to 20 of SEQ ID NO: 33, amino acids 1 to 20 of SEQ ID NO: 34, amino acids 1 to 20 of SEQ ID NO: 35, amino acids 1 to 16 of SEQ ID NO: 36, amino acids 1 to 18 of SEQ ID NO: 37, amino acids 1 to 20 of SEQ ID NO: 38, amino acids
  • polynucleotide of paragraph 22 further comprising a polynucleotide encoding a propeptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 22, amino acids 1 to 20 of SEQ ID NO: 23, amino acids 1 to 20 of SEQ ID NO: 24, amino acids 1 to 20 of SEQ ID NO: 25, amino acids 1 to 20 of SEQ ID NO: 26, amino acids 1 to 20 of SEQ ID NO: 27, amino acids 1 to 20 of SEQ ID NO: 28, amino acids 1 to 20 of SEQ ID NO: 29, amino acids 1 to 20 of SEQ ID NO: 30, amino acids 1 to 21 of SEQ ID NO: 31 , amino acids 1 to 20 of SEQ ID NO: 32, amino acids 1 to 20 of SEQ ID NO: 33, amino acids 1 to 20 of SEQ ID NO: 34, amino acids 1 to 20 of SEQ ID NO: 35, amino acids 1 to 16 of SEQ ID NO: 36, amino acids 1 to 18 of SEQ ID NO: 37, amino acids 1 to 20 of SEQ ID NO: 38, amino acids 1
  • a nucleic acid construct or expression vector comprising the polynucleotide of paragraph 22 or 23.
  • a recombinant host cell comprising a nucleic acid construct or expression vector of paragraph 24.
  • a method of producing a protein comprising cultivating the recombinant host cell of paragraph 25 under conditions conducive for production of the protein.
  • An isolated or purified polynucleotide encoding a propeptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 22, amino acids 1 to 20 of SEQ ID NO: 23, amino acids 1 to 20 of SEQ ID NO: 24, amino acids 1 to 20 of SEQ ID NO: 25, amino acids 1 to 20 of SEQ ID NO: 26, amino acids 1 to 20 of SEQ ID NO: 27, amino acids 1 to 20 of SEQ ID NO: 28, amino acids 1 to 20 of SEQ ID NO: 29, amino acids 1 to 20 of SEQ ID NO: 30, amino acids 1 to 21 of SEQ ID NO: 31 , amino acids 1 to 20 of SEQ ID NO: 32, amino acids 1 to 20 of SEQ ID NO: 33, amino acids 1 to 20 of SEQ ID NO: 34, amino acids 1 to 20 of SEQ ID NO: 35, amino acids 1 to 16 of SEQ ID NO: 36, amino acids 1 to 18 of SEQ ID NO: 37, amino acids 1 to 20 of SEQ ID NO: 38, amino acids 1 to 21 of SEQ ID NO: 39, amino acids
  • a nucleic acid construct or expression vector comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 28, wherein the gene is heterologous to the polynucleotide encoding the propeptide.
  • a recombinant host cell comprising a nucleic acid construct or expression vector of paragraph 29.
  • a method of producing a protein comprising cultivating the recombinant host cell of paragraph 30 under conditions conducive for production of the protein.
  • protease is Subtilisin protease with at least 90% sequence identity to SEQ ID NO:43.
  • the enzyme composition of paragraph 33 further comprising a Cellulase composition comprised an Aspergillus aculeatus GH10 xylanase and a T choderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase and Thermoascus aurantiacus GH61A polypeptide.
  • a method of treating sludge comprising:
  • protease is Subtilisin protease with at least 90% sequence identity to SEQ ID NO:43.
  • the enzyme composition of paragraph 38 further comprising a Cellulase composition comprised an Aspergillus aculeatus GH10 xylanase and a T choderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase and Thermoascus aurantiacus GH61A polypeptide.
  • a method of treating sludge comprising i) adding an effective amount of one or more CHAP enzymes, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids and ii) separating the coagulated and flocculated solids from the water.
  • a method for treating sludge comprising:
  • a method for improving sludge flocculation comprising i) adding one or more CHAP enzymes, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids.
  • a method for reducing polymer consumption comprising i) adding or more CHAP enzymes, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids.
  • a method of treating sludge comprising the addition of a protease and a cellulase to said sludge characterized in the further addition of an amidase comprising a CHAP- domain.
  • amidase comprising a CHAP- domain is a polypeptide as defined by any of embodiments 1 to 8.
  • PDA plates were composed of 39 grams of potato dextrose agar and deionized water to
  • YG agar plates were composed of 5 g of yeast extract, 10 g of glucose, 20 g of agar, and deionized water to 1 liter.
  • YPG medium was composed of 0.4% yeast extract, 0.1% KH 2 P0 4 , 0.05% MgS0 4 -7H 2 0, and 1.5% glucose in deionized water.
  • Horikoshi medium was prepared by adding 10g glucose, 5g polypeptone, 5g yeast extract, 1g K2HP04, MgS047H20, 15g agar in 900ml of distilled water, then Autoclaving at 121°C for 15mins and afterautoclaving aseptically adding 100ml of sterile 10% Na2C03, finally adjusting to pH 10 with 1mM NaOH.
  • LB plates were composed of 10g of Bacto-tryptone, 5g of yeast extract, 10g of sodium chloride, 15g of Bacto-agar, and deionized water to 1 liter.
  • LB medium was composed of 10g of Bacto-tryptone, 5g of yeast extract, and 10g of sodium chloride, and deionized water to 1 liter.
  • COVE-plate/slant medium was composed of 30g of sucrose, 20ml of COVE salt solution, 20g of agar, and deionized water up to 1 liter. Autoclave at 121 °C for 20mins.
  • COVE salt solution was composed of 26g of potassium chloride, 26g of magnesium sulfate heptahydrate, 76g of monopotassium phosphate, 50ml of COVE trace metal solution, and deionized water up to 1 liter.
  • COVE trace metal solution was composed of 0.04g of sodium tetraborate decahydrate, 0.4g of copper (II) sulfate pentahydrate, 0.8g of ferrous sulfate heptahydrate, 0.8g of manganese sulfate monohydrate, 0.8g of sodium molybdate dihydrate, 8g of zinc sulfate heptahydrate, and deionized water up to 1 liter.
  • amdS selection medium resolve the Cove medium and add 10ml of 1M acetamide (filter sterilized).
  • pyrG selection medium resolve the Cove medium and add 10ml of 1 M sodium nitrate (filter sterilized).
  • DAP4C-1 medium was composed of 0.5g yeast extract, 10g maltose, 20g glucose, 11 g magnesium sulfate heptahydrate, 1g monopotassium phosphate, 2.2g citric acid monohydrate, 5.2g potassium phosphate tribasic monohydrate, supplemented with 0.5ml of AMG Trace element solution, and deionized water up to 1 liter. Stir to resolve. Aliquot 400ml to a shake flask of 2L. Add 1 tablet of 0.5g calcium carbonate to each flask. After autoclave at 121 °C for 20mins, 3.3ml of 20% lactic acid and 9.3ml of 50% ammonium monohydric phosphate, both sterile, were added to each flask.
  • AMG Trace element solution was composed of 6.8g of zinc chloride, 2.5g of copper (II) sulfate pentahydrate, 0.5g of nickle chloride hexahydrate, 13.9g of ferrous sulfate heptahydrate, 8.225g of Manganese (II) sulfate monohydrate, 3g of citric acid and deionized water up to 1 liter.
  • the fungal strain NN046871 was isolated from soil samples collected from China, in 1998 by the dilution plate method with YG medium pH7, 37°C. It was then purified by transferring a single conidium onto a PDA agar plate.
  • the strain NN046871 was identified as Zopfiella sp. t180-6, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN075160 was isolated from soil samples collected from Gansu province, China, in 2016 by the dilution plate method with Horilkoshi medium at pH10, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate.
  • the strain NN075160 was isolated from soil samples collected from Gansu province, China, in 2016 by the dilution plate method with Horilkoshi medium at pH10, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain
  • NN075160 was identified as Subramaniula anamorphosa, based on both morphological characteristics and ITS rDNA sequence.
  • the strain NN071244 was isolated from soil samples collected from Shandong province, China, in 2015 by the dilution plate method with Horilkoshi medium at pH 10, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN071244 was identified as Staphylotrichum boninense, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN057881 was obtained from Prof. Cai Lei in Institute of Microbiology, CAS, in 2014.
  • the strain NN057881 was identified as Chaetomium megalocarpum, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN057922 was obtained from Prof. Cai Lei in Institute of Microbiology, CAS, in 2014.
  • the strain NN057922 was identified as Chaetomium sp. ZY089, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN057872 was obtained from Prof. Cai Lei in Institute of Microbiology, CAS, in 2014.
  • the strain NN057872 was identified as Thielavia sp. ZY346, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN057892 was obtained from Prof. Cai Lei in Institute of Microbiology, CAS, in 2014.
  • the strain NN057892 was identified as Chaetomium jodhpurense, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN046924 was isolated from soil samples collected from China, in 1998 by the dilution plate method with YG medium pH7, 45°C. It was then purified by transferring a single conidium onto a PDA agar plate.
  • the strain NN046924 was identified as Taifanglania major, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN009318 was from CBS with access number as CBS540.82.
  • the strain NN009318 was identified as Thermothelomyces hinnulea, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN000837 was from CBS with access number as CBS454.80.
  • the strain NN000837 was identified as Humicola hyalothermophila, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN000308 was from CBS with access number as CBS174.70.
  • the strain NN000308 was identified as Crassicarpon thermophilum, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN075508 was isolated from soil samples collected from Guangdong province, China, in 2016 by the dilution plate method with PDA medium, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate.
  • the strain NN075508 was identified as Zopfiella latipes, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN053773 was obtained from a collaboration with the Institute of Microbiology, CAS in 2011 , by the dilution plate method with PDA medium pH7, 10°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN053773 was identified as Trichocladium asperum, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN043500 was isolated from litter samples collected from China in 1998 by the dilution plate method with Horilkoshi medium at pH10, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate.
  • the strain NN043500 was identified as Fusarium neocosmosporiellum, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN047801 was isolated from litter samples collected from China, in 1998 by the dilution plate method with PDA medium pH7, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate.
  • the strain NN047801 was identified as Sporormia fimetaria, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN046572 was isolated from soil samples collected from China, in 1998 by the dilution plate method with PDA medium, pH7, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate.
  • the strain NN046572 was identified as Simplicillium obclavatum, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN057920 was obtained through a collaboration with Professor Cai Lei in Institude of Microbiology, CAS, in 2014. The strain was collected from China. It was identified as Chaetomium sp. ZY474, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN057890 was obtained through a collaboration with Professor Cai Lei in Institude of Microbiology, CAS, in 2014. The strain was collected from China. It was identified as Geastrales sp. LC1927, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN057353 was from CBS with access number as CBS 320.62.
  • the strain NN057353 was identified as Geomyces vinaceus, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN071728 was isolated from the enviromental samples collected from Jilin province, China, in 2015 by the dilution plate method with Horikoshi medium, pH10, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate.
  • the strain NN071728 was identified as Gliomastix sp-71728, based on both morphological characteristics and ITS rDNA sequence.
  • the fungal strain NN057128 was isolated from the enviromental samples collected from Jilin province, China, in 2013 by the dilution plate method with Horikoshi medium, pH10, 10°C. It was then purified by transferring a single conidium onto a PDA agar plate.
  • the strain NN057128 was identified as Sarocladium sp. XZ2014, based on both morphological characteristics and ITS rDNA sequence.
  • Strain Zopfiella sp. t180-6 was inoculated onto a PDA plate and incubated for 7 days at 37°C in the darkness.
  • Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • the mycelia were collected by scraping from agar plate with the sterilized scalpel and transferred to Lysing Matrix A tube (MP Biomedicals GmbH, Eschwege, Germany) and frozen under liquid nitrogen. Frozen mycelia were ground by MiniG1600 (SPEX SamplePrep LLC, New Jersey, United States), to a fine powder, and genomic DNA was isolated using DNeasy® Plant Mini Kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction. Genomic DNA extraction from strains of Staphylotrichum boninense
  • Strain Staphylotrichum boninense was inoculated onto a PDA plate and incubated for several days at 25°C in the darkness.
  • Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • Strain Thielavia sp. ZY346 was inoculated onto a PDA plate and incubated for several days at 37°C in the darkness.
  • Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • Strain Taifanglania major was inoculated onto a PDA plate and incubated for several days at 45°C in the darkness.
  • Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 45°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • Thermothelomyces hinnulea was inoculated onto a PDA plate and incubated for several days at 37°C in the darkness.
  • Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • Strain Humicola hyalothermophila was inoculated onto a PDA plate and incubated for several days at 25°C in the darkness.
  • Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • Strain Crassicarpon thermophilum was inoculated onto a PDA plate and incubated for several days at 37°C in the darkness.
  • Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 3 days at 37°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using Biospin Fungus Genomic DNA KIT (Bioer Technology Co. Ltd., Hangzhou, China) following the manufacturer’s instruction.
  • Strain Zopfiella latipes was inoculated onto a PDA plate and incubated for several days at 25°C in the darkness.
  • the mycelia were collected by scraping from agar plate with the sterilized scalpel and transferred to Lysing Matrix A tube (MP Biomedicals GmbH, Eschwege, Germany) and frozen under liquid nitrogen. Frozen mycelia were ground by MiniG1600 (SPEX SamplePrep LLC, New Jersey, United States), to a fine powder, and genomic DNA was isolated using DNeasy® Plant Mini Kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • Strain Trichocladium asperum was inoculated onto a PDA plate and incubated for 7 days at 15°C in the darkness.
  • Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 15°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction. Genomic DNA extraction from strains of Fusarium neocosmosporiellum
  • Strain Fusarium neocosmosporiellum was inoculated onto a PDA plate and incubated for 7 days at 25°C in the darkness.
  • mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • Strain Sporormia fimetaria was inoculated onto a PDA plate and incubated for 7 days at 28°C in the darkness.
  • Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 28°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using MP Fast DNA spin kit for soil (MP Biomedicals, Santa Ana, California, USA) following the manufacturer’s instruction.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24)
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • the mycelia were collected by scraping from agar plate with the sterilized scalpel and transferred to Lysing Matrix A tube (MP Biomedicals GmbH, Eschwege, Germany) and frozen under liquid nitrogen. Frozen mycelia were ground by MiniG1600 (SPEX SamplePrep LLC, New Jersey, United States), to a fine powder, and genomic DNA was isolated using DNeasy® Plant Mini Kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • Strain Gliomastix sp-71728 was inoculated onto a PDA plate and incubated for 7 days at 25°C in the darkness.
  • Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25°C with shaking at 160 rpm.
  • the mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
  • the mycelia were collected by scraping from agar plate with the sterilized scalpel and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using MP Fast DNA spin kit for soil (MP Biomedicals, Santa Ana, California, USA) following the manufacturer’s instruction.
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC
  • Bioinformatics, 7:263) and SignalP program were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • Novozymes A/S for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et al., 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of Staphylotrichum boninense was delivered to
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of Chaetomium sp. ZY089 was delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA).
  • the raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai, 2012, Journal of Computational Biology, 19(5): 455-477).
  • the assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction.
  • GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction.
  • Blastall version 2.2.25 Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of Thielavia sp. ZY346 was delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA).
  • the raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai., 2012, Journal of Computational Biology, 19(5): 455-477).
  • the assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction.
  • GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction.
  • Blastall version 2.2.25 Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides.
  • Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights. Genome sequencing, assembly and annotation of strain Chaetomium jodhpurense
  • the extracted genomic DNA sample of Chaetomium jodhpurense was delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA).
  • the raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai, 2012, Journal of Computational Biology, 19(5): 455-477).
  • the assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction.
  • GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction.
  • Blastall version 2.2.25 Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC
  • Bioinformatics, 7:263) and SignalP program were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights. Genome sequencing, assembly and annotation of strain Thermothelomyces hinnulea
  • the extracted genomic DNA sample of Thermothelomyces hinnulea was delivered to Fastens (Switzerland) for genome sequencing using an ILLUMINA® HiSeq 2000 System (lllumina,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC
  • Bioinformatics, 7:263) and SignalP program were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of Humicola hyalothermophila was delivered to Fastens (Switzerland) for genome sequencing using an ILLUMINA® HiSeq 2000 System (lllumina,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC
  • Bioinformatics, 7:263) and SignalP program were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • Genome sequencing, assembly and annotation of strain Crassicarpon thermophilum The extracted genomic DNA sample of Crassicarpon thermophilum was delivered to Beijing Genome Institute (BGI, Shenzhen, China) for genome sequencing using an ILLUMINA® GA2 System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at BGI using program SOAPdenovo (Li et ai, 2010, Genome Research, 20: 265-72). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and functional prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et a!., 2008, Genome Research, 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et at., 1990, Journal of Molecular Biology, 215(3): 403-410,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of Zopfiella latipes was delivered to Novozymes A/S (Denmark) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA).
  • the raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai., 2012, Journal of Computational Biology, 19(5): 455-477).
  • the assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction.
  • GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction.
  • Blastall version 2.2.25 Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of Trichocladium asperum was delivered to Fastens (Switzerland) for genome sequencing using an ILLUMINA® HiSeq 2000 System (lllumina, Inc., San Diego, CA, USA).
  • the raw reads were assembled at Novozymes Denmark using program Idba (Peng, Yu et al. , 2010, Research in Computational Molecular Biology, 6044:426- 440. Springer Berlin Heidelberg).
  • the assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction.
  • GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction.
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC
  • Bioinformatics, 7:263) and SignalP program were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of Fusarium neocosmosporiellum was delivered to
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of Sporormia fimetaria was delivered to Beijing Genome Institute (BGI, Shenzhen, China) for genome sequencing using an ILLUMINA® GA2 System (lllumina, Inc., San Diego, CA, USA).
  • the raw reads were assembled at BGI using program
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai, 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai, 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of Simplicillium obclavatum was delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA).
  • the raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai., 2012, Journal of Computational Biology, 19(5): 455-477).
  • the assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction.
  • GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction.
  • Blastall version 2.2.25 Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of strain Geastrales sp. LC1927 was delivered to
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of strain Geomyces vinaceus was delivered to Novozymes A/S (Denmark) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA).
  • the raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et al., 2012, Journal of Computational Biology, 19(5): 455-477).
  • the assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction.
  • GeneMark-ES fungal version (Ter-Hovhannisyan V et a!., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction.
  • Blastall version 2.2.25 Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • the extracted genomic DNA sample of strain Gliomastix sp-71728 was delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA).
  • the raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai., 2012, Journal of Computational Biology, 19(5): 455-477).
  • the assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction.
  • GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction.
  • Blastall version 2.2.25 Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • HMMER version 2.1.1 National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
  • CHAP genes were selected for In-Fusion cloning, including Amd_Zop, Amd_Suan, Amd_Stbo, Amd_Chme, Amd_Chaet, Amd_Thie, Amd_Chjo, Amd_Tama, Amd_Thin, Amd_Huhy, Amd_Crth, Amd_Zola, Amd_Tras, Amd_Fune, Amd_Spfi, Amd_Siob, Amd_Chaet474, CHAP_Geas, CHAP_Gevin, CHAP_Gliom and CHAP_Saroc. In-Fusion cloning primers were designed and ordered from GENEWIZ Suzhou, China (see list in table below).
  • Lowercase characters of the forward primer represent the 5’ of the coding region of the gene and lowercase characters of the reverse primer represent the 3’ of the coding region (all the genes except Amd_Chaet474) or the downstream flanking region of the gene for
  • PCR amplifications of genes encoding for these polypeptides were carried out using Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, Massachusetts, United States) in a 50pL volume reaction.
  • the PCR reaction mixes were consisting of 10pL Phusion HF reaction buffer (5x); 1 pL each of the forward and reverse primer (10mM); 1 ul each of 2.5 mM dATP, dTTP, dGTP, and dCTP; 1-2ul of the genomic DNA; 0.3pL Phusion High-Fidelity DNA Polymerase #M0530L (2000U/mL); and PCR grade water up to 50pL.
  • PCR reactions were incubated on a C1000 Thermal Cycler (Biorad, Hercules, California, USA). The following programs were used:
  • the purified PCR products of CHAP gene coding sequences were then ligated to the vector pCaHj505 (for Amd_Zop and Amd_Chaet474) or pDau724 (all the other genes except Amd_Zop and Amd_Chaet474), both linearized with BamHI & Xhol with In-Fusion® HD cloning kit (Takara Bio USA, Inc., formerly known as Clontech Laboratories, Inc., Mountain View, California, USA). Briefly, for each ligation reaction, 1 ul of 5x In-Fusion HD Enzyme Premix was added to 0.3ul of linearized pCaHj505 or pDau724, and 3.7ul of DNA fragment. Reactions were incubated at 50°C for 15min and kept on ice prior to E. coli transformation.
  • E. coli transformation 5ul of the ligation solution was added to 50 pi of frozen-thawed E. coli TOP 10 competent cells (TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China) and kept on ice for 30 minutes. Then the cells were heat-shocked at 42°C for 1 min, and placed on ice for 2 min. Next, 200ul of LB medium were added to the cells and incubated at 37°C for 50 min shaking at 350rpm. Finally, all the cells were spread on LB plate containing 100ug/ml of ampicillin and incubated at 37°C overnight.
  • the resulting plasmid of expression constructs were named as p505-Amd_Zop, pDau724- Amd_Suan, pDau724-Amd_Stbo, pDau724-Amd_Chme, pDau724-Amd_Chaet, pDau724- Amd_Thie, pDau724-Amd_Chjo, pDau724-Amd_Tama, pDau724-Amd_Thin, pDau724- Amd_Huhy, pDau724-Amd_Crth, pDau724-Amd_Zola, pDau724-Amd_Tras, pDau724- Amd_Fune, pDau724-Amd_Spfi and pDau724-Amd_Siob, p505-Amd_Chaet474, pDau7
  • the final plasmids were individually transformed into an Aspergillus oryzae expression host MT3568 (described in W02014026630A1 , example 2, page 29) for Amd_Zop and
  • Transformants were selected during regeneration from protoplasts based on the ability to utilize acetamide ((for Amd_Zop and Amd_Chaet474) or NaN0 3 (for all the other genes except Amd_Zop and Amd_Chaet474) as a nitrogen source conferred by a selectable marker in the expression vectors respectively.
  • Four transformants of each transformation were selected and inoculated to 3 ml of DAP4C-1 medium in 24-well plate and incubated at 30°C, 150 rpm. After 3-4 days incubation, 20 pi of supernatant from each transformant were analyzed on NuPAGE Novex 4-12% Bis-Tris Gel w/MES according to the manufacturer's
  • Amd_Chjo Amd_Tama, Amd_Thin, Amd_Huhy, Amd_Crth, Amd_Zola, Amd_Tras, Amd_Fune Amd_Chaet474, CHAP_Geas, CHAP_Gevin, CHAP_Gliom and CHAP_Saroc, and 16-18KD for Amd_Spfi, Amd_Siob and CHAP_Geas.
  • the recombinant Aspergillus oryzae strains with the strongest protein band were selected for shaking flask culturing and designated as: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42 corresponding to SEQ ID 22 to 42.
  • Each expression strain was inoculated on slants and incubated at 37C for 6-7 days. When strains were well grown to fully sporulated, they were inoculated to shaking flasks of 2L each containing 400ml of DAP4C-1 medium, several flasks for each strain. Flasks were shaking at 80rpm, 30C. Cultures were harvested on day 3 or day 4 and filtered using a 0.45 pm
  • a PCR amplification was performed by using the Phusion Hing-Fidelity DNA polymerase in a 50uL volume reaction with exp_SYZv1_F and exp_SYZv1_R as the primer pair and the synthetic DNA as template.
  • the following PCR program was used: initial denaturation of 1min at 98°C followed by 10 cycles of 30sec at 98°C, 30sec at 65°C with 1 °C decrease each cycle, 30sec at 72°C, then another 25 cycles of 30sec at 98°C, 30sec at 56°C, 30sec at 72°C, and ending up by a final elongation of 7min at 72°C, 15°C for ever.
  • the PCR resulted in a DNA fragment of ⁇ 750bp.
  • the fragment was purified by using an ILLUSTRATM GFXTM PCR DNA and Gel Band Purification Kit and was ligated to the vector of pDau724 linearized by BamHI & Xhol.
  • the transformation of TOP10 competent cells with the ligation solution resulted in E. coli transformants on LB+ampicillin plate. Two transformants were picked up for sequencing and the one with correct insertion was inoculated for plasmid DNA p724-CHSYZv1 preparation.
  • the plasmid DNA p724-CHSYZv1 was then transformed into Dau785.
  • transformants were inoculated to 3ml of DAP4C-1 medium in 24-well plate and incubated at 30°C, 150 rpm for 3 days. Supernatant of each transformant was analyzed on NuPAGE Novex 4-12% Bis-Tris Gel w/MES. Most transformants showed a band at -30KD.
  • the recombinant strain with the strongest protein band was selected for shaking flask culturing and designated as 084B68. 084B68 was inoculated on a slant made and incubated at 37°C for 6 days. When the strain was well grown to fully sporulated, it was inoculated to 4 shaking flasks of 2L containing 400ml of DAP4C-1 medium. Flasks were shaking at 80rpm, 30°C. Cultures were harvested on day 4. The culture broth was filtered using a 0.45 pm DURAPORE membrane and used for purification.
  • the culture supernatant of SEQ ID NO: 22 was added by ammonium sulfate with the conductivity to about 185 mS/cm, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM Bis-Tris at pH6.5 with 1.8M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for enzyme activity.
  • the fractions with enzyme activity and flow-through fraction were pooled together, and the conductivity was adjusted to 200 mS/cm, then re-loaded into HIC column equilibrated with 20mM Bis-Tris at pH6.5 with 2M ammonium sulfate added. Elution was applied by gradient concentration decrease of ammonium sulfate.
  • the fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM Bis-Tris at pH6.0. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 23 was added with ammonium sulfate to final conductivity about 200 mS/cm, and loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added.
  • a gradient decrease of ammonium sulfate concentration was applied as elution buffer from 2M to zero, and then elution fractions and flow-through fraction were collected to detect OD drop activity.
  • the fractions with OD drop activity were pooled together, analyzed by SDS-PAGE, concentrated, and then diafiltrated by 20mM Tris-HCI at pH7.0.
  • the protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 24 was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20mM PBS at pH7.0. The solution was filtered with 0.45um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20mM PBS at pH7.0. A gradient of NaCI concentration was applied as elution buffer from zero to 1 M, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were pooled and analyzed by SDS-PAGE, and then concentrated for further evaluation. The protein concentration was determined by Qubit ®
  • the culture supernatant of SEQ ID NO: 25 was added by ammonium sulfate with final concentration of 1.7M, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.7M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
  • the fractions with OD drop activity were loaded into SP Fast Flow column equilibrated with 20mM PBS at pH7.0. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit ® Protein Assay Kit
  • the culture supernatant of SEQ ID NO: 26 was added by ammonium sulfate with final concentration of 1.6M, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.6M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
  • the fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH6.5, then loaded into SP Fast Flow column equilibrated with 20mM PBS at pH6.5. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH6.5. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 27 was added with ammonium sulfate to final concentration of 1.7M, and loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20mM PBS at pH7.0 with 1.7M ammonium sulfate added.
  • a gradient decrease of ammonium sulfate concentration was applied as elution buffer from 1.7M to zero, and then elution fractions and flow-through fraction were collected to detect OD drop activity.
  • the fractions with OD drop activity were pooled together, analyzed by SDS-PAGE, concentrated, and then diafiltrated by 20mM PBS at pH7.0.
  • the protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 28 was added with ammonium sulfate to final conductivity about 200 mS/cm, and loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added.
  • a gradient decrease of ammonium sulfate concentration was applied as elution buffer from 2M to zero, and then elution fractions and flow-through fraction were collected to detect OD drop activity.
  • the fractions with OD drop activity were pooled together, analyzed by SDS-PAGE, concentrated, and then diafiltrated by 20mM Tris-HCI at pH7.0.
  • the protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 29 was added by ammonium sulfate with final concentration of 1.6M, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.6M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
  • the fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH6.5, then loaded into SP Fast Flow column equilibrated with 20mM PBS at pH6.5. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH6.5. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 30 was added by ammonium sulfate with final concentration of 1.8M, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.8M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
  • the fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH7.0, then loaded into SP Fast Flow column equilibrated with 20mM PBS at pH7.0. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212). Purification of mature polypeptide of SEQ ID NO: 31
  • the culture supernatant ofSEQ ID NO: 31 was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20mM PBS at pH7.0. The solution was filtered with 0.45um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20mM PBS at pH7.0. A gradient of NaCI concentration was applied as elution buffer from zero to 1 M, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were analyzed by SDS-PAGE, and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 32 was added with ammonium sulfate to final conductivity about 210 mS/cm, and loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added.
  • a gradient decrease of ammonium sulfate concentration was applied as elution buffer from 2M to zero, and then elution fractions and flow-through fraction were collected to detect OD drop activity.
  • the fractions with OD drop activity were analyzed by SDS-PAGE, concentrated, and then diafiltrated by 20mM Tris-HCI at pH7.0.
  • the protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 33 was added with ammonium sulfate to final conductivity about 180 mS/cm, and loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added.
  • a gradient decrease of ammonium sulfate concentration was applied as elution buffer from 2M to zero, and then elution fractions and flow-through fraction were collected to detect OD drop activity.
  • the fractions with OD drop activity were analyzed by SDS-PAGE, concentrated, and then diafiltrated by 20mM Tris-HCI at pH7.0.
  • the protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant ofSEQ ID NO: 34 was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20mM PBS at pH7.0. The solution was filtered with 0.45um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20mM PBS at pH7.0. A gradient of NaCI concentration was applied as elution buffer from zero to 1 M, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were analyzed by SDS-PAGE, concentrated, and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 35 was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20mM PBS at pH7.5. The solution was filtered with 0.45um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20mM PBS at pH7.5. A gradient of NaCI concentration was applied as elution buffer from zero to 1 M, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were analyzed by SDS-PAGE, concentrated, and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 36 was added by ammonium sulfate with final conductivity of 200 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM Tris-HCI at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
  • the fractions with OD drop activity were pooled together, adjusted conductivity to 200 mS/cm, then reloaded into Phenyl HP column equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added. Elution was applied by gradient decrease of ammonium sulfate concentration. The fractions with OD drop activity were analyzed by SDS-PAGE, pooled together, concentrated and diafiltrated with 20mM Tris-HCI at pH7.0. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 37 was added by ammonium sulfate with final conductivity of 200 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM Tris-HCI at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
  • the fractions with OD drop activity were pooled together, adjusted conductivity to 200 mS/cm, then reloaded into Phenyl Sepharose High Performance column equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added. Elution was applied by gradient decrease of ammonium sulfate concentration. The fractions with OD drop activity were analyzed by SDS-PAGE, pooled together, concentrated and diafiltrated with 20mM Tris-HCI at pH7.0. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 38 was added by ammonium sulfate with a final conductivity to about 140 mS/cm and loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM NaAc at pH5.5 with 1.2M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration from 1 2M to 0 was set up as elution condition. The fractions and flow-through faction were tested for enzyme activity.
  • the fractions with enzyme activity and flow-through fraction were pooled together, and the conductivity of mixture was adjusted to about 185 mS/cm, and then were loaded on Phenyl column again, which was equilibrated with 20mM NaAc at pH5.5 with 1.8M ammonium sulfate. A gradient decrease of ammonium sulfate concentration was applied as elution buffer, and the fractions with OD drop activity was pooled and assayed by SDS-PAGE. Finally, the pooled sample was diafiltrated with 20mM PBS at pH6.0. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 39 was added by ammonium sulfate with final conductivity of 200 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 40 was added by ammonium sulfate with final concentration of 1.7M, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.7M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
  • the fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH7.0, then loaded into SP Fast Flow column equilibrated with 20mM PBS at pH7.0. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 41 was added by ammonium sulfate with final conductivity of 195 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit ® Protein Assay Kit (Invitrogen, cat Q33212).
  • the culture supernatant of SEQ ID NO: 42 was added by ammonium sulfate with final conductivity of 195 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
  • the fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH7.0, then loaded into Capto SP column equilibrated with 20mM PBS at pH7.0. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0.
  • the culture supernatant of SEQ ID NO: 45 was added by ammonium sulfate with final conductivity of 200 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
  • the fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH7.0, then loaded into MonoQ column equilibrated with 20mM PBS at pH7.0. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0.
  • Control is set by adding 10 pi of 60 mM KH 2 P0 4 at pH 6.0 or pH 4.0 buffer to replace protein sample.
  • Bacterial strains Exiguobacterium sp. isolated from soil was picked up as substrate. The bacterial strain was washed and suspended in 60mM KH2P04 buffer at pH 6.0 with final concentration of 1 % (w/v) as substrate stock.
  • the substrate stock was diluted with near 60mM Citric acid-Na2HP04 buffer at pH 6.0 or 60mM PBS buffer at pH 8.0 until Abs.450nm approximately reach 1.
  • 20 mI protein at 50 pg/ml (except several which was used at stocked concentration: SEQ ID NO: 39 0.5mg/ml, SEQ ID NO: 41 3.2mg/ml, SEQ ID NO: 42 0.2mg/ml, 064VZ8 0.3mg/ml) and 200 mI diluted bacterial strain solution were added into 96-well plate, mixed and read OD450. Then the plate was incubated at 37°C, 300 rpm for 1hour, read OD450 again.
  • OD difference between 1 hour to initial read showed the OD drop activity for these proteins.
  • Blank was set by adding 20ul MQ water, and each sample was measured in triplicate.
  • Table 2 shown the OD drop result of all CHAP proteins at pH6.0 and pH 8.0. Such pH ranges are typical for sludge.
  • Example 5 Use of SEQ ID NO: 30 for improvinq sludqe flocculation
  • Cellulase composition comprised an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a T choderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656)
  • Flocculent Cationic polyacrylamide was obtained from the wastewater plant. Prepare the polymer solution by mixing the polymer into required amount of de-ionized water to a 0.2% polymer solution. The polymer could be stored for no longer than 1 week.
  • Enzyme were added into the sludge sample. Polypeptide with CHAP domain was added on top of protease+celluase composition.
  • the polypeptide was used in amount of 50 ug protein/t-DS, protease was used in amount of 0.5 kg/t-DS, and cellulase composition was used in amount of 0.5 kg/t-DS.
  • the results (Table 3 and Fig.1 ) shown that the addition of protease and cellulase composition can improve the sludge flocculation and save polymer to achieve the optimum flocculation.
  • SEQ ID NO: 30 which contains CHAP domain, on top of protease and cellulase composition brought in a better flocculation and clearer bulk water, and could achieve 27% reductions of optimum polymer dosage for sludge 1 , and 12% reductions for sludge 2. And this performance was obtained on two kinds of digested sludge.
  • Example 6 Use of polypeptides with CHAP domain for improvina sludae flocculation
  • polypeptides with CHAP domain SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, respectively
  • the other polypeptides with CHAP domain SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, respectively
  • the polypeptide was used in amount of 50 ug protein/t-DS
  • protease was used in amount of 0.5 kg/t-DS
  • cellulase composition was used in amount of 0.5 kg/t-DS.
  • the optimum polymer demanded for different polypeptide molecules were shown in Table 4 and the filtrate absorbance were shown in Figure 2. Adding one of 5 amidases of the enzyme in addition to a protease and cellulase composition all brought in a better flocculation and clearer bulk water.
  • the polymer demanded for the optimum flocculation was 3.8-4 kg/t-TS, corresponding to 20-24% polymer reduction compared to the sludge only, and 10-14% more saving compared to protease and cellulase composition.
  • Enzyme were added to the sludge sample. For each sample all the active protein added were the same. SEC ID NO: 30 was added to replace cellulase composition or protease + cellulase composition with the same amount of active protein.
  • Cellulase composition comprised an Aspergillus aculeatus GH10 xylanase (WO
  • Protease was Subtilisin protease of WO 2011/036263.
  • Polypeptide sample was SEQ ID NO: 30
  • Flocculent Cationic polyacrylamide was obtained from the wastewater plant. Prepare the polymer solution by mixing the polymer into required amount of de-ionized water to a 0.2% polymer solution. The polymer could be stored for no longer than 1 week.
  • protease 21ug/g-TS
  • cellulase composition 100ug/g-TS
  • the mixture of protease, cellulase composition and SEQ ID NO: 30 could bring in 17% polymer savings, while 8% savings for only use of protease and cellulase composition.
  • addition of SEQ ID NO: 30 increased the cake dryness by 1.7% at the optimum polymer dose, which was 1 % higher than protease and cellulase composition.
  • Example 9 Use of polypeptides with CHAP domain for improving sludge flocculation
  • Cellulase composition comprised an Aspergillus aculeatus GH10 xylanase (WO
  • Polypeptide sample was SEQ ID NO:35, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:38, SEQ ID NO:29, SEQ ID NO:25, SEQ ID NO:34, SEQ ID NO:41 , SEQ ID NO:39 or SEQ ID NO:40.
  • Flocculent Cationic polyacrylamide was obtained from the wastewater plant. Prepare the polymer solution by mixing the polymer into required amount of de-ionized water to a 0.2% polymer solution. The polymer could be stored for no longer than 1 week.
  • Enzyme were added into the sludge sample. Polypeptide with CHAP domain was added on top of protease+celluase composition.
  • the polymer demanded for the optimum flocculation was 3.6- 4.1 kg/t-TS, corresponding to 10-22% polymer reduction compared to the sludge only, and 2- 14% reduction compared to protease+ cellulase composition.
  • the polymer demanded for the optimum flocculation was 4.8-4.9 kg/t- TS, corresponding to about 14% polymer reduction compared to the sludge only, and 11% more saving compared to protease plus cellulase composition.
  • Example 10 Use of polypeptides with CHAP domain for reducing the polymer consumption
  • Enzyme were added into the sludge sample. Polypeptide with CHAP domain was added on top of protease+celluase composition.
  • Example 11 CST test for the polypeptides with CHAP domain.
  • Enzyme were added into the sludge sample. Polypeptide with CHAP domain was added on top of protease+celluase composition.
  • Capillary suction time was a simple and precise measure of the rate at which water was released from a sludge matrix. This measure of sludge dewaterability was used to optimize the performance and operation of sludge dewatering processes. Sludges that released water quickly had a low CST (see Progress in Filtration and Separation, Edition: 1 , Chapter: 17. Capillary Suction Time (CST), Publisher: Academic Press, Editors: E.S. Tarleton, pp.659-670).
  • the polypeptide was used in amount of 50 ug protein/t-DS, protease was used in amount of 0.5 kg/t-DS, and cellulase composition was used in amount of 0.5 kg/t-DS.
  • the addition of SEQ ID Nos:35, 38, 41. 42 and 45 on top of protease and cellulase composition significantly improved the dewaterability of the digested sludge by 22%, 12%, 22%, 12% and 12% respectively compared with protease + cellulase composition.

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Abstract

Isolated polypeptides with CHAP domain having amidase activity,nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing the polypeptides and their use in treating sludge are provided.

Description

POLYPEPTIDES WITH CHAP DOMAIN AND THEIR USE FOR TREATING SLUDGE
Reference to a Sequence Listing
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
Field of the Invention
The present invention relates to polypeptides with amidase activity comprising a CHAP domain and polynucleotides encoding the polypeptides, and to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. The invention also relates to the use of the polypeptides with amidase activity and with CHAP domain in sludge/wastewater treatment.
Background of the Invention
Sludge, generated during the course of conventional wastewater treatment, is usually dewatered or concentrated prior to disposal by incineration, land application, land filling, composting, etc. A basic dewatering scenario involves forming strong, shear-resistant sludge floes through the addition of a conditioning agent such as ferric sulphate and/or a flocculating agent (e.g. polyelectrolyte) followed by mechanical solid/liquid separation across gravity belt thickeners, belt filter presses, or centrifuges. By dewatering sludge, the wastewater treatment plant (WWTP) enhances the amount of solids per volumetric unit of sludge (i.e. cake solids) that ultimately must be disposed of. The benefits of higher cake solids include: reduced dewatered sludge volume (less sludge to be“managed” by the plant); lower annual
transportation costs (shipping the sludge to landfills or sites of land application); less water to be evaporated before sludge can be incinerated (increasing the net energy value of the sludge when incineration is used for cogeneration purposes); a more concentrated feed to digesters; and/or reduced volume of sludge to be landfilled or land applied.
A wider range of enzymes have been used in sludge/wastewater treatment industry. A number of papers exist describing the use of enzymes for selective hydrolysis within the EPS to reduce the sludge volume, with varying results. See e.g., DE10249081 , W09110723, and DE3713739. Prior art of interest includes U.S. Patent Publication No. US-2008-0190845 (herein incorporated by reference in its entirety) to DeLozier et al. relating to methods for enhancing the dewaterability of residuals (i.e. sludge) generated by conventional wastewater treatment operations. WO 1999/027082 and WO 2003/006602 (both herein incorporated by reference in their entirety) are of interest as they relate to proteases and variants thereof. As sludge remains problematic and dewatering difficult, there is a continuous need to improve methods for enhancing the dewaterability of residuals (i.e. sludge) generated by conventional wastewater treatment operations.
The CHAP domain is termed for the region of cysteine, histidine-dependent
amidohydrolase /peptidases. The CHAP domain is associated with several families of amidases, which suggests that many of these proteins have multiple peptidoglycan hydrolytic activities. Experiments on glutathionylspermidine (GSP) amidase and other enzymes suggests that all the members of CHAP utilize a catalytic cysteine residue in a nucleophilic-attack mechanism.
It is therefore an object of the present invention to provide several polypeptides with CHAP domain, having amidase activity. The use of the polypeptides of the present invention improve the flocculation performance of the sludge and thereby reduce the amount of polymer, and enhance the amount of solids per volumetric unit of sludge.
Summary of the Invention
An aspect of the disclosure is directed to a method of treating sludge comprising the use of one or more amidase enzyme having a CHAP domain. The invention provides for a method of treating sludge comprising the addition of one or more amidase enzyme having a CHAP domain, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids. The invention provides for a method for improving sludge flocculation comprising i) adding one or more CHAP enzymes, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids. The invention provides for a method for reducing polymer consumption, comprising i) adding or more CHAP enzymes, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids.
The present invention provides for a method of treating sludge comprising i) adding an effective amount of one or more amidase enzyme having a CHAP domain, typically further comprising adding one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids and separating the coagulated and flocculated solids from the water. The present invention further provides isolated or purified polypeptides with CHAP domain and polynucleotides encoding the polypeptides.
In one embodiment, the present invention relates to an isolated or purified polypeptide having amidase activity, selected from the group consisting of:
(a) a polypeptide having at least 60% sequence identity to mature polypeptide of any of SEQ ID NOs: 22-42; (b) a polypeptide encoded by a polynucleotide that hybridizes under medium, medium-high, high or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA sequence thereof;
(e) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NOs: 1-21 ;
(f) a fragment of the polypeptide of (a), (b), (c), (d), or (e) that has amidase activity.
In one embodiment, the purified polypeptide has CHAP domain.
In another embodiment, the present invention still relates to the isolated polynucleotide encoding the above-mentioned polypeptides, the expression vectors or recombined host cells and their use in the method for producing the above-mentioned polypeptides.
In one embodiment, the present invention relates to an enzyme composition comprising a protease and any one of the above-mentioned polypeptides. In another embodiment, the present invention also relates to the method for treating sludge with said enzyme composition.
In one embodiment, the present invention relates to an enzyme composition comprising a protease and an enzyme with CHAP domain. In another embodiment, the present invention also relates to the method for treating sludge with said enzyme composition.
Brief Description of the Figures
Figure 1 shows the flocculation result of the polypeptide (SEQ ID NO: 30) in
combination with the protease and cellulase composition. The performance was conducted on two kinds of digested sludges. Wherein, the polypeptide was used in amount of 50 ug protein/t- DS, protease was used in amount of 0.5 kg/t-DS, and cellulase composition was used in amount of 0.5 kg/t-DS.
Figure 2 shows the flocculation results of several polypeptides with CHAP domain in combination with the protease and cellulase composition. Each was tested in triplicated.
Wherein, the polypeptide was used in amount of 50 ug protein/t-DS, protease was used in amount of 0.5 kg/t-DS, and cellulase composition was used in amount of 0.5 kg/t-DS.
Figure 3 shows the Flocculation result of the sludge with the same enzyme dosage. The enzyme dose was maintained as 121 g protein/t-DS, wherein, the protease was fixed in amount of 21 g/t-DS. Shifting the amount of SEQ ID NO: 30 and cellulase composition from 0 to 100 g/t-DS respectively. Each condition was carried out in triplicated.
Figure 4 shows the different flocculation status under score 1-5. The flocculation status was classified into five groups according to the floes appearance and the clarity of bulk water. As shown in figure 4, groups 4 and 5 represented the desired flocculation status and thereby were scored as 4 and 5 respectively. Detailed Description of the Invention
In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.
Reference to“about” a value or parameter herein includes aspects that are directed to that value or parameter per se. For example, description referring to“about X” includes the aspect“X”.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term“amidase” means a kind of hydrolase which mainly acts on intramolecular C-N bond and catalyzes the hydrolysis of amide to produce corresponding carboxylic acid and ammonia. For purposes of the present invention, amidase activity is determined according to the procedure described in the Examples.
The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
The term "cellulase composition” means a blend of enzyme with cellulase or hemicelluase activity. In one embodiment, the cellulase composition comprises xylanase, cellulase, beta-glucosidase and a GH61A polypeptide. The term“xylanase” means a 1 ,4-beta- D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans. Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6. The term“cellulase” means one or more ( e.g ., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The term“beta-glucosidase” means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose. Beta-glucosidase activity can be determined using p-nitrophenyl-beta-D- glucopyranoside as substrate according to the procedure of Venturi et ai, 2002, J. Basic Microbiol. 42: 55-66. And the term "GH61" means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat B., 1991 , A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1 ,4-beta-D-glucanase activity in one family member. The structure and mode of action of these enzymes are non-canonical and they cannot be considered as bona fide glycosidases. However, they are kept in the CAZy classification on the basis of their capacity to enhance the breakdown of lignocellulose when used in conjunction with a cellulase or a mixture of cellulases.
The term "CHAP domain” is a term which defines the region of cysteine, histidine- dependent amidohydrolase /peptidases. This domain is between 110 and 140 amino acid in length and contains two invariant residues, a cysteine and a histidine. These residues form part of the active site of these proteins. A large family of proteins has been unified by the presence of CHAP domain. The CHAP domain is present in proteins containing at least three types of amidase domains.
The CHAP domain of the polypeptides of the present invention may be represented by amino acids 126 to 212 of SEQ ID NO: 22, amino acid 128 to 214 of SEQ ID NO:23, amino acids 129 to 215 of SEQ ID NO: 24, amino acid 125 to 211 of SEQ ID NO:25, amino acids 131 to 213 of SEQ ID NO: 26, amino acid 127 to 213 of SEQ ID NO:27, amino acids 125 to 211 of SEQ ID NO: 28, amino acid 126 to 212 of SEQ ID NO:29, amino acids 129 to 213 of SEQ ID NO: 30, amino acid 132 to 217 of SEQ ID NO:31 , amino acids 121 to 207 of SEQ ID NO: 32, amino acid 129 to 214 of SEQ ID NO:33, amino acids 127 to 213 of SEQ ID NO: 34, amino acid 128 to 211 of SEQ ID NO:35, amino acids 54 to 138 of SEQ ID NO: 36, amino acid 56 to 139 of SEQ ID NO:37, amino acids 136 to 218 of SEQ ID NO: 38, amino acid 141 to 227 of SEQ ID NO:39, amino acids 126 to 211 of SEQ ID NO: 40, amino acid 122 to 208 of SEQ ID NO:41 , amino acids 131 to 218 of SEQ ID NO: 42 or amino acids 129 to 214 of SEQ ID NO: 45.
The term“coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
The term“control sequences” means nucleic acid sequences necessary for expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or heterologous (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or heterologous to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
The term“expression” means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
The term“expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
The term“fragment” means a polypeptide, a catalytic domain, or a binding module having one or more ( e.g ., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has amidase activity.
The term "host cell" means any microbial or plant cell into which a nucleic acid construct or expression vector comprising a polynucleotide of the present invention has been introduced. Methods for introduction include but are not limited to protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. In some embodiments, the host cell is an isolated recombinant host cell that is partially or completely separated from at least one other component with, including but not limited to, proteins, nucleic acids, cells, etc.
The term“hybrid polypeptide” means a polypeptide comprising domains from two or more polypeptides, e.g., a binding module from one polypeptide and a catalytic domain from another polypeptide. The domains may be fused at the N-terminus or the C-terminus.
The term "hybridization" means the pairing of substantially complementary strands of nucleic acids, using standard Southern blotting procedures. Hybridization may be performed under medium, medium-high, high or very high stringency conditions. Medium stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 55°C. Medium-high stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 60°C. High stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 65°C. Very high stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 70°C. The term“isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component with which it is naturally associated as found in nature, including but not limited to, for example, other proteins, nucleic acids, cells, etc. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide.
The term“mature polypeptide” means a polypeptide in its mature form following N-terminal processing (e.g., removal of signal peptide). In one aspect, the mature polypeptide is amino acids 21-236 of SEQ ID NO:22 based on the SignalP 3.0 program (Bendtsen et al., 2004, J.
Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 22 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-238 of SEQ ID NO:23 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 23 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-239 of SEQ ID NO:24 based on the SignalP 3.0 program
(Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 24 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-235 of SEQ ID NO:25 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783- 795) that predicts amino acids 1 to 20 of SEQ ID NO: 25 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-237 of SEQ ID NO:26 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 26 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21- 237 of SEQ ID NO:27 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 27 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-235 of SEQ ID NO:28 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 28 are a signal peptide. In one aspect, the mature polypeptide is amino acids
21-236 of SEQ ID NO:29 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 29 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-236 of SEQ ID NO:30 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 30 are a signal peptide. In one aspect, the mature polypeptide is amino acids
22-239 of SEQ ID NO:31 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 21 of SEQ ID NO: 31 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-230 of SEQ ID NO:32 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 32 are a signal peptide. In one aspect, the mature polypeptide is amino acids 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 33 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-239 of SEQ ID NO:34 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 34 are a signal peptide. In one aspect, the mature polypeptide is amino acids
21-235 of SEQ ID NO:35 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 35 are a signal peptide. In one aspect, the mature polypeptide is amino acid 17-161 of SEQ ID NO:36 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 16 of SEQ ID NO: 36 are a signal peptide. In one aspect, the mature polypeptide is amino acids
19-163 of SEQ ID NO:37 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 18 of SEQ ID NO: 37 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-242 of SEQ ID NO:38 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 38 are a signal peptide. In one aspect, the mature polypeptide is amino acids
22-251 of SEQ ID NO:39 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 21 of SEQ ID NO: 39 are a signal peptide. In one aspect, the mature polypeptide is amino acids 21-235 of SEQ ID NO:40 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 40 are a signal peptide. In one aspect, the mature polypeptide is amino acids
20-230 of SEQ ID NO:41 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 19 of SEQ ID NO: 41 are a signal peptide. In one aspect, the mature polypeptide is amino acids 20-242 of SEQ ID NO:42 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 19 of SEQ ID NO: 42 are a signal peptide. In one aspect, the mature polypeptide is amino acids
21-235 of SEQ ID NO:45 based on the SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 45 are a signal peptide. Mass spectrum analysis was conducted for SEQ ID Nos. 22, 30 and 32. The results are matched with those predicted by SignalP, which means that the predicted mature polypeptide is matched with the actual mature polypeptide.
The term“mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having amidase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 711 of SEQ ID NO: 1. Nucleotides 1-60 of SEQ ID NO:1 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:2 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 403, 477-793 of SEQ ID NO: 3 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:3 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 391 , 456 to 772 of SEQ ID NO: 4 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:4 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 397, 470 to 786 of SEQ ID NO: 5 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:5 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 391 , 455 to 771 of SEQ ID NO: 6 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:6 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 391 , 452-768 of SEQ ID NO: 7 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:7 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 394, 455 to 771 of SEQ ID NO: 8 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:8 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 397, 475 to 788 of SEQ ID NO: 9 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:9 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 64 to 411 , 492 to 800 of SEQ ID NO: 10 or cDNA sequences thereof. Nucleotides 1-63 of SEQ ID NO:10 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 379, 482-795 of SEQ ID NO: 11 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:11 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 717 of SEQ ID NO: 12. Nucleotides 1- 60 of SEQ ID NO: 12 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 720 of SEQ ID NO: 13. Nucleotides 1-60 of SEQ ID NO:13 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 708 of SEQ ID NO: 14. Nucleotides 1-60 of SEQ ID NO:14 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 49 to 486 of SEQ ID NO: 15. Nucleotides 1-48 of SEQ ID NO: 15 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 55 to 492 of SEQ ID NO: 16. Nucleotides 1-54 of SEQ ID NO:16 are signal. In one aspect, the mature
polypeptide coding sequence is nucleotides 61 to 412, 463-779 of SEQ ID NO: 17 or cDNA sequences thereof. Nucleotides 1-60 of SEQ ID NO:17 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 64 to 756 of SEQ ID NO: 18. Nucleotides 1-63 of SEQ ID NO:18 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 708 of SEQ ID NO: 19. Nucleotides 1-60 of SEQ ID NO:19 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 58 to 693 of SEQ ID NO: 20. Nucleotides 1-57 of SEQ ID NO:20 are signal. In one aspect, the mature polypeptide coding sequence is nucleotides 58 to 729 of SEQ ID NO: 21. Nucleotides 1-57 of SEQ ID NO:21 are signal.
The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences. The term“operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3). Suitable protease in accordance with the present disclosure includes enzymes capable of cleaving the amide linkages in protein, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H.
Freeman and Company, San Francisco, Chapter 3).
The term“purified” means a nucleic acid or polypeptide that is substantially free from other components as determined by analytical techniques well known in the art ( e.g ., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.
The term "recombinant," when used in reference to a cell, nucleic acid, protein or vector, means that it has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.
Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g., a heterologous promoter in an expression vector. Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences. A vector comprising a nucleic acid encoding a polypeptide is a recombinant vector. The term“recombinant” is synonymous with“genetically modified” and“transgenic”.
The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter“sequence identity”.
For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of“longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line.
For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of“longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line.
The term“variant” means a polypeptide having amidase activity comprising an alteration, i.e. , a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.
Polypeptides Having Amidase Activity and a CHAP Domain
The invention relates, in one aspect to amidase enzymes having a CHAP domain. CHAP is the amidase domain of bifunctional Escherichia coli glutathionylspermidine
synthetase/amidase, and it catalyses the hydrolysis of Gsp (glutathionylspermidine) into glutathione and spermidine. In molecular biology, the CHAP domain is a region between 110 and 140 amino acids that is found in proteins from bacteria, bacteriophages, archaea and eukaryotes of the family Trypanosomidae. The domain is named after the acronym cysteine, histidine-dependent amidohydrolases/peptidases. Many of these proteins are uncharacterised, but it has been proposed that they may function mainly in peptidoglycan hydrolysis. The CHAP domain is found in a wide range of protein architectures; it is commonly associated with bacterial type SH3 domains and with several families of amidase domains. It has been suggested that CHAP domain containing proteins utilise a catalytic cysteine residue in a nucleophilic-attack mechanism.
The CHAP domain contains two invariant residues, a cysteine and a histidine. These residues form part of the putative active site of CHAP domain containing proteins. Secondary structure predictions show that the CHAP domain belongs to the alpha + beta structural class, with the N-terminal half largely containing predicted alpha helices and the C-terminal half principally composed of predicted beta strands.
Some proteins known to contain a CHAP domain are listed below:
• Bacterial and trypanosomal glutathionylspermidine amidases.
• A variety of bacterial autolysins.
• A Nocardia aerocolonigenes putative esterase.
• Streptococcus pneumoniae choline-binding protein D.
• Methanosarcina mazei protein MM2478, a putative chloride channel.
• Several phage-encoded peptidoglycan hydrolases.
• Cysteine peptidases belonging to MEROPS peptidase family C51 (D-alanyl- glycyl endopeptidase, clan CA).
The polypeptides of SEQ ID Nos:22-42 all contain CHAP domain. In one embodiment, the present invention relates to a CHAP domain having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to amino acids 126 to 212 of SEQ ID NO: 22, amino acid 128 to 214 of SEQ ID NO:23, amino acids 129 to 215 of SEQ ID NO: 24, amino acid 125 to 211 of SEQ ID NO:25, amino acids 131 to 213 of SEQ ID NO: 26, amino acid 127 to 213 of SEQ ID NO:27, amino acids 125 to 211 of SEQ ID NO: 28, amino acid 126 to 212 of SEQ ID NO:29, amino acids 129 to 213 of SEQ ID NO: 30, amino acid 132 to 217 of SEQ ID NO:31 , amino acids 121 to 207 of SEQ ID NO: 32, amino acid 129 to 214 of SEQ ID NO:33, amino acids 127 to 213 of SEQ ID NO: 34, amino acid 128 to 211 of SEQ ID NO:35, amino acids 54 to 138 of SEQ ID NO: 36, amino acid 56 to 139 of SEQ ID NO:37, amino acids 136 to 218 of SEQ ID NO: 38, amino acid 141 to 227 of SEQ ID NO:39, amino acids 126 to 211 of SEQ ID NO: 40, amino acid 122 to 208 of SEQ ID NO:41 , amino acids 131 to 218 of SEQ ID NO: 42 or amino acids 129 to 214 of SEQ ID NO: 45.
The amidase enzyme having a CHAP domain is an isolated or purified polypeptide having amidase activity, selected from the group consisting of:
a) a polypeptide having at least 60% sequence identity to polypeptide of any of SEQ ID NOs: 22-42;
(b) a polypeptide encoded by a polynucleotide that hybridizes under medium, medium-high, high or very high stringency conditions with the full-length complement of the mature
polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA sequence thereof; (e) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1-21 ;
(f) a fragment of the polypeptide of (a), (b), or (e) that has amidase activity.
The fragment of the polypeptide of (a), (b), or (e) that has amidase activity typically as a chain length of at least 40% the chain length of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 80% of the amino acids of the CHAP domain, such as a chain length of at least 50% the chain length of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 80% of the amino acids of the CHAP domain, such as chain length of at least 60% of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 80% of the amino acids of the CHAP domain, such as chain length of at least 70% of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 80% of the amino acids of the CHAP domain, such as chain length of at least 80% of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 80% of the amino acids of the CHAP domain, such as chain length of at least 80% of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 90% of the amino acids of the CHAP domain, such as chain length of at least 90% of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 90% of the amino acids of the CHAP domain, such as chain length of at least 90% of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 95% of the amino acids of the CHAP domain, such as chain length of at least 90% of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 98% of the amino acids of the CHAP domain, and such as chain length of at least 95% of any one the polypeptide of (a), (b), or (e), whilst having amidase activity and whilst comprising at least 95% of the amino acids of the CHAP domain.
In one aspect, the invention is directed to the use of a polypeptide with amidase activity comprising a CHAP domain for the treatment of sludge. In an aspect of the invention, the present invention relates to isolated or purified polypeptides having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the mature polypeptide of SEQ ID NOs: 22-42, which have amidase activity. In one aspect, the polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NOs: 22-42. a related aspect, the disclosure is directed to an isolated or purified polypeptide having amidase activity and a CHAP domain is selected from the group consisting of
i. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 22 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids
126 to 212 of SEQ ID NO: 22,
ii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 23 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids
128 to 214 of SEQ ID NO:23,
iii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 24 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids
129 to 215 of SEQ ID NO: 24,
iv. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 25 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 125 to 21 1 of SEQ ID NO:25,
v. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 26 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 131 to 213 of SEQ ID NO: 26,
vi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid
127 to 213 of SEQ ID NO:27,
vii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 28 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 125 to 21 1 of SEQ ID NO: 28, viii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 29 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid
126 to 212 of SEQ ID NO:29,
ix. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 30 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 129 to 213 of SEQ ID NO: 30,
x. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 31 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 132 to 217 of SEQ ID NO:31 ,
xi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 32 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 121 to 207 of SEQ ID NO: 32,
xii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 33 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 129 to 214 of SEQ ID NO:33,
xiii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 34 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids
127 to 213 of SEQ ID NO: 34,
xiv. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 35 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid
128 to 21 1 of SEQ ID NO:35,
xv. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 36 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 54 to 138 of SEQ ID NO: 36,
xvi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 37 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 56 to 139 of SEQ ID NO:37,
xvii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 38 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 136 to 218 of SEQ ID NO: 38,
xviii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 39 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 141 to 227 of SEQ ID NO:39,
xix. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 40 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 126 to 21 1 of SEQ ID NO: 40,
xx. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 41 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 122 to 208 of SEQ ID NO:41 , and
xxi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 42 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 131 to 218 of SEQ ID NO: 42.
In a related aspect, the disclosure is directed to an isolated or purified polypeptide having amidase activity and a CHAP domain is selected from the group consisting of : i. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 236 of SEQ ID NO: 22 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 126 to 212 of SEQ ID NO: 22,
ii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 238 of SEQ ID NO: 23 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 128 to 214 of SEQ ID NO:23,
iii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 239 of SEQ ID NO: 24 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 129 to 215 of SEQ ID NO: 24,
iv. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 25 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 125 to 21 1 of SEQ ID NO:25,
v. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 237 of SEQ ID NO: 26 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 131 to 213 of SEQ ID NO: 26,
vi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 237 of SEQ ID NO: 27 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 127 to 213 of SEQ ID NO:27,
vii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 28 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 125 to 21 1 of SEQ ID NO: 28,
viii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 236 of SEQ ID NO: 29 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 126 to 212 of SEQ ID NO:29,
ix. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 236 of SEQ ID NO: 30 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 129 to 213 of SEQ ID NO: 30,
x. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 22 to 239 of SEQ ID NO: 31 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 132 to 217 of SEQ ID NO:31 ,
xi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 230 of SEQ ID NO: 32 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 121 to 207 of SEQ ID NO: 32,
xii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 238 of SEQ ID NO: 33 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 129 to 214 of SEQ ID NO:33,
xiii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 239 of SEQ ID NO: 34 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 127 to 213 of SEQ ID NO: 34,
xiv. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 35 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 128 to 21 1 of SEQ ID NO:35,
xv. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 17 to 161 of SEQ ID NO: 36 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 54 to 138 of SEQ ID NO: 36,
xvi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 19 to 163 of SEQ ID NO: 37 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 56 to 139 of SEQ ID NO:37,
xvii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 242 of SEQ ID NO: 38 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 136 to 218 of SEQ ID NO: 38, xviii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 22 to 251 of SEQ ID NO: 39 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 141 to 227 of SEQ ID NO:39,
xix. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 40 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 126 to 21 1 of SEQ ID NO: 40, xx. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 20 to 230 of SEQ ID NO: 41 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 122 to 208 of SEQ ID NO:41 , and xxi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 20 to 242 of SEQ ID NO: 42 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 131 to 218 of SEQ ID NO: 42.
In a related embodiment, the isolated or purified polypeptide having amidase activity and a CHAP domain is selected from the group consisting of :
i. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 236 of SEQ ID NO: 22 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 126 to 212 of SEQ ID NO: 22, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.22, such as a chain length 95% to 105% of SEQ ID N0.22;
ii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 238 of SEQ ID NO: 23 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 128 to 214 of SEQ ID N0:23, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.23, such as a chain length 95% to 105% of SEQ ID N0.23;
iii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 239 of SEQ ID NO: 24 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 129 to 215 of SEQ ID NO: 24, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.24, such as a chain length 95% to 105% of SEQ ID N0.24;
iv. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 25 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 125 to 21 1 of SEQ ID NO:25, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.25, such as a chain length 95% to 105% of SEQ ID N0.25;
v. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 237 of SEQ ID NO: 26 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 131 to 213 of SEQ ID NO: 26, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.26, such as a chain length 95% to 105% of SEQ ID N0.26;
vi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 237 of SEQ ID NO: 27 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 127 to 213 of SEQ ID NO:27, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.27, such as a chain length 95% to 105% of SEQ ID N0.27;
vii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 28 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 125 to 21 1 of SEQ ID NO: 28, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.28, such as a chain length 95% to 105% of SEQ ID N0.28;
viii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 236 of SEQ ID NO: 29 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 126 to 212 of SEQ ID NO:29, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.29, such as a chain length 95% to 105% of SEQ ID N0.29;
ix. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 236 of SEQ ID NO: 30 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 129 to 213 of SEQ ID NO: 30, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.30, such as a chain length 95% to 105% of SEQ ID NO.30;
x. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 22 to 239 of SEQ ID NO: 31 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 132 to 217 of SEQ ID NO:31 , wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.31 , such as a chain length 95% to 105% of SEQ ID N0.31 ;
xi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 230 of SEQ ID NO: 32 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 121 to 207 of SEQ ID NO: 32, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.32, such as a chain length 95% to 105% of SEQ ID N0.32;
xii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 238 of SEQ ID NO: 33 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 129 to 214 of SEQ ID NO:33, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.33, such as a chain length 95% to 105% of SEQ ID N0.33;
xiii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 239 of SEQ ID NO: 34 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 127 to 213 of SEQ ID NO: 34, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.34, such as a chain length 95% to 105% of SEQ ID N0.34;
xiv. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 35 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 128 to 21 1 of SEQ ID NO:35, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.35, such as a chain length 95% to 105% of SEQ ID N0.35;
xv. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 17 to 161 of SEQ ID NO: 36 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 54 to 138 of SEQ ID NO: 36, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.36, such as a chain length 95% to 105% of SEQ ID N0.36;
xvi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 19 to 163 of SEQ ID NO: 37 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 56 to 139 of SEQ ID NO:37, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.37, such as a chain length 95% to 105% of SEQ ID N0.37; xvii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 242 of SEQ ID NO: 38 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 136 to 218 of SEQ ID NO: 38, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.38, such as a chain length 95% to 105% of SEQ ID N0.38;
xviii. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 22 to 251 of SEQ ID NO: 39 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 141 to 227 of SEQ ID N0:39, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.39, such as a chain length 95% to 105% of SEQ ID N0.39;
xix. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 21 to 235 of SEQ ID NO: 40 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 126 to 21 1 of SEQ ID NO: 40, wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.40, such as a chain length 95% to 105% of SEQ ID NO.40;
xx. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 20 to 230 of SEQ ID NO: 41 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acid 122 to 208 of SEQ ID NO:41 , wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.41 , such as a chain length 95% to 105% of SEQ ID N0.41 ; and
xxi. a polypeptide having at least 70%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to amino acids 20 to 242 of SEQ ID NO: 42 wherein said polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to amino acids 131 to 218 of SEQ ID NO: 42 wherein the polypeptide has a chain length 90% to 1 10% of SEQ ID NO.42, such as a chain length 95% to 105% of SEQ ID N0.42. The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 22-42 or the mature polypeptide thereof; or is a fragment thereof having amidase activity. Preferably the fragment comprises or contains amino acids 21- 236 of SEQ ID NO:22, amino acids 21-238 of SEQ ID NO:23, amino acids 21-239 of SEQ ID NO:24, amino acids 21-235 of SEQ ID NO:25, amino acids 21-237 of SEQ ID NO:26, amino acids 21-237 of SEQ ID NO:27, amino acids 21-235 of SEQ ID NO:28, amino acids 21-236 of SEQ ID NO:29, amino acids 21-236 of SEQ ID NO:30, amino acids 22-239 of SEQ ID NO:31 , amino acids 21-230 of SEQ ID NO:32, amino acids 21-238 of SEQ ID NO:33, amino acid 21- 239 of SEQ ID NO:34, amino acids 21-235 of SEQ ID NO:35, amino acid 17-161 of SEQ ID NO:36, amino acids 19-163 of SEQ ID NO:37, amino acid 21-242 of SEQ ID NO:38, amino acids 22-251 of SEQ ID NO:39, amino acid 21-235 of SEQ ID NO:40, amino acids 20-230 of SEQ ID NO:41 , or amino acid 20-242 of SEQ ID NO:42, wherein the fragment has amidase activity. The fragment of the polypeptide of the invention may comprise 1 to 30, typically 1 to 20, such as 1 to 10 amino acid substitutions, deletions and/or insertions within the sequence defined by any one of amino acids 21-236 of SEQ ID NO:22, amino acids 21-238 of SEQ ID NO:23, amino acids 21-239 of SEQ ID NO:24, amino acids 21-235 of SEQ ID NO:25, amino acids 21-237 of SEQ ID NO:26, amino acids 21-237 of SEQ ID NO:27, amino acids 21-235 of SEQ ID NO:28, amino acids 21-236 of SEQ ID NO:29, amino acids 21-236 of SEQ ID NO:30, amino acids 22-239 of SEQ ID NO:31 , amino acids 21-230 of SEQ ID NO:32, amino acids 21- 238 of SEQ ID NO:33, amino acid 21-239 of SEQ ID NO:34, amino acids 21-235 of SEQ ID NO:35, amino acid 17-161 of SEQ ID NO:36, amino acids 19-163 of SEQ ID NO:37, amino acid 21-242 of SEQ ID NO:38, amino acids 22-251 of SEQ ID NO:39, amino acid 21-235 of SEQ ID NO:40, amino acids 20-230 of SEQ ID NO:41 , or amino acid 20-242 of SEQ ID NO:42, wherein the fragment has amidase activity. Furthermore, the fragment of the polypeptide of the invention may comprise 1 to 30 amino acid amino- or carboxyl-terminal extensions to the sequence defined by any one of amino acids 21-236 of SEQ ID NO:22, amino acids 21-238 of SEQ ID NO:23, amino acids 21-239 of SEQ ID NO:24, amino acids 21-235 of SEQ ID NO:25, amino acids 21-237 of SEQ ID NO:26, amino acids 21-237 of SEQ ID NO:27, amino acids 21- 235 of SEQ ID NO:28, amino acids 21-236 of SEQ ID NO:29, amino acids 21-236 of SEQ ID NO:30, amino acids 22-239 of SEQ ID NO:31 , amino acids 21-230 of SEQ ID NO:32, amino acids 21-238 of SEQ ID NO:33, amino acid 21-239 of SEQ ID NO:34, amino acids 21-235 of SEQ ID NO:35, amino acid 17-161 of SEQ ID NO:36, amino acids 19-163 of SEQ ID NO:37, amino acid 21-242 of SEQ ID NO:38, amino acids 22-251 of SEQ ID NO:39, amino acid 21-235 of SEQ ID NO:40, amino acids 20-230 of SEQ ID NO:41 , or amino acid 20-242 of SEQ ID NO:42, wherein the fragment has amidase activity. In some embodiments, the present invention relates to isolated or purified polypeptides having amidase activity encoded by polynucleotides that hybridize under medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of SEQ ID NOs: 1-21 or the cDNA thereof (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).
In some embodiments, the present invention relates to variants of any one of the mature polypeptides of SEQ ID NOs: 22-42 comprising a substitution, deletion, and/or insertion at one or more ( e.g ., several) positions. In one aspect, the number of amino acid substitutions, deletions and/or insertions introduced into any of the mature polypeptides of SEQ ID NO: 22-42 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding module.
Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for amidase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899- 904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127). Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et ai., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
The polypeptide may be a hybrid polypeptide or a fusion polypeptide.
The polypeptides of the present invention have CHAP domain.
Sources of Polypeptides Having Amidase Activity
A polypeptide having amidase activity of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term“obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.
In one aspect, the polypeptide of SEQ ID NO:22 is a Zopfiella polypeptide, e.g., a polypeptide obtained from Zopfiella sp. t180-6. In one aspect, the polypeptide of SEQ ID NO:23 is a Subramaniula anamorphosa polypeptide. In one aspect, the polypeptide of SEQ ID NO:24 is a Staphylotrichum boninense polypeptide. In one aspect, the polypeptide of SEQ ID NO:25 is a Chaetomium megalocarpum polypeptide. In one aspect, the polypeptide of SEQ ID NO:26 is a Chaetomium polypeptide, e.g., a polypeptide obtained from Chaetomium sp. ZY089. In one aspect, the polypeptide of SEQ ID NO:27 is Thieiavia polypeptide, e.g., a polypeptide obtained from Thieiavia sp. ZY346. In one aspect, the polypeptide of SEQ ID NO:28 is a Chaetomium jodhpurense polypeptide. In one aspect, the polypeptide of SEQ ID NO:29 is a Taifanglania major polypeptide. In one aspect, the polypeptide of SEQ ID NO:30 is a Thermothelomyces hinnulea polypeptide. In one aspect, the polypeptide of SEQ ID NO:31 is a Humicola hyalothermophila polypeptide. In one aspect, the polypeptide of SEQ ID NO:32 is a
Crassicarpon thermophilum, polypeptide. In one aspect, the polypeptide of SEQ ID NO:33 and 45 are Zopfiella latipes polypeptides. In one aspect, the polypeptide of SEQ ID NO:34 is a Trichocladium asperum polypeptide. In one aspect, the polypeptide of SEQ ID NO:35 is a Fusarium neocosmosporiellum polypeptide. In one aspect, the polypeptide of SEQ ID NO:36 is a Sporormia fimetaria polypeptide. In one aspect, the polypeptide of SEQ ID NO:37 is a Simplicillium obclavatum polypeptide. In one aspect, the polypeptide of SEQ ID NO:38 is a Chaetomium polypeptide, e.g., a polypeptide obtained from Chaetomium sp. ZY474. In one aspect, the polypeptide of SEQ ID NO:39 is a Geastrales polypeptide, e.g., a polypeptide obtained from Geastrales sp. LC1927. In one aspect, the polypeptide of SEQ ID NO:40 is a
Geomyces vinaceus polypeptide. In one aspect, the polypeptide of SEQ ID NO:41 is a
Gliomastix polypeptide, e.g., a polypeptide obtained from Gliomastix sp-71728. And in one aspect, the polypeptide of SEQ ID NO:42 is a Sarocladium polypeptide, e.g., a polypeptide obtained from Sarocladium sp. XZ2014.
It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
The polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above- mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
Polynucleotides
The present invention also relates to isolated polynucleotides encoding a polypeptide of the present invention, as described herein.
In some embodiments, the present invention relates to isolated or purified polypeptides having amidase activity encoded by polynucleotides that hybridize under medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of SEQ ID NOs: 1-21 or the cDNA thereof (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).
The polynucleotides of SEQ ID NOs: 1-21 or a subsequence thereof, as well as the mature polypeptides of SEQ ID NOs: 22-42 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having amidase activity from strains of different genera or species according to methods well known in the art. Such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the
corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.
A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having amidase activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or another suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NOs: 1-21 or a subsequence thereof, the carrier material is used in a Southern blot.
For purposes of the present invention, hybridization indicates that the polynucleotides hybridize to a labeled nucleic acid probe corresponding to (i) SEQ ID NOs: 1-21 ; (ii) the mature polypeptide coding sequences of SEQ ID NOs: 1-21 ; (iii) the cDNA sequence thereof; (iv) the full-length complement thereof; or (v) a subsequence thereof; under medium to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
In some aspect, the nucleic acid probe is a polynucleotide that encodes the mature polypeptide of any of SEQ ID NOs: 22-42; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1 or nucleotides 61 to 711 of SEQ ID NO: 1 , SEQ ID NO: 2 or nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2, SEQ ID NO: 3 or nucleotides 61 to 403, 477-793 of SEQ ID NO: 3, SEQ ID NO: 4 or nucleotides 61 to 391 , 456 to 772 of SEQ ID NO:
4, SEQ ID NO: 5 or nucleotides 61 to 397, 470 to 786 of SEQ ID NO: 5, SEQ ID NO: 6 or nucleotides 61 to 391 , 455 to 771 of SEQ ID NO: 6, SEQ ID NO: 7 or nucleotides 61 to 391 , 452-768 of SEQ ID NO: 7, SEQ ID NO: 8 or nucleotides 61 to 394, 455 to 771 of SEQ ID NO:
8, SEQ ID NO: 9 or nucleotides 61 to 397, 475 to 788 of SEQ ID NO: 9, SEQ ID NO: 10 or nucleotides 64 to 41 1 , 492 to 800 of SEQ ID NO: 10, SEQ ID NO: 11 or nucleotides 61 to 379, 482-795 of SEQ ID NO: 11 , SEQ ID NO: 12 or nucleotides 61 to 717 of SEQ ID NO: 12, SEQ ID NO: 13 or nucleotides 61 to 720 of SEQ ID NO: 13, SEQ ID NO: 14 or nucleotides 61 to 708 of SEQ ID NO: 14, SEQ ID NO: 15 or nucleotides 49 to 486 of SEQ ID NO: 15, SEQ ID NO: 16 or nucleotides 55 to 492 of SEQ ID NO: 16, SEQ ID NO: 17 or nucleotides 61 to 412, 463-779 of SEQ ID NO: 17, SEQ ID NO: 18 or nucleotides 64 to 756 of SEQ ID NO: 18, SEQ ID NO: 19 or nucleotides 61 to 708 of SEQ ID NO: 19, SEQ ID NO: 20 or nucleotides 58 to 693 of SEQ ID NO: 20, SEQ ID NO: 21 or nucleotides 58 to 729 of SEQ ID NO: 21.
In some embodiments, the present invention relates to isolated polypeptides having amidase activity encoded by polynucleotides having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the mature polypeptide coding sequence of SEQ ID
NOs: 1-21 or the cDNA sequence thereof.
The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et ai, 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used. The polynucleotides may be cloned from a strain of Zopfiella sp. t180-6, Subramaniula anamorphosa, Staphylothchum boninense, Chaetomium megalocarpum, Chaetomium sp. ZY089, Thieiavia sp. ZY346, Chaetomium jodhpurense, Taifanglania major, Thermothelomyces hinnulea, Humicola hyalothermophila, Crassicarpon thermophilum, Zopfiella latipes, Trichocladium asperum, Fusahum neocosmospohellum, Sporormia fimetaria, Simplicillium obclavatum, Chaetomium sp. ZY474, Geastrales sp.
LC1927, Geomyces vinaceus, Gliomastix sp-71728 and Sarocladium sp. XZ2014, or a related organism and thus, for example, may be a species variant of the polypeptide encoding region of the polynucleotide.
Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary for synthesizing polypeptides substantially similar to the polypeptide. The term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like. The variants may be constructed on the basis of the polynucleotide presented as the mature polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA sequences thereof, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et ai, 1991 , Protein Expression and Purification 2: 95-107.
Nucleic Acid Constructs
The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene ( amyQ ), Bacillus licheniformis alpha-amylase gene ( amyL ), Bacillus licheniformis penicillinase gene ( penP ), Bacillus stearothermophilus
maltogenic amylase gene ( amyM ), Bacillus subtilis levansucrase gene ( sacB ), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994,
Molecular Microbiology 13: 97-107), E. col i lac operon, E. coli trc promoter (Egon et ai, 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene ( dagA ), and prokaryotic beta- lactamase gene (Villa-Kamaroff et ai., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et ai., 1980, Scientific American 242: 74-94; and in Sambrook et ai., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835. Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase ( glaA ), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae those phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Flhizomucor miehei lipase,
Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei x ylanase I, Trichoderma reesei xylanase II, Trichoderma reesei x ylanase III, Trichoderma reesei beta-xylosidase, and
Trichoderma reesei translation elongation factor, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha- amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Patent No. 6,011 ,147.
In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1 , ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et ai, 1992, Yeast 8: 423-488.
The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease ( aprH ), Bacillus licheniformis alpha-amylase ( amyL ), and Escherichia coli ribosomal RNA ( rrnB ).
Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei x ylanase I, Trichoderma reesei xylanase II, Trichoderma reesei x ylanase III, Trichoderma reesei beta-xylosidase, and
Trichoderma reesei translation elongation factor.
Preferred terminators for yeast host cells are obtained from the genes for
Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et ai, 1992, supra.
The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et ai., 1995, J. Bacteriol. 177: 3465-3471).
The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.
Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans those phosphate isomerase.
Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase,
Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990. The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell’s secretory pathway. The 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5’-end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a heterologous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus
stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases ( nprT , nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiol. Rev. 57: 109-137.
Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
Useful signal peptides for yeast host cells are obtained from the genes for
Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et ai, 1992, supra.
The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease ( aprE ), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the
polynucleotide encoding the polypeptide would be operably linked to the regulatory sequence.
Expression Vectors
The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector ( e.g ., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial
chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosyl- aminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5’-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene. Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.
The selectable marker may be a dual selectable marker system as described in WO 2010/039889. In one aspect, the dual selectable marker is a hph-tk dual selectable marker system.
The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the
corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term“origin of replication” or“plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and rAMb1 permitting replication in Bacillus.
Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1 , ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et at., 1991 , Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163- 9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO
00/24883.
More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the
polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
Host Cells
The present invention also relates to recombinant host cells, comprising a
polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. In some embodiments, the polypeptide is heterologous to the recombinant host cell.
In some embodiments, at least one of the one or more control sequences is
heterologous to the polynucleotide encoding the polypeptide.
In some embodiments, the recombinant host cell comprises at least two copies, e.g., three, four, or five, of the polynucleotide of the present invention.
The host cell may be any microbial or plant cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.
The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus,
Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas,
Salmonella, and Ureaplasma.
The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.
The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
The introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987,
J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or
electroporation (see, e.g., Dower et ai, 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et ai, 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g., Mazodier et ai., 1989, J. Bacteriol. 171 : 3583-3585), or transduction (see, e.g., Burke et ai, 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a
Pseudomonas cell may be effected by electroporation (see, e.g., Choi et ai., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.
Microbiol. 71 : 51-57). The introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981 , Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991 , Microbios 68: 189-207),
electroporation (see, e.g., Buckley et ai, 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981 , Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.
The host cell may be a fungal cell.“Fungi” as used herein includes the phyla
Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
The fungal host cell may be a yeast cell.“Yeast” as used herein includes
ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,
Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kiuyveri, Saccharomyces norbensis,
Saccharomyces oviformis, or Yarrowia lipolytica cell.
The fungal host cell may be a filamentous fungal cell.“Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
For example, the filamentous fungal host cell may be an Aspergillus awamori,
Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa,
Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium iucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookweiiense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thieiavia terrestris, Trametes villosa, Trametes versicolor,
Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et ai, 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
Methods of Production
The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.
The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.
The host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid-state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published compositions ( e.g ., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
The polypeptide may be detected using methods known in the art that are specific for the polypeptides. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide
The polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the fermentation medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a whole fermentation broth comprising the polypeptide is recovered.
The polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
Fermentation Broth Formulations or Cell Compositions
The present invention also relates to a fermentation broth formulation or a cell composition comprising a polypeptide of the present invention. The fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding the polypeptide of the present invention which are used to produce the polypeptide of interest), cell debris, biomass, fermentation media and/or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.
The term "fermentation broth" as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.
In an embodiment, the fermentation broth formulation and cell compositions comprise a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof. In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid,
4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris. In one embodiment, the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these
components.
The fermentation broth formulations or cell compositions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
The cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon- limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.
A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673. Enzyme Compositions
The present invention also relates to compositions comprising a polypeptide with amidase activity comprising a CHAP domain. In one aspect, the enzyme composition of the present invention further comprises a protease.
In one embodiment, the protease is a serine proteases. A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 "Principles of Biochemistry," Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753).
In one embodiments, the protease is a subtilase. Subtilase refers to a sub-group of the serine proteases as proposed by Siezen et ai, Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et ai. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al.( 1997).
One subgroup of the subtilases for use in accordance with the present disclosure includes, I-S1 or“true” subtilisins, comprises the "classical" subtilisins, such as subtilisin 168 (BSS168), subtilisin BPN', subtilisin Carlsberg (ALCALASE®, NOVOZYMES A/S), and subtilisin DY (BSSDY).
A further subgroup of the subtilases for use in accordance with the present disclosure includes the subtilases, I-S2 or high alkaline subtilisins, as recognized by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprises enzymes such as subtilisin PB92 (BAALKP) (MAXACAL®, Gist-Brocades NV), subtilisin 309
(SAVINASE®, NOVOZYMES A/S), subtilisin 147 (BLS147) (ESPERASE®, NOVOZYMES A/S), and alkaline elastase YaB (BSEYAB). In embodiments, the suitable protease used in the present invention is subtilisin protease from B. Lentus as shown in WO 1999/027082 or WO 2003/006602, both of which are incorporated by reference in their entirety.
In embodiments, the enzyme composition comprises a protease at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to a protease as shown in SEQ ID NO:43.
Protease may be applied under conditions suitable to the sludge processing conditions, such as, for example, temperatures from 20 to 60°C, pH conditions from 4 to 10, and for a treatment time of 1 to 100 hours, 16 to 72 hours, or 1 , 2, 3, 4, 5, 6, 7 days.
In another embodiment, the enzyme composition further comprises a cellulase composition comprising xylanase and a cellulase preparation comprising beta-glucosidase and GH61A polypeptide.
In another embodiment, the one or more (e.g., several) cellulase composition include a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present disclosure include, for example, CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S), and ULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAM IN EX™ (Genencor Int.), SPEZYME™ CP (Genencor Int.), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Rohm GmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR (Dyadic
International, Inc.), or VISCOSTAR® 150L (Dyadic International, Inc.). In further embodiment, the cellulase composition comprises an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a T choderma reesei cellulase preparation containing Aspergillus fumigatus beta- glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656). The cellulase enzymes are added in amounts effective from about 0.005 wt % of solids, e.g., about 0.01 wt % of solids or about 0.1 wt % of solids. The cellulase enzymes are added in amounts effective from about 0.005 to 0.1 wt % of solids. Besides, the cellulase composition may be applied under conditions suitable to the sludge processing conditions, such as, for example, temperatures from 20 to 60°C, pH conditions from 4 to 10, and for a treatment time of 1 to 100 hours, 16 to 72 hours, or 1 , 2, 3, 4, 5, 6, 7 days.
Further, the enzyme composition of the present invention may contain enzymatic component, e.g., a mono-component composition. Alternatively, the compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or
transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta- galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,
polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. The compositions may be stabilized in accordance with methods known in the art. The dosage of the composition and other conditions under which the composition is used may be determined on the basis of methods known in the art.
Sludge Treatment
The present disclosure relates to an enzymatic method to facilitate and/or improve the process of dewatering sludge, such as sludge generated during conventional wastewater treatment. An important aspect of the disclosure is directed to a method of treating sludge comprising the use of one or more amidase enzyme having a CHAP domain.
The various processes to treat industrial and municipal wastewater often generate sludge as a by-product of proper operation. Sludges generated by the wastewater treatment industry are classified not only by the source of wastewater (e.g. municipal or industrial) but also by specific stages of the wastewater treatment process. In the broadest classification, sludge is considered primary, secondary or tertiary. Primary sludges are usually considered “raw” as they are often the result of settling of solids from raw wastewater influent passed across primary clarifiers. In most instances, the clarified water is then sent to activated sludge basins (ASBs) in which suspended floes of microorganisms remove soluble contaminants from the water. As the microorganisms replicate, they must be periodically removed from the ASB to avoid overgrowth. Their removal takes place at a secondary clarifier receiving influent from the ASB. This“secondary sludge” is considered“waste activated sludge” (WAS) and has a relatively universal presence at WWTPs employing biological nutrient removal (BNR) systems. To reduce the volume of (and stabilize) this secondary sludge, the sludge may be sent to aerobic (ambient aeration or pure oxygen) or anaerobic digesters which may be operated under either mesophilic or thermophilic conditions. The resultant“tertiary” sludge is then known as “digested sludge” and may be further classified according to the specifics of digestion (e.g. thermophilic aerobically digested sludge). So, as can be seen, innumerable sludge types are produced during the treatment of wastewater. However, they can be loosely grouped as:
1. Primary or raw sludge; 2. Secondary or waste activated sludge; and
3. Tertiary, stabilized or digested sludge
Regardless of the means by which it was generated, sludge produced during
wastewater treatment operations, usually employing some means of biological nutrient removal, will contain substances that serve as substrates for enzymatic hydrolysis. In most instances, this substrate is present as a component of the extracellular polymeric substances (EPS) that comprise the majority of the sludge solids. The composition of EPS varies from sludge to sludge depending upon a number of variables including the nature of the wastewater to be treated, the treatment process employed and the treatment conditions. Specific
monosaccharides (e.g. glucose, mannose, galactose, etc.) tend to be universally present within sludge EPS. Considering this, although the overall composition of the EPS of sludge(s) may differ greatly, there is some degree of similarity in the type of glycosidic linkages present in the sludge components.
An aspect of the invention is directed to a method of treating sludge comprising i) adding an effective amount of one or more CHAP enzymes, or an enzyme composition comprising an effective amount of one or more CHAP enzymes, typically further comprising adding and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids and ii) separating the coagulated and flocculated solids from the water. A suitable embodiment of the invention relates to method for treating sludge, comprising:
(a) Contacting the sludge with one or more CHAP enzymes, or an enzyme composition comprising an effective amount of one or more CHAP enzymes, and
(b) Removing water from the sludge.
According to the present disclosure, the method of the invention is suitable for all sludge(s) associated with conventional wastewater treatment specifically to improve
dewaterability. In a preferred embodiment, the enzyme compositions are applied to tertiary sludge(s) generated during treatment of industrial and municipal waste water. In embodiments, the enzyme compositions of the present disclosure are applied to digested sludge form such as anaerobically or aerobically digested sludge. A purpose of the present disclosure is to facilitate or improve the process of sludge dewatering including treating sludge with a combination of the polypeptides with CHAP domain and protease, prior to conventional sludge conditioning and dewatering operations.
One embodiment of the process to enhance the dewaterability of sludge according to the present disclosure comprises or consists of the following steps:
a) generating or obtaining sludge, such as, during conventional wastewater treatment; b) treating the sludge with a polypeptides with amidase activity comprising a CHAP domain or an enzyme composition comprising a polypeptides with amidase activity comprising a CHAP domain;
c) optionally, conditioning the sludge with coagulating and/or flocculating additives; d) dewatering the enzyme treated sludge with conventional equipment.
In addition to above steps further optional steps may be included, such as, for example, treating the sludge with enzymes post digestion and before dewatering stages. In
embodiments, enzyme composition of the present disclosure is contacted with sludge before mechanical dewatering of sludge in the waste water process stream.
In one embodiment, the method of the invention and the enzyme composition in accordance with the present disclosure further comprise a protease together with polypeptides with amidase activity. In another embodiment, the enzyme composition in accordance with the present disclosure comprises a protease and polypeptides with CHAP domain. In still another embodiment, the enzyme composition in accordance with the present disclosure further comprises a cellulase composition.
In one embodiment, suitable amounts of the polypeptide with amidase activity of the present invention include 0.002 to 0.4 g protein per kg of total suspended solids, 0.01 to 0.1 g of protein per kg of total suspended solids, 0.02 to 0.08 g of protein per kg of total suspended solids. In embodiments, said polypeptide is dosed at about 0.05g/kg TS (-50 ppm).
In one embodiment, suitable amounts of the polypeptide with CHAP domain of the present invention include 0.002 to 0.4 g protein per kg of total suspended solids, 0.01 to 0.1 g of protein per kg of total suspended solids, 0.02 to 0.08 g of protein per kg of total suspended solids. In embodiments, said polypeptide is dosed at about 0.05g/kg TS (-50 ppm).
In one embodiment, suitable amounts of protease include 0.004 to 0.09 g protein per kg of total suspended solids, 0.005 to 0.03 g of protein per kg of total suspended solids, 0.010 to 0.025 g of protein per kg of total suspended solids. In embodiments, suitable protease is dosed at 0.011-0.025g/kg TS.
In one embodiments, suitable amounts of cellulase composition used to combine with protease include 0.002 to 0.4 g protein per kg of total suspended solids, 0.01 to 0.1 g of protein per kg of total suspended solids, 0.04 to 0.09 g of protein per kg of total suspended solids. In embodiments, said cellulase composition is dosed at 0.045-0.09 g/kg TS.
The enzyme composition in accordance with this disclosure may be applied under conditions suitable to the sludge processing conditions, such as, for example, temperatures from 20 to 60°C, pH conditions from 4 to 10, and for a treatment time of 1 to 100 hours, 16 to 72 hours, or 1 , 2, 3, 4, 5, 6, 7 days. Signal Peptide and propeptide
The present invention also relates to an isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 22, amino acids 1 to 20 of SEQ ID NO: 23, amino acids 1 to 20 of SEQ ID NO: 24, amino acids 1 to 20 of SEQ ID NO: 25, amino acids 1 to 20 of SEQ ID NO: 26, amino acids 1 to 20 of SEQ ID NO: 27, amino acids 1 to 20 of SEQ ID NO: 28, amino acids 1 to 20 of SEQ ID NO: 29, amino acids 1 to 20 of SEQ ID NO: 30, amino acids 1 to 21 of SEQ ID NO: 31 , amino acids 1 to 20 of SEQ ID NO: 32, amino acids 1 to 20 of SEQ ID NO: 33, amino acids 1 to 20 of SEQ ID NO: 34, amino acids 1 to 20 of SEQ ID NO: 35, amino acids 1 to 16 of SEQ ID NO: 36, amino acids 1 to 18 of SEQ ID NO: 37, amino acids 1 to 20 of SEQ ID NO: 38, amino acids 1 to 21 of SEQ ID NO: 39, amino acids 1 to 20 of SEQ ID NO: 40, amino acids 1 to 19 of SEQ ID NO: 41 , amino acids 1 to 19 of SEQ ID NO: 42, or amino acids 1 to 20 of SEQ ID NO:45. which is operably linked to a polynucleotide encoding a polypeptide which is heterologous to the signal peptide.
The present invention also relates to an isolated polynucleotide encoding a propeptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 22, amino acids 1 to 20 of SEQ ID NO: 23, amino acids 1 to 20 of SEQ ID NO: 24, amino acids 1 to 20 of SEQ ID NO: 25, amino acids 1 to 20 of SEQ ID NO: 26, amino acids 1 to 20 of SEQ ID NO: 27, amino acids 1 to 20 of SEQ ID NO: 28, amino acids 1 to 20 of SEQ ID NO: 29, amino acids 1 to 20 of SEQ ID NO: 30, amino acids 1 to 21 of SEQ ID NO: 31 , amino acids 1 to 20 of SEQ ID NO: 32, amino acids 1 to 20 of SEQ ID NO: 33, amino acids 1 to 20 of SEQ ID NO: 34, amino acids 1 to 20 of SEQ ID NO: 35, amino acids 1 to 16 of SEQ ID NO: 36, amino acids 1 to 18 of SEQ ID NO: 37, amino acids 1 to 20 of SEQ ID NO: 38, amino acids 1 to 21 of SEQ ID NO: 39, amino acids 1 to 20 of SEQ ID NO: 40, amino acids 1 to 19 of SEQ ID NO: 41 , or amino acids 1 to 19 of SEQ ID NO: 42. The polynucleotides may further comprise a gene encoding a protein, which is operably linked to the signal peptide and/or propeptide. The protein is preferably heterologous to the signal peptide and/or propeptide. In one aspect, the polynucleotide encoding the signal peptide is nucleotides 61 to 711 of SEQ ID NO: 1 , nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2, nucleotides 61 to 403, 477-793 of SEQ ID NO: 3, nucleotides 61 to 391 , 456 to 772 of SEQ ID NO: 4, nucleotides 61 to 397, 470 to 786 of SEQ ID NO: 5, nucleotides 61 to 391 , 455 to 771 of SEQ ID NO: 6, nucleotides 61 to 391 , 452-768 of SEQ ID NO: 7, nucleotides 61 to 394, 455 to 771 of SEQ ID NO: 8, nucleotides 61 to 397, 475 to 788 of SEQ ID NO: 9, nucleotides 64 to 41 1 , 492 to 800 of SEQ ID NO: 10, nucleotides 61 to 379, 482-795 of SEQ ID NO: 1 1 , nucleotides 61 to 717 of SEQ ID NO: 12, nucleotides 61 to 720 of SEQ ID NO: 13, nucleotides 61 to 708 of SEQ ID NO: 14, nucleotides 49 to 486 of SEQ ID NO: 15, nucleotides 55 to 492 of SEQ ID NO: 16, nucleotides 61 to 412, 463-779 of SEQ ID NO: 17, nucleotides 64 to 756 of SEQ ID NO: 18, nucleotides 61 to 708 of SEQ ID NO: 19, nucleotides 58 to 693 of SEQ ID NO: 20, nucleotides 58 to 729 of SEQ ID NO: 21. In another aspect, the polynucleotide encoding the propeptide is nucleotides 61 to 711 of SEQ ID NO: 1 , nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2, nucleotides 61 to 403, 477-793 of SEQ ID NO: 3, nucleotides 61 to 391 , 456 to 772 of SEQ ID NO: 4, nucleotides 61 to 397, 470 to 786 of SEQ ID NO: 5, nucleotides 61 to 391 , 455 to 771 of SEQ ID NO: 6, nucleotides 61 to 391 , 452-768 of SEQ ID NO: 7, nucleotides 61 to 394, 455 to 771 of SEQ ID NO: 8, nucleotides 61 to 397, 475 to 788 of SEQ ID NO: 9, nucleotides 64 to 411 , 492 to 800 of SEQ ID NO: 10, nucleotides 61 to 379, 482-795 of SEQ ID NO: 11 , nucleotides 61 to 717 of SEQ ID NO: 12, nucleotides 61 to 720 of SEQ ID NO: 13, nucleotides 61 to 708 of SEQ ID NO: 14, nucleotides 49 to 486 of SEQ ID NO: 15, nucleotides 55 to 492 of SEQ ID NO: 16, nucleotides 61 to 412, 463-779 of SEQ ID NO: 17, nucleotides 64 to 756 of SEQ ID NO: 18, nucleotides 61 to 708 of SEQ ID NO: 19, nucleotides 58 to 693 of SEQ ID NO: 20, nucleotides 58 to 729 of SEQ ID NO: 21.
The present invention also relates to nucleic acid constructs, expression vectors and recombinant host cells comprising such polynucleotides.
The present invention also relates to methods of producing a protein, comprising (a) cultivating a recombinant host cell comprising such polynucleotide; and optionally (b) recovering the protein.
The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and polypeptides. The term“protein” also encompasses two or more polypeptides combined to form the encoded product. The proteins also include hybrid polypeptides and fused polypeptides.
Preferably, the protein is a hormone, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter. For example, the protein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase,
carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The gene may be obtained from any prokaryotic, eukaryotic, or other source.
The invention is further defined in the following embodiments:
1. An isolated or purified polypeptide having amidase activity, selected from the group consisting of:
(a) a polypeptide having at least 60% sequence identity to mature polypeptide of any of SEQ ID NOs: 22-42; (b) a polypeptide encoded by a polynucleotide that hybridizes under medium, medium-high, high or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA sequence thereof;
(e) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1-21 ;
(f) a fragment of the polypeptide of (a), (b), (c), (d), or (e) that has amidase activity.
2. The polypeptide of paragraph 1 , having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to mature polypeptide of any of SEQ ID NOs: 22-42.
3. The polypeptide of paragraphs 1 , which is encoded by a polynucleotide that hybridizes under medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA thereof.
4. The polypeptide of any one of paragraphs 1-3, which is encoded by a
polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA thereof.
5. The polypeptide of any one of paragraphs 1-4, which is a variant of the mature polypeptide of any of SEQ ID NOs: 22-42 comprising a substitution, deletion, and/or insertion at one or more positions.
6. The polypeptide of any one of paragraphs 1-5, comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 22-42 or a mature polypeptide thereof.
7. The polypeptide of any one of paragraphs 1-9, which is a fragment of any of SEQ ID NOs: 22-42 or the mature polypeptide thereof, wherein the fragment preferably contains amino acids 21-236 of SEQ ID NO:22, amino acids 21-238 of SEQ ID NO:23, amino acids 21-239 of SEQ ID NO:24, amino acids 21-235 of SEQ ID NO:25, amino acids 21-237 of SEQ ID NO:26, amino acids 21-237 of SEQ ID NO:27, amino acids 21-235 of SEQ ID NO:28, amino acids 21-236 of SEQ ID NO:29, amino acids 21-236 of SEQ ID NO:30, amino acids 22- 239 of SEQ ID NO:31 , amino acids 21-230 of SEQ ID NO:32, amino acids 21-238 of SEQ ID NO:33, amino acid 21-239 of SEQ ID NO:34, amino acids 21-235 of SEQ ID NO:35, amino acid 17-161 of SEQ ID NO:36, amino acids 19-163 of SEQ ID NO:37, amino acid 21-242 of SEQ ID NO:38, amino acids 22-251 of SEQ ID NO:39, amino acid 21-235 of SEQ ID NQ:40, amino acids 20-230 of SEQ ID NO:41 , or amino acid 20-242 of SEQ ID NO:42, and wherein the fragment has amidase activity.
8. An isolated or purified polynucleotide encoding the polypeptide of any one of paragraphs 1-7.
9. The polynucleotide of claim 8, which comprises SEQ ID NO: 1 or nucleotides 61 to 711 of SEQ ID NO: 1 , SEQ ID NO: 2 or nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2, SEQ ID NO: 3 or nucleotides 61 to 403, 477-793 of SEQ ID NO: 3, SEQ ID NO: 4 or nucleotides 61 to 391 , 456 to 772 of SEQ ID NO: 4, SEQ ID NO: 5 or nucleotides 61 to 397,
470 to 786 of SEQ ID NO: 5, SEQ ID NO: 6 or nucleotides 61 to 391 , 455 to 771 of SEQ ID NO: 6, SEQ ID NO: 7 or nucleotides 61 to 391 , 452-768 of SEQ ID NO: 7, SEQ ID NO: 8 or nucleotides 61 to 394, 455 to 771 of SEQ ID NO: 8, SEQ ID NO: 9 or nucleotides 61 to 397,
475 to 788 of SEQ ID NO: 9, SEQ ID NO: 10 or nucleotides 64 to 411 , 492 to 800 of SEQ ID NO: 10, SEQ ID NO: 11 or nucleotides 61 to 379, 482-795 of SEQ ID NO: 11 , SEQ ID NO: 12 or nucleotides 61 to 717 of SEQ ID NO: 12, SEQ ID NO: 13 or nucleotides 61 to 720 of SEQ ID NO: 13, SEQ ID NO: 14 or nucleotides 61 to 708 of SEQ ID NO: 14, SEQ ID NO: 15 or nucleotides 49 to 486 of SEQ ID NO: 15, SEQ ID NO: 16 or nucleotides 55 to 492 of SEQ ID NO: 16, SEQ ID NO: 17 or nucleotides 61 to 412, 463-779 of SEQ ID NO: 17, SEQ ID NO: 18 or nucleotides 64 to 756 of SEQ ID NO: 18, SEQ ID NO: 19 or nucleotides 61 to 708 of SEQ ID NO: 19, SEQ ID NO: 20 or nucleotides 58 to 693 of SEQ ID NO: 20, SEQ ID NO: 21 or nucleotides 58 to 729 of SEQ ID NO: 21.
10. A nucleic acid construct or expression vector comprising the polynucleotide of paragraph 8 or 9, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.
11. A recombinant host cell comprising the polynucleotide of paragraph 8 or 9 operably linked to one or more control sequences that direct the production of the polypeptide.
12. The recombinant host cell of paragraph 11 , wherein the polypeptide is heterologous to the recombinant host cell.
13. The recombinant host cell of paragraph 11 or 12, wherein at least one of the one or more control sequences is heterologous to the polynucleotide encoding the polypeptide.
14. The recombinant host cell of any one of paragraphs 11-13, which comprises at least two copies, e.g., three, four, or five, of the polynucleotide of paragraph 8 or 9.
15. The recombinant host cell of any one of paragraphs 11-14, which is a yeast recombinant host cell, e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. 16. The recombinant host cell of any one of paragraphs 11-13, which is a
filamentous fungal recombinant host cell, e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum,
Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
17. The recombinant host cell of any one of paragraphs 11-13, which is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,
Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E. coli, Flavobacterium, Fusobacterium,
Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus
licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes,
Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells. 18. A method of producing the polypeptide of any one of paragraphs 1-7, comprising cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide.
19. The method of paragraph 18, further comprising recovering the polypeptide.
20. A method of producing a polypeptide having amidase activity, comprising cultivating the recombinant host cell of any one of paragraphs 1 1-17 under conditions conducive for production of the polypeptide.
21. The method of paragraph 20, further comprising recovering the polypeptide.
22. An isolated or purified polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 22, amino acids 1 to 20 of SEQ ID NO: 23, amino acids 1 to 20 of SEQ ID NO: 24, amino acids 1 to 20 of SEQ ID NO: 25, amino acids 1 to 20 of SEQ ID NO: 26, amino acids 1 to 20 of SEQ ID NO: 27, amino acids 1 to 20 of SEQ ID NO: 28, amino acids 1 to 20 of SEQ ID NO: 29, amino acids 1 to 20 of SEQ ID NO: 30, amino acids 1 to 21 of SEQ ID NO: 31 , amino acids 1 to 20 of SEQ ID NO: 32, amino acids 1 to 20 of SEQ ID NO: 33, amino acids 1 to 20 of SEQ ID NO: 34, amino acids 1 to 20 of SEQ ID NO: 35, amino acids 1 to 16 of SEQ ID NO: 36, amino acids 1 to 18 of SEQ ID NO: 37, amino acids 1 to 20 of SEQ ID NO: 38, amino acids 1 to 21 of SEQ ID NO: 39, amino acids 1 to 20 of SEQ ID NO: 40, amino acids 1 to 19 of SEQ ID NO: 41 , or amino acids 1 to 19 of SEQ ID NO: 42, which is operably linked to a polynucleotide encoding a polypeptide which is heterologous to the signal peptide.
23. The polynucleotide of paragraph 22, further comprising a polynucleotide encoding a propeptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 22, amino acids 1 to 20 of SEQ ID NO: 23, amino acids 1 to 20 of SEQ ID NO: 24, amino acids 1 to 20 of SEQ ID NO: 25, amino acids 1 to 20 of SEQ ID NO: 26, amino acids 1 to 20 of SEQ ID NO: 27, amino acids 1 to 20 of SEQ ID NO: 28, amino acids 1 to 20 of SEQ ID NO: 29, amino acids 1 to 20 of SEQ ID NO: 30, amino acids 1 to 21 of SEQ ID NO: 31 , amino acids 1 to 20 of SEQ ID NO: 32, amino acids 1 to 20 of SEQ ID NO: 33, amino acids 1 to 20 of SEQ ID NO: 34, amino acids 1 to 20 of SEQ ID NO: 35, amino acids 1 to 16 of SEQ ID NO: 36, amino acids 1 to 18 of SEQ ID NO: 37, amino acids 1 to 20 of SEQ ID NO: 38, amino acids 1 to 21 of SEQ ID NO: 39, amino acids 1 to 20 of SEQ ID NO: 40, amino acids 1 to 19 of SEQ ID NO: 41 , or amino acids 1 to 19 of SEQ ID NO: 42.
24. A nucleic acid construct or expression vector comprising the polynucleotide of paragraph 22 or 23.
25. A recombinant host cell comprising a nucleic acid construct or expression vector of paragraph 24. 26. A method of producing a protein, comprising cultivating the recombinant host cell of paragraph 25 under conditions conducive for production of the protein.
27. The method of paragraph 26, further comprising recovering the protein.
28. An isolated or purified polynucleotide encoding a propeptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 22, amino acids 1 to 20 of SEQ ID NO: 23, amino acids 1 to 20 of SEQ ID NO: 24, amino acids 1 to 20 of SEQ ID NO: 25, amino acids 1 to 20 of SEQ ID NO: 26, amino acids 1 to 20 of SEQ ID NO: 27, amino acids 1 to 20 of SEQ ID NO: 28, amino acids 1 to 20 of SEQ ID NO: 29, amino acids 1 to 20 of SEQ ID NO: 30, amino acids 1 to 21 of SEQ ID NO: 31 , amino acids 1 to 20 of SEQ ID NO: 32, amino acids 1 to 20 of SEQ ID NO: 33, amino acids 1 to 20 of SEQ ID NO: 34, amino acids 1 to 20 of SEQ ID NO: 35, amino acids 1 to 16 of SEQ ID NO: 36, amino acids 1 to 18 of SEQ ID NO: 37, amino acids 1 to 20 of SEQ ID NO: 38, amino acids 1 to 21 of SEQ ID NO: 39, amino acids 1 to 20 of SEQ ID NO: 40, amino acids 1 to 19 of SEQ ID NO: 41 , or amino acids 1 to 19 of SEQ ID NO: 42, which is operably linked to a polynucleotide encoding a polypeptide which is heterologous to the propeptide.
29. A nucleic acid construct or expression vector comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 28, wherein the gene is heterologous to the polynucleotide encoding the propeptide.
30. A recombinant host cell comprising a nucleic acid construct or expression vector of paragraph 29.
31. A method of producing a protein, comprising cultivating the recombinant host cell of paragraph 30 under conditions conducive for production of the protein.
32. The method of paragraph 31 , further comprising recovering the protein.
33. An enzyme composition comprising a protease and the polypeptide of any of paragraph 1-7.
34. The enzyme composition of paragraph 33, wherein, the protease is Subtilisin protease with at least 90% sequence identity to SEQ ID NO:43.
35. The enzyme composition of paragraph 33 further comprising a Cellulase composition comprised an Aspergillus aculeatus GH10 xylanase and a T choderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase and Thermoascus aurantiacus GH61A polypeptide.
36. The use of the enzyme composition of paragraph 33-35 in enhancing the dewaterability of sludge.
37. A method of treating sludge, comprising:
(a) Contacting the sludge with the enzyme composition of any of paragraphs 33-35, and
(b) Removing water from the sludge. 38. An enzyme composition comprising an enzyme with CHAP domain and a protease.
39. The enzyme composition of paragraph 38, wherein, the protease is Subtilisin protease with at least 90% sequence identity to SEQ ID NO:43.
40. The enzyme composition of paragraph 38 further comprising a Cellulase composition comprised an Aspergillus aculeatus GH10 xylanase and a T choderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase and Thermoascus aurantiacus GH61A polypeptide.
41. The enzyme composition of paragraph 38, wherein, the enzyme with CHAP domain comprising the polypeptide of any of paragraph 1-7.
42. A method of treating sludge comprising i) adding an effective amount of one or more CHAP enzymes, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids and ii) separating the coagulated and flocculated solids from the water.
43. A method for treating sludge, comprising:
(a) Contacting the sludge with the enzyme composition of any of paragraphs 38-41 , and
(b) Removing water from the sludge.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
44. A method for improving sludge flocculation comprising i) adding one or more CHAP enzymes, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids.
45. A method for reducing polymer consumption, comprising i) adding or more CHAP enzymes, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids.
46. A method of treating sludge comprising the addition of a protease and a cellulase to said sludge characterized in the further addition of an amidase comprising a CHAP- domain.
47. A method according to claim 46 wherein the amidase comprising a CHAP- domain is a polypeptide as defined by any of embodiments 1 to 8.
48. The method according to embodiment 44, further comprising the addition of a combination of a protease and a cellulase.
49. The method according to embodiment 45, further comprising the addition of a combination of a protease and a cellulase. EXAMPLES
Example 1
Medium:
PDA plates were composed of 39 grams of potato dextrose agar and deionized water to
1 liter.
YG agar plates were composed of 5 g of yeast extract, 10 g of glucose, 20 g of agar, and deionized water to 1 liter.
YPG medium was composed of 0.4% yeast extract, 0.1% KH2P04, 0.05% MgS04-7H20, and 1.5% glucose in deionized water.
Horikoshi medium was prepared by adding 10g glucose, 5g polypeptone, 5g yeast extract, 1g K2HP04, MgS047H20, 15g agar in 900ml of distilled water, then Autoclaving at 121°C for 15mins and afterautoclaving aseptically adding 100ml of sterile 10% Na2C03, finally adjusting to pH 10 with 1mM NaOH.
LB plates were composed of 10g of Bacto-tryptone, 5g of yeast extract, 10g of sodium chloride, 15g of Bacto-agar, and deionized water to 1 liter.
LB medium was composed of 10g of Bacto-tryptone, 5g of yeast extract, and 10g of sodium chloride, and deionized water to 1 liter.
COVE-plate/slant medium was composed of 30g of sucrose, 20ml of COVE salt solution, 20g of agar, and deionized water up to 1 liter. Autoclave at 121 °C for 20mins.
COVE salt solution was composed of 26g of potassium chloride, 26g of magnesium sulfate heptahydrate, 76g of monopotassium phosphate, 50ml of COVE trace metal solution, and deionized water up to 1 liter.
COVE trace metal solution was composed of 0.04g of sodium tetraborate decahydrate, 0.4g of copper (II) sulfate pentahydrate, 0.8g of ferrous sulfate heptahydrate, 0.8g of manganese sulfate monohydrate, 0.8g of sodium molybdate dihydrate, 8g of zinc sulfate heptahydrate, and deionized water up to 1 liter.
amdS selection medium: resolve the Cove medium and add 10ml of 1M acetamide (filter sterilized).
pyrG selection medium: resolve the Cove medium and add 10ml of 1 M sodium nitrate (filter sterilized).
DAP4C-1 medium was composed of 0.5g yeast extract, 10g maltose, 20g glucose, 11 g magnesium sulfate heptahydrate, 1g monopotassium phosphate, 2.2g citric acid monohydrate, 5.2g potassium phosphate tribasic monohydrate, supplemented with 0.5ml of AMG Trace element solution, and deionized water up to 1 liter. Stir to resolve. Aliquot 400ml to a shake flask of 2L. Add 1 tablet of 0.5g calcium carbonate to each flask. After autoclave at 121 °C for 20mins, 3.3ml of 20% lactic acid and 9.3ml of 50% ammonium monohydric phosphate, both sterile, were added to each flask. AMG Trace element solution was composed of 6.8g of zinc chloride, 2.5g of copper (II) sulfate pentahydrate, 0.5g of nickle chloride hexahydrate, 13.9g of ferrous sulfate heptahydrate, 8.225g of Manganese (II) sulfate monohydrate, 3g of citric acid and deionized water up to 1 liter.
Strain information:
The fungal strain NN046871 was isolated from soil samples collected from China, in 1998 by the dilution plate method with YG medium pH7, 37°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN046871 was identified as Zopfiella sp. t180-6, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN075160 was isolated from soil samples collected from Gansu province, China, in 2016 by the dilution plate method with Horilkoshi medium at pH10, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain
NN075160 was identified as Subramaniula anamorphosa, based on both morphological characteristics and ITS rDNA sequence.
The strain NN071244 was isolated from soil samples collected from Shandong province, China, in 2015 by the dilution plate method with Horilkoshi medium at pH 10, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN071244 was identified as Staphylotrichum boninense, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN057881 was obtained from Prof. Cai Lei in Institute of Microbiology, CAS, in 2014. The strain NN057881 was identified as Chaetomium megalocarpum, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN057922 was obtained from Prof. Cai Lei in Institute of Microbiology, CAS, in 2014. The strain NN057922 was identified as Chaetomium sp. ZY089, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN057872 was obtained from Prof. Cai Lei in Institute of Microbiology, CAS, in 2014. The strain NN057872 was identified as Thielavia sp. ZY346, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN057892 was obtained from Prof. Cai Lei in Institute of Microbiology, CAS, in 2014. The strain NN057892 was identified as Chaetomium jodhpurense, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN046924 was isolated from soil samples collected from China, in 1998 by the dilution plate method with YG medium pH7, 45°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN046924 was identified as Taifanglania major, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN009318 was from CBS with access number as CBS540.82. The strain NN009318 was identified as Thermothelomyces hinnulea, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN000837 was from CBS with access number as CBS454.80. The strain NN000837 was identified as Humicola hyalothermophila, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN000308 was from CBS with access number as CBS174.70. The strain NN000308 was identified as Crassicarpon thermophilum, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN075508 was isolated from soil samples collected from Guangdong province, China, in 2016 by the dilution plate method with PDA medium, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN075508 was identified as Zopfiella latipes, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN053773 was obtained from a collaboration with the Institute of Microbiology, CAS in 2011 , by the dilution plate method with PDA medium pH7, 10°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN053773 was identified as Trichocladium asperum, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN043500 was isolated from litter samples collected from China in 1998 by the dilution plate method with Horilkoshi medium at pH10, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN043500 was identified as Fusarium neocosmosporiellum, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN047801 was isolated from litter samples collected from China, in 1998 by the dilution plate method with PDA medium pH7, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN047801 was identified as Sporormia fimetaria, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN046572 was isolated from soil samples collected from China, in 1998 by the dilution plate method with PDA medium, pH7, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN046572 was identified as Simplicillium obclavatum, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN057920 was obtained through a collaboration with Professor Cai Lei in Institude of Microbiology, CAS, in 2014. The strain was collected from China. It was identified as Chaetomium sp. ZY474, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN057890 was obtained through a collaboration with Professor Cai Lei in Institude of Microbiology, CAS, in 2014. The strain was collected from China. It was identified as Geastrales sp. LC1927, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN057353 was from CBS with access number as CBS 320.62. The strain NN057353 was identified as Geomyces vinaceus, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN071728 was isolated from the enviromental samples collected from Jilin province, China, in 2015 by the dilution plate method with Horikoshi medium, pH10, 25°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN071728 was identified as Gliomastix sp-71728, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN057128 was isolated from the enviromental samples collected from Jilin province, China, in 2013 by the dilution plate method with Horikoshi medium, pH10, 10°C. It was then purified by transferring a single conidium onto a PDA agar plate. The strain NN057128 was identified as Sarocladium sp. XZ2014, based on both morphological characteristics and ITS rDNA sequence.
Example 1 : Genomic DNA preparations:
Genomic DNA extraction from strains of Zopfiella sp. t180-6
Strain Zopfiella sp. t180-6 was inoculated onto a PDA plate and incubated for 7 days at 37°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Subramaniula anamorphosa
Strain Subramaniula anamorphosa was inoculated onto a PDA plate and incubated for several days at 25°C in the darkness.
The mycelia were collected by scraping from agar plate with the sterilized scalpel and transferred to Lysing Matrix A tube (MP Biomedicals GmbH, Eschwege, Germany) and frozen under liquid nitrogen. Frozen mycelia were ground by MiniG1600 (SPEX SamplePrep LLC, New Jersey, United States), to a fine powder, and genomic DNA was isolated using DNeasy® Plant Mini Kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction. Genomic DNA extraction from strains of Staphylotrichum boninense
Strain Staphylotrichum boninense was inoculated onto a PDA plate and incubated for several days at 25°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Chaetomium meqalocarpum
Strain Chaetomium megalocarpum was inoculated onto a PDA plate and incubated for several days at 37°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Chaetomium sp. ZY089
Strain Chaetomium sp. ZY089 was inoculated onto a PDA plate and incubated for several days at 37°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Thielavia so. ZY346
Strain Thielavia sp. ZY346 was inoculated onto a PDA plate and incubated for several days at 37°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm. The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Chaetomium iodhpurense
Strain Chaetomium jodhpurense was inoculated onto a PDA plate and incubated for several days at 37°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Taifanqlania major
Strain Taifanglania major was inoculated onto a PDA plate and incubated for several days at 45°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 45°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Thermothelomyces hinnulea
Strain Thermothelomyces hinnulea was inoculated onto a PDA plate and incubated for several days at 37°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Humicola hyalothermophila
Strain Humicola hyalothermophila was inoculated onto a PDA plate and incubated for several days at 25°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Crassicarpon thermophilum
Strain Crassicarpon thermophilum was inoculated onto a PDA plate and incubated for several days at 37°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 3 days at 37°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using Biospin Fungus Genomic DNA KIT (Bioer Technology Co. Ltd., Hangzhou, China) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Zopfiella latipes
Strain Zopfiella latipes was inoculated onto a PDA plate and incubated for several days at 25°C in the darkness.
The mycelia were collected by scraping from agar plate with the sterilized scalpel and transferred to Lysing Matrix A tube (MP Biomedicals GmbH, Eschwege, Germany) and frozen under liquid nitrogen. Frozen mycelia were ground by MiniG1600 (SPEX SamplePrep LLC, New Jersey, United States), to a fine powder, and genomic DNA was isolated using DNeasy® Plant Mini Kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Figure imgf000062_0001
Strain Trichocladium asperum was inoculated onto a PDA plate and incubated for 7 days at 15°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 15°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction. Genomic DNA extraction from strains of Fusarium neocosmosporiellum
Strain Fusarium neocosmosporiellum was inoculated onto a PDA plate and incubated for 7 days at 25°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Sporormia fimetaria
Strain Sporormia fimetaria was inoculated onto a PDA plate and incubated for 7 days at 28°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 28°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strains of Simplicillium obclavatum
Strain Simplicillium obclavatum was inoculated onto a PDA plate and incubated for 7 days at 37°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using MP Fast DNA spin kit for soil (MP Biomedicals, Santa Ana, California, USA) following the manufacturer’s instruction.
Figure imgf000063_0001
Strain Chaetomium sp. ZY474 was inoculated onto a PDA plate and incubated for 7 days at 37°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24)
(QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strain of Geastrales sp. LC1927
Strain Geastrales sp. LC1927 was inoculated onto a PDA plate and incubated for 7 days at 37°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 37°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Genomic DNA extraction from strain of Geomyces vinaceus
Strain Geomyces vinaceus was inoculated onto a PDA plate and incubated for several days at 25°C in the darkness.
The mycelia were collected by scraping from agar plate with the sterilized scalpel and transferred to Lysing Matrix A tube (MP Biomedicals GmbH, Eschwege, Germany) and frozen under liquid nitrogen. Frozen mycelia were ground by MiniG1600 (SPEX SamplePrep LLC, New Jersey, United States), to a fine powder, and genomic DNA was isolated using DNeasy® Plant Mini Kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Figure imgf000064_0001
Strain Gliomastix sp-71728 was inoculated onto a PDA plate and incubated for 7 days at 25°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 4 days at 25°C with shaking at 160 rpm.
The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instruction.
Figure imgf000064_0002
Strain Sarocladium sp. XZ2014 was inoculated onto a PDA plate and incubated for several days at 15°C in the darkness.
The mycelia were collected by scraping from agar plate with the sterilized scalpel and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using MP Fast DNA spin kit for soil (MP Biomedicals, Santa Ana, California, USA) following the manufacturer’s instruction.
Genome sequencing, assembly and annotation
Genome sequencing, assembly and annotation of strain Zopfiella sp. 1180-6
The extracted genomic DNA sample of Zopfiella sp. t180-6 was delivered to Fastens
(Switzerland) for genome sequencing using an ILLUMINA® HiSeq 2000 System (lllumina,
Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Idba (Peng, Yu et al. , 2010, Research in Computational Molecular Biology, 6044:426- 440. Springer Berlin Heidelberg). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410, ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC
Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Subramaniula anamorphosa
The extracted genomic DNA sample of Subramaniula anamorphosa was delivered to
Novozymes A/S (Denmark) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et al., 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Staphylotrichum boninense
The extracted genomic DNA sample of Staphylotrichum boninense was delivered to
Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System
(lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai, 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et at, 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Chaetomium megalocarpum
The extracted genomic DNA sample of Chaetomium megalocarpum was delivered to
Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System
(lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai., 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et ai, 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et ai., 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Chaetomium sp. ZY089
The extracted genomic DNA sample of Chaetomium sp. ZY089 was delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai, 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Thielavia sp. ZY346
The extracted genomic DNA sample of Thielavia sp. ZY346 was delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai., 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights. Genome sequencing, assembly and annotation of strain Chaetomium jodhpurense
The extracted genomic DNA sample of Chaetomium jodhpurense was delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai, 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Taifanglania major
The extracted genomic DNA sample of Taifanglania major was delivered to Exiqon A/S
(Denmark) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Idba (Peng, Yu et al. , 2010, Research in Computational Molecular Biology, 6044:426-440. Springer Berlin Heidelberg). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter- Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410, ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC
Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights. Genome sequencing, assembly and annotation of strain Thermothelomyces hinnulea
The extracted genomic DNA sample of Thermothelomyces hinnulea was delivered to Fastens (Switzerland) for genome sequencing using an ILLUMINA® HiSeq 2000 System (lllumina,
Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Idba (Peng, Yu et al. , 2010, Research in Computational Molecular Biology, 6044:426- 440. Springer Berlin Heidelberg). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410, ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC
Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Humicola hyalothermophila
The extracted genomic DNA sample of Humicola hyalothermophila was delivered to Fastens (Switzerland) for genome sequencing using an ILLUMINA® HiSeq 2000 System (lllumina,
Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Idba (Peng, Yu et al., 2010, Research in Computational Molecular Biology, 6044:426- 440. Springer Berlin Heidelberg). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410, ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC
Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Crassicarpon thermophilum The extracted genomic DNA sample of Crassicarpon thermophilum was delivered to Beijing Genome Institute (BGI, Shenzhen, China) for genome sequencing using an ILLUMINA® GA2 System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at BGI using program SOAPdenovo (Li et ai, 2010, Genome Research, 20: 265-72). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and functional prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et a!., 2008, Genome Research, 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et at., 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Zopfiella latipes
The extracted genomic DNA sample of Zopfiella latipes was delivered to Novozymes A/S (Denmark) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai., 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Trichocladium asperum
The extracted genomic DNA sample of Trichocladium asperum was delivered to Fastens (Switzerland) for genome sequencing using an ILLUMINA® HiSeq 2000 System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Idba (Peng, Yu et al. , 2010, Research in Computational Molecular Biology, 6044:426- 440. Springer Berlin Heidelberg). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410, ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC
Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Fusarium neocosmosporiellum
The extracted genomic DNA sample of Fusarium neocosmosporiellum was delivered to
Fastens (Switzerland) for genome sequencing using an ILLUMINA® HiSeq 2000 System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Idba (Peng, Yu et al., 2010, Research in Computational Molecular Biology, 6044:426-440. Springer Berlin Heidelberg). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410, ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Sporormia fimetaria
The extracted genomic DNA sample of Sporormia fimetaria was delivered to Beijing Genome Institute (BGI, Shenzhen, China) for genome sequencing using an ILLUMINA® GA2 System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at BGI using program
SOAPdenovo (Li et al., 2010, Genome Research, 20: 265-72). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and functional prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et a!., 2008, Genome
Research, 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai, 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai, 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Simplicillium obclavatum
The extracted genomic DNA sample of Simplicillium obclavatum was delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai., 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Chaetomium sp. ZY474
The extracted genomic DNA sample of strain Chaetomium sp. ZY474 was delivered to
Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System
(lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et al., 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Geastrales sp. LC1927
The extracted genomic DNA sample of strain Geastrales sp. LC1927 was delivered to
Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System
(lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et al., 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et al., 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et al., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Geomyces vinaceus
The extracted genomic DNA sample of strain Geomyces vinaceus was delivered to Novozymes A/S (Denmark) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et al., 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et a!., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Gliomastix sp-71728
The extracted genomic DNA sample of strain Gliomastix sp-71728 was delivered to Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System (lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Spades (Anton Bankevich et ai., 2012, Journal of Computational Biology, 19(5): 455-477). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et ai., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410,
ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et ai., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Genome sequencing, assembly and annotation of strain Sarocladium sp. XZ2014
The extracted genomic DNA sample of strain Sarocladium sp. XZ2014 was delivered to
Novozymes Davis (USA) for genome sequencing using an ILLUMINA® MiSeq System
(lllumina, Inc., San Diego, CA, USA). The raw reads were assembled at Novozymes Denmark using program Idba (Peng, Yu et ai., 2010, Research in Computational Molecular Biology, 6044:426-440. Springer Berlin Heidelberg). The assembled sequences were analyzed using standard bioinformatics methods for gene identification and function prediction. GeneMark-ES fungal version (Ter-Hovhannisyan V et a!., 2008, Genome Research 18(12): 1979-1990) was used for gene prediction. Blastall version 2.2.25 (Altschul et ai, 1990, Journal of Molecular Biology, 215(3): 403-410, ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.25/) and HMMER version 2.1.1 (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to predict function based on structural homology. The CHAP domain was identified directly by analysis of the Blast results. The Agene program (Munch and Krogh, 2006, BMC Bioinformatics, 7:263) and SignalP program (Nielsen et ai., 1997, Protein Engineering, 10: 1-6) were used to identify start codons. SignalP program was further used to predict signal peptides. Pepstats (Rice et al., 2000, Trends Genet, 16(6): 276-277) was used to predict isoelectric points and molecular weights.
Table 1 : Summary of the Genes
Figure imgf000075_0001
Example 2: Cloning, expression and cultivation of genes
Based on the gene sequences identified by genome mining in Zopfiella sp. t180-6, Subramaniula anamorphosa, Staphylotrichum boninense, Chaetomium megalocarpum, Chaetomium sp. ZY089, Thielavia sp. ZY346, Chaetomium jodhpurense, Taifanglania major, Thermothelomyces hinnulea, Humicola hyalothermophila, Crassicarpon thermophilum, Zopfiella latipes, Trichocladium asperum, Fusarium neocosmosporiellum, Sporormia fimetaria,
Simplicillium obclavatum, Chaetomium sp. ZY474, Geastrales sp. LC1927, Geomyces vinaceus, Gliomastix sp-71728 and Sarocladium sp. XZ2014. 21 CHAP genes were selected for In-Fusion cloning, including Amd_Zop, Amd_Suan, Amd_Stbo, Amd_Chme, Amd_Chaet, Amd_Thie, Amd_Chjo, Amd_Tama, Amd_Thin, Amd_Huhy, Amd_Crth, Amd_Zola, Amd_Tras, Amd_Fune, Amd_Spfi, Amd_Siob, Amd_Chaet474, CHAP_Geas, CHAP_Gevin, CHAP_Gliom and CHAP_Saroc. In-Fusion cloning primers were designed and ordered from GENEWIZ Suzhou, China (see list in table below).
Table 2: In-Fusion cloning primers
Figure imgf000076_0001
Figure imgf000077_0001
Lowercase characters of the forward primer represent the 5’ of the coding region of the gene and lowercase characters of the reverse primer represent the 3’ of the coding region (all the genes except Amd_Chaet474) or the downstream flanking region of the gene for
Amd_Chaet474. while capitalized characters represent a region homologous to insertion sites of pCaHj505 (Described in WO2013/029496) or pDau724 (Described in WO 2016/026938). The 4 letters underlined in the forward primers represent the Kozark sequence as the initiation of translation process.
PCR amplifications of genes encoding for these polypeptides were carried out using Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, Massachusetts, United States) in a 50pL volume reaction. The PCR reaction mixes were consisting of 10pL Phusion HF reaction buffer (5x); 1 pL each of the forward and reverse primer (10mM); 1 ul each of 2.5 mM dATP, dTTP, dGTP, and dCTP; 1-2ul of the genomic DNA; 0.3pL Phusion High-Fidelity DNA Polymerase #M0530L (2000U/mL); and PCR grade water up to 50pL. PCR reactions were incubated on a C1000 Thermal Cycler (Biorad, Hercules, California, USA). The following programs were used:
For Amd_Zop, initial denaturation of 1min at 98°C followed by 10 cycles of 15sec at 98°C, 30sec at 70°C with 1°C decrease each cycle, 1 min at 72°C, then another 25 cycles of 15sec at 98°C, 30sec at 60°C, 1min at 72°C, and ending up by a final elongation of 7min at 72°C;
For Amd_Tras & Amd_Fune, initial denaturation of 1min at 98°C followed by 10 cycles of 15sec at 98°C, 30sec at 68°C with 1°C decrease each cycle, 1min at 72°C, then another 25 cycles of 15sec at 98°C, 30sec at 58°C, 1min at 72°C, and ending up by a final elongation of 7min at 72°C;
For Amd_Chaet474, initial denaturation of 1 min at 98°C followed by 10 cycles of 15sec at 98°C, 30sec at 70°C with 1°C decrease each cycle, 1 min at 72°C, then another 25 cycles of 15sec at 98°C, 30sec at 60°C, 1min at 72°C, and ending up by a final elongation of 7min at 72°C;
For CHAP_Geas and CHAP_Gevin, initial denaturation of 1min at 98°C followed by 10 cycles of 15sec at 98°C, 30sec at 65°C with 1 °C decrease each cycle, 2.5min at 72°C, then another 25 cycles of 15sec at 98°C, 30sec at 58°C, 2.5min at 72°C, and ending up by a final elongation of 7min at 72°C.
For other genes, initial denaturation of 1min at 98°C followed by 10 cycles of 30sec at 98°C, 30sec at 68°C with 1°C decrease each cycle, 1 min at 72°C, then another 25 cycles of 30sec at 98°C, 30sec at 58°C, 1min at 72°C, and ending up by a final elongation of 7min at 72°C. PCR amplicons were purified using an ILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) following the manufacturer's
instructions.
The purified PCR products of CHAP gene coding sequences were then ligated to the vector pCaHj505 (for Amd_Zop and Amd_Chaet474) or pDau724 (all the other genes except Amd_Zop and Amd_Chaet474), both linearized with BamHI & Xhol with In-Fusion® HD cloning kit (Takara Bio USA, Inc., formerly known as Clontech Laboratories, Inc., Mountain View, California, USA). Briefly, for each ligation reaction, 1 ul of 5x In-Fusion HD Enzyme Premix was added to 0.3ul of linearized pCaHj505 or pDau724, and 3.7ul of DNA fragment. Reactions were incubated at 50°C for 15min and kept on ice prior to E. coli transformation.
In E. coli transformation, 5ul of the ligation solution was added to 50 pi of frozen-thawed E. coli TOP 10 competent cells (TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China) and kept on ice for 30 minutes. Then the cells were heat-shocked at 42°C for 1 min, and placed on ice for 2 min. Next, 200ul of LB medium were added to the cells and incubated at 37°C for 50 min shaking at 350rpm. Finally, all the cells were spread on LB plate containing 100ug/ml of ampicillin and incubated at 37°C overnight.
For each gene 2 colonies were picked up for sequencing using 3730XL DNA Analyzers (Applied Biosystems Inc, Foster City, CA, USA). After the sequences were confirmed, the colony with correct insertion was inoculated for plasmid DNA extraction with a QIAPREP® Spin Miniprep Kit (QIAGEN GmbH, Hilden, Germany) by following the manufacturer’s instruction.
The resulting plasmid of expression constructs were named as p505-Amd_Zop, pDau724- Amd_Suan, pDau724-Amd_Stbo, pDau724-Amd_Chme, pDau724-Amd_Chaet, pDau724- Amd_Thie, pDau724-Amd_Chjo, pDau724-Amd_Tama, pDau724-Amd_Thin, pDau724- Amd_Huhy, pDau724-Amd_Crth, pDau724-Amd_Zola, pDau724-Amd_Tras, pDau724- Amd_Fune, pDau724-Amd_Spfi and pDau724-Amd_Siob, p505-Amd_Chaet474, pDau724- CHAP_Geas, pDau724-CHAP_Gevin, p724acc-CHAP_Gliom and p724acc-CHAP_Saroc, according to SEQ ID: 1-21 , respectively.
The final plasmids were individually transformed into an Aspergillus oryzae expression host MT3568 (described in W02014026630A1 , example 2, page 29) for Amd_Zop and
Amd_Chaet474, Dau785 (described in WO 2018/113745, page 293) for other genes, by the methods described in Christensen et al., 1988, Biotechnology 6, 1419-1422 and WO
04/032648. Transformants were selected during regeneration from protoplasts based on the ability to utilize acetamide ((for Amd_Zop and Amd_Chaet474) or NaN03 (for all the other genes except Amd_Zop and Amd_Chaet474) as a nitrogen source conferred by a selectable marker in the expression vectors respectively. Four transformants of each transformation were selected and inoculated to 3 ml of DAP4C-1 medium in 24-well plate and incubated at 30°C, 150 rpm. After 3-4 days incubation, 20 pi of supernatant from each transformant were analyzed on NuPAGE Novex 4-12% Bis-Tris Gel w/MES according to the manufacturer's
instructions. The resulting gel was stained with Instant Blue. SDS-PAGE profiles of the cultures showed that all CHAP genes were expressed with the major protein band detected at 28-30KD for Amd_Zop, Amd_Suan, Amd_Stbo, Amd_Chme, Amd_Chaet, Amd_Thie,
Amd_Chjo, Amd_Tama, Amd_Thin, Amd_Huhy, Amd_Crth, Amd_Zola, Amd_Tras, Amd_Fune Amd_Chaet474, CHAP_Geas, CHAP_Gevin, CHAP_Gliom and CHAP_Saroc, and 16-18KD for Amd_Spfi, Amd_Siob and CHAP_Geas. The recombinant Aspergillus oryzae strains with the strongest protein band were selected for shaking flask culturing and designated as: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42 corresponding to SEQ ID 22 to 42.
Each expression strain was inoculated on slants and incubated at 37C for 6-7 days. When strains were well grown to fully sporulated, they were inoculated to shaking flasks of 2L each containing 400ml of DAP4C-1 medium, several flasks for each strain. Flasks were shaking at 80rpm, 30C. Cultures were harvested on day 3 or day 4 and filtered using a 0.45 pm
DURAPORE Membrane and used for purification.
A synthetic DNA encoding the variant sequence of SEQ ID NO. 33, with mutations of A25G, S27T, D94S, A95G, K107R and T113S, was ordered from GENEWIZ Suzhou, China., named SEQ ID NO:44 (exp_SYZvl). Primers for DNA amplification were also ordered from GENEWIZ (see exp_SYZv1_F and exp_SYZv1_R in table 2).
A PCR amplification was performed by using the Phusion Hing-Fidelity DNA polymerase in a 50uL volume reaction with exp_SYZv1_F and exp_SYZv1_R as the primer pair and the synthetic DNA as template. The following PCR program was used: initial denaturation of 1min at 98°C followed by 10 cycles of 30sec at 98°C, 30sec at 65°C with 1 °C decrease each cycle, 30sec at 72°C, then another 25 cycles of 30sec at 98°C, 30sec at 56°C, 30sec at 72°C, and ending up by a final elongation of 7min at 72°C, 15°C for ever. The PCR resulted in a DNA fragment of ~750bp. The fragment was purified by using an ILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit and was ligated to the vector of pDau724 linearized by BamHI & Xhol. The transformation of TOP10 competent cells with the ligation solution resulted in E. coli transformants on LB+ampicillin plate. Two transformants were picked up for sequencing and the one with correct insertion was inoculated for plasmid DNA p724-CHSYZv1 preparation.
The plasmid DNA p724-CHSYZv1 was then transformed into Dau785. Four
transformants were inoculated to 3ml of DAP4C-1 medium in 24-well plate and incubated at 30°C, 150 rpm for 3 days. Supernatant of each transformant was analyzed on NuPAGE Novex 4-12% Bis-Tris Gel w/MES. Most transformants showed a band at -30KD. The recombinant strain with the strongest protein band was selected for shaking flask culturing and designated as 084B68. 084B68 was inoculated on a slant made and incubated at 37°C for 6 days. When the strain was well grown to fully sporulated, it was inoculated to 4 shaking flasks of 2L containing 400ml of DAP4C-1 medium. Flasks were shaking at 80rpm, 30°C. Cultures were harvested on day 4. The culture broth was filtered using a 0.45 pm DURAPORE membrane and used for purification.
Example 3: Purification of mature polypeptides
Purification of mature polypeptide of SEQ ID NO: 22
The culture supernatant of SEQ ID NO: 22 was added by ammonium sulfate with the conductivity to about 185 mS/cm, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM Bis-Tris at pH6.5 with 1.8M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for enzyme activity.
The fractions with enzyme activity and flow-through fraction were pooled together, and the conductivity was adjusted to 200 mS/cm, then re-loaded into HIC column equilibrated with 20mM Bis-Tris at pH6.5 with 2M ammonium sulfate added. Elution was applied by gradient concentration decrease of ammonium sulfate. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM Bis-Tris at pH6.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 23
The culture supernatant of SEQ ID NO: 23 was added with ammonium sulfate to final conductivity about 200 mS/cm, and loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution buffer from 2M to zero, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were pooled together, analyzed by SDS-PAGE, concentrated, and then diafiltrated by 20mM Tris-HCI at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 24
The culture supernatant of SEQ ID NO: 24 was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20mM PBS at pH7.0. The solution was filtered with 0.45um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20mM PBS at pH7.0. A gradient of NaCI concentration was applied as elution buffer from zero to 1 M, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were pooled and analyzed by SDS-PAGE, and then concentrated for further evaluation. The protein concentration was determined by Qubit®
Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 25
The culture supernatant of SEQ ID NO: 25 was added by ammonium sulfate with final concentration of 1.7M, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.7M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
The fractions with OD drop activity were loaded into SP Fast Flow column equilibrated with 20mM PBS at pH7.0. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit
(Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 26
The culture supernatant of SEQ ID NO: 26 was added by ammonium sulfate with final concentration of 1.6M, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.6M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
The fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH6.5, then loaded into SP Fast Flow column equilibrated with 20mM PBS at pH6.5. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH6.5. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 27
The culture supernatant of SEQ ID NO: 27 was added with ammonium sulfate to final concentration of 1.7M, and loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20mM PBS at pH7.0 with 1.7M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution buffer from 1.7M to zero, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were pooled together, analyzed by SDS-PAGE, concentrated, and then diafiltrated by 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 28
The culture supernatant of SEQ ID NO: 28 was added with ammonium sulfate to final conductivity about 200 mS/cm, and loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution buffer from 2M to zero, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were pooled together, analyzed by SDS-PAGE, concentrated, and then diafiltrated by 20mM Tris-HCI at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 29
The culture supernatant of SEQ ID NO: 29 was added by ammonium sulfate with final concentration of 1.6M, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.6M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
The fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH6.5, then loaded into SP Fast Flow column equilibrated with 20mM PBS at pH6.5. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH6.5. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 30
The culture supernatant of SEQ ID NO: 30 was added by ammonium sulfate with final concentration of 1.8M, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.8M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
The fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH7.0, then loaded into SP Fast Flow column equilibrated with 20mM PBS at pH7.0. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212). Purification of mature polypeptide of SEQ ID NO: 31
The culture supernatant ofSEQ ID NO: 31 was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20mM PBS at pH7.0. The solution was filtered with 0.45um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20mM PBS at pH7.0. A gradient of NaCI concentration was applied as elution buffer from zero to 1 M, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were analyzed by SDS-PAGE, and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 32
The culture supernatant of SEQ ID NO: 32 was added with ammonium sulfate to final conductivity about 210 mS/cm, and loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution buffer from 2M to zero, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were analyzed by SDS-PAGE, concentrated, and then diafiltrated by 20mM Tris-HCI at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 33
The culture supernatant of SEQ ID NO: 33 was added with ammonium sulfate to final conductivity about 180 mS/cm, and loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution buffer from 2M to zero, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were analyzed by SDS-PAGE, concentrated, and then diafiltrated by 20mM Tris-HCI at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 34
The culture supernatant ofSEQ ID NO: 34 was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20mM PBS at pH7.0. The solution was filtered with 0.45um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20mM PBS at pH7.0. A gradient of NaCI concentration was applied as elution buffer from zero to 1 M, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were analyzed by SDS-PAGE, concentrated, and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 35
The culture supernatant of SEQ ID NO: 35 was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20mM PBS at pH7.5. The solution was filtered with 0.45um filter and then loaded into Capto SP column (GE Healthcare) equilibrated with 20mM PBS at pH7.5. A gradient of NaCI concentration was applied as elution buffer from zero to 1 M, and then elution fractions and flow-through fraction were collected to detect OD drop activity. The fractions with OD drop activity were analyzed by SDS-PAGE, concentrated, and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 36
The culture supernatant of SEQ ID NO: 36 was added by ammonium sulfate with final conductivity of 200 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM Tris-HCI at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
The fractions with OD drop activity were pooled together, adjusted conductivity to 200 mS/cm, then reloaded into Phenyl HP column equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added. Elution was applied by gradient decrease of ammonium sulfate concentration. The fractions with OD drop activity were analyzed by SDS-PAGE, pooled together, concentrated and diafiltrated with 20mM Tris-HCI at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 37
The culture supernatant of SEQ ID NO: 37 was added by ammonium sulfate with final conductivity of 200 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM Tris-HCI at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
The fractions with OD drop activity were pooled together, adjusted conductivity to 200 mS/cm, then reloaded into Phenyl Sepharose High Performance column equilibrated with 20mM Tris-HCI at pH7.0 with 2M ammonium sulfate added. Elution was applied by gradient decrease of ammonium sulfate concentration. The fractions with OD drop activity were analyzed by SDS-PAGE, pooled together, concentrated and diafiltrated with 20mM Tris-HCI at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 38
The culture supernatant of SEQ ID NO: 38 was added by ammonium sulfate with a final conductivity to about 140 mS/cm and loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM NaAc at pH5.5 with 1.2M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration from 1 2M to 0 was set up as elution condition. The fractions and flow-through faction were tested for enzyme activity.
The fractions with enzyme activity and flow-through fraction were pooled together, and the conductivity of mixture was adjusted to about 185 mS/cm, and then were loaded on Phenyl column again, which was equilibrated with 20mM NaAc at pH5.5 with 1.8M ammonium sulfate. A gradient decrease of ammonium sulfate concentration was applied as elution buffer, and the fractions with OD drop activity was pooled and assayed by SDS-PAGE. Finally, the pooled sample was diafiltrated with 20mM PBS at pH6.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 39
The culture supernatant of SEQ ID NO: 39 was added by ammonium sulfate with final conductivity of 200 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 40
The culture supernatant of SEQ ID NO: 40 was added by ammonium sulfate with final concentration of 1.7M, then loaded into Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.7M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
The fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH7.0, then loaded into SP Fast Flow column equilibrated with 20mM PBS at pH7.0. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 41
The culture supernatant of SEQ ID NO: 41 was added by ammonium sulfate with final conductivity of 195 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 42
The culture supernatant of SEQ ID NO: 42 was added by ammonium sulfate with final conductivity of 195 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
The fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH7.0, then loaded into Capto SP column equilibrated with 20mM PBS at pH7.0. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein
concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Purification of mature polypeptide of SEQ ID NO: 45
The culture supernatant of SEQ ID NO: 45 was added by ammonium sulfate with final conductivity of 200 mS/cm, then loaded into Phenyl Sepharose High Performance column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 2.0M ammonium sulfate added. A gradient decrease of ammonium sulfate concentration was applied as elution condition. The fractions and flow-through fraction were assayed for OD drop activity.
The fractions with OD drop activity were pooled together, dialyzed with 20mM PBS at pH7.0, then loaded into MonoQ column equilibrated with 20mM PBS at pH7.0. Elution was applied by gradient increase of NaCI concentration. The fractions with OD drop activity were pooled together, concentrated and diafiltrated with 20mM PBS at pH7.0. The protein
concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
Example 4: Determination of amidase activity and characterization
Amidase activity detection during purification Two bacterial strains, Micrococcus lysodeikticus ATCC No. 4698 (Sigma) or Exiguobacterium sp. (isolated from soil), were picked up as substrates, separately. The bacterial strain was suspended in 60mM KH2P04 buffer at pH6.0 with final concentration of 1% as substrate stock. Before activity detection, the concentration of substrate was diluted into 0.035% by 60 mM KH2P04 pH 4.0 or pH 6.0 buffer. 10ul of protein sample and 190ul of 0.035% substrate were added into 96-well plate, and then read OD450. If the protein sample was purified by HIC, 5ul sample was replaced by Milli-Q water. The plate was incubated for 30 or 60 minutes at 37°C, and the plate was read OD450 again. The OD drop showed enzyme activity. Control is set by adding 10 pi of 60 mM KH2P04 at pH 6.0 or pH 4.0 buffer to replace protein sample.
Amidase characterization
Bacterial strains Exiguobacterium sp. (isolated from soil) was picked up as substrate. The bacterial strain was washed and suspended in 60mM KH2P04 buffer at pH 6.0 with final concentration of 1 % (w/v) as substrate stock.
The substrate stock was diluted with near 60mM Citric acid-Na2HP04 buffer at pH 6.0 or 60mM PBS buffer at pH 8.0 until Abs.450nm approximately reach 1. 20 mI protein at 50 pg/ml (except several which was used at stocked concentration: SEQ ID NO: 39 0.5mg/ml, SEQ ID NO: 41 3.2mg/ml, SEQ ID NO: 42 0.2mg/ml, 064VZ8 0.3mg/ml) and 200 mI diluted bacterial strain solution were added into 96-well plate, mixed and read OD450. Then the plate was incubated at 37°C, 300 rpm for 1hour, read OD450 again. The OD difference between 1 hour to initial read showed the OD drop activity for these proteins. Blank was set by adding 20ul MQ water, and each sample was measured in triplicate. Table 2 shown the OD drop result of all CHAP proteins at pH6.0 and pH 8.0. Such pH ranges are typical for sludge.
Table 2. the OD drop result of CHAP amidases
Figure imgf000088_0001
Figure imgf000089_0001
Example 5: Use of SEQ ID NO: 30 for improvinq sludqe flocculation
Materials
Digested sludge 1 from a municipal wastewater plant in China. This sludge was generated by mesothermal digestion of wasted activated sludge.
Digested sludge 2 from another municipal wastewater plant in China. This sludge was processed by thermal hydrolysis by 120-180°C for 20-60 min before anaerobic digestion.
Cellulase composition comprised an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a T choderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656)
Protease was Subtilisin protease of WO 1999/027082 and WO 2003/006602.
Flocculent: Cationic polyacrylamide was obtained from the wastewater plant. Prepare the polymer solution by mixing the polymer into required amount of de-ionized water to a 0.2% polymer solution. The polymer could be stored for no longer than 1 week.
Method
1. 50 mL sludge was weight and put into a 100ml_ beaker.
2. Enzyme were added into the sludge sample. Polypeptide with CHAP domain was added on top of protease+celluase composition.
3. Sealed the reaction beaker and put into a shaker, incubated overnight at 35°C in a shaker with slow speed at 90 rpm. 4. The sludge was taken out from shaker and a known volume of polymer solution was added, and stirred immediately using magnetic stirring at a 150 rpm for 15s. and then stirred very slowly for 60s.
5. Took photo and observed the flocculation status. Calculated the optimum dosage according to the floe size and clarity of bulk water. Poured the flocculated sludge onto a 7um filter cloth, and measured the absorbance of the filtrate at 450 nm.
Results
Each condition was carried out in triplicated. The polypeptide was used in amount of 50 ug protein/t-DS, protease was used in amount of 0.5 kg/t-DS, and cellulase composition was used in amount of 0.5 kg/t-DS. The results (Table 3 and Fig.1 ) shown that the addition of protease and cellulase composition can improve the sludge flocculation and save polymer to achieve the optimum flocculation. Added SEQ ID NO: 30 which contains CHAP domain, on top of protease and cellulase composition brought in a better flocculation and clearer bulk water, and could achieve 27% reductions of optimum polymer dosage for sludge 1 , and 12% reductions for sludge 2. And this performance was obtained on two kinds of digested sludge.
Table 3. Optimum polymer dosage for two kinds of sludge
Figure imgf000090_0001
Example 6. Use of polypeptides with CHAP domain for improvina sludae flocculation
Materials
Digested sludge from a municipal wastewater plant in China. This sludge was generated by mesothermal digestion of wasted activated sludge. TS=1.7%
Results
The other polypeptides with CHAP domain (SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, respectively) were tested using the same procedure of Example 5. Each condition was carried out in triplicated. The polypeptide was used in amount of 50 ug protein/t-DS, protease was used in amount of 0.5 kg/t-DS, and cellulase composition was used in amount of 0.5 kg/t-DS.
The optimum polymer demanded for different polypeptide molecules were shown in Table 4 and the filtrate absorbance were shown in Figure 2. Adding one of 5 amidases of the enzyme in addition to a protease and cellulase composition all brought in a better flocculation and clearer bulk water. The polymer demanded for the optimum flocculation was 3.8-4 kg/t-TS, corresponding to 20-24% polymer reduction compared to the sludge only, and 10-14% more saving compared to protease and cellulase composition.
Table 4. Optimum polymer dosage for different CHAP molecules
Figure imgf000091_0001
Example 7. Flocculation result of sludge with the same enzyme dosage
Materials
Digested sludge from a municipal wastewater plant in China. This sludge was generated by mesothermal digestion of wasted activated sludge. TS=2.0%
Method
1. 50 mL sludge was weight and put into a 100ml_ beaker.
2. Enzyme were added to the sludge sample. For each sample all the active protein added were the same. SEC ID NO: 30 was added to replace cellulase composition or protease + cellulase composition with the same amount of active protein.
3. Seal the reaction beaker and put into a shaker, incubated overnight at 35°C in a shaker with slow speed at 90 rpm.
4. The sludge was taken out from shaker and a known volume of polymer solution was added. Stirred immediately using magnetic stirring at a 150 rpm for 15s. and then stirred very slowly for 60s.
5. Took photo and observed the flocculation status. Collected the bulk water and measured the absorbance at 600 nm. Results
Each condition was carried out in triplicated. The result shown in Table 5 and Fig.3. The mixture of protease, cellulase composition and SEQ ID NO: 30 could achieve a better flocculation of sludge at the same protein dosage. The best result was happened at the 50% replacement of cellulase composition, with brought in 18% polymer savings, while 7% polymer savings for protease + cellulase composition.
Table 5. Optimum polymer dosage for different CHAP molecules.
Figure imgf000092_0001
Example 8. Use of polypeptides with CHAP domain for increasinq dryness Materials
Digested sludge from a municipal wastewater plant in China. This sludge was generated by mesothermal digestion of wasted activated sludge. TS=1.8%
Cellulase composition comprised an Aspergillus aculeatus GH10 xylanase (WO
94/021785) and a T choderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656)
Protease was Subtilisin protease of WO 2011/036263. Polypeptide sample was SEQ ID NO: 30
Flocculent: Cationic polyacrylamide was obtained from the wastewater plant. Prepare the polymer solution by mixing the polymer into required amount of de-ionized water to a 0.2% polymer solution. The polymer could be stored for no longer than 1 week.
Method
1. 120 mL sludge was weight and put into a 250 ml_ beaker.
Protease and cellulase composition were added to the sludge sample polypeptide was added on top of protease and cellulase composition. Enzyme dose: protease: 21ug/g-TS, cellulase composition: 100ug/g-TS, SEQ ID NO: 30 50ug/g-TS.
2. Sealed the reaction beaker and put into a shaker, incubated overnight at 35°C in a shaker with slow speed at 90 rpm.
3. The sludge was taken out from shaker and a known volume of polymer solution was added. Stirred immediately using magnetic stirring at a 150 rpm for 15s. and then stirred very slowly for 60s.
4. Took photo and observed the flocculation status.
5. Poured the sludge to the vacuum system at the vacuum of 0.08 MPa and tested the cake dryness after 10 min vacuum filtration.
Results
As shown in Table 6, the mixture of protease, cellulase composition and SEQ ID NO: 30 could bring in 17% polymer savings, while 8% savings for only use of protease and cellulase composition. Besides, addition of SEQ ID NO: 30 increased the cake dryness by 1.7% at the optimum polymer dose, which was 1 % higher than protease and cellulase composition.
Table 6. Optimum polymer dose and cake dryness after vacuum filtration
Figure imgf000093_0001
Example 9. Use of polypeptides with CHAP domain for improving sludge flocculation
Materials
Digested sludge from a municipal wastewater plant in China. This sludge was generated by mesothermal digestion of wasted activated sludge. TS=1.7%
Cellulase composition comprised an Aspergillus aculeatus GH10 xylanase (WO
94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656)
Protease was Subtilisin protease of WO 1999/027082 and WO 2003/006602.
Polypeptide sample was SEQ ID NO:35, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:38, SEQ ID NO:29, SEQ ID NO:25, SEQ ID NO:34, SEQ ID NO:41 , SEQ ID NO:39 or SEQ ID NO:40.
Flocculent: Cationic polyacrylamide was obtained from the wastewater plant. Prepare the polymer solution by mixing the polymer into required amount of de-ionized water to a 0.2% polymer solution. The polymer could be stored for no longer than 1 week.
Method
1. 50 mL sludge was weight and put into a 100ml_ beaker.
2. Enzyme were added into the sludge sample. Polypeptide with CHAP domain was added on top of protease+celluase composition.
3. Sealed the reaction beaker and put into a shaker, incubated overnight at 35°C in a shaker with slow speed at 90 rpm.
4. The sludge was taken out from shaker and a known volume of polymer solution was added, and stirred immediately using magnetic stirring at a 150 rpm for 15s. and then stirred very slowly for 60s.
5. Took photo and observed the flocculation status in light of Figure 4. The optimum polymer dosage used to achieve the optimum flocculation status (at least score 4) was recorded.
Results
Each condition was carried out in triplicated. And each polypeptide was tested in duplicate. The polypeptide was used in amount of 50 ug protein/t-DS, protease was used in amount of 0.5 kg/t-DS, and cellulase composition was used in amount of 0.5 kg/t-DS. The results were shown in Table 7-9. Seen from Table 7, SEQ ID NO:35 shown the best performance. The polymer demanded for the optimum flocculation was 5.2 kg/t-TS, corresponding to 13% polymer reduction compared to the sludge only, and 7% reduction compared to protease + cellulase composition.
Seen from Table 8, SEQ ID NOs: 38, 27, 34 and 41 shown better performance on reducing polymer consumption. The polymer demanded for the optimum flocculation was 3.6- 4.1 kg/t-TS, corresponding to 10-22% polymer reduction compared to the sludge only, and 2- 14% reduction compared to protease+ cellulase composition.
Seen from Table 9, SEQ ID NOs: 39, 25 and 40 shown better performance on reducing polymer consumption. The polymer demanded for the optimum flocculation was 4.8-4.9 kg/t- TS, corresponding to about 14% polymer reduction compared to the sludge only, and 11% more saving compared to protease plus cellulase composition.
Table 7. Optimum polymer dosage for different CHAP molecules
Figure imgf000095_0001
Table 8. Optimum polymer dosage for different CHAP molecules
Figure imgf000095_0002
Figure imgf000096_0002
Table 9. Optimum polymer dosage for different CHAP molecules
Figure imgf000096_0001
Example 10: Use of polypeptides with CHAP domain for reducing the polymer consumption
Materials
Digested sludge from a municipal wastewater plant in China. This sludge was generated by mesothermal digestion of wasted activated sludge.
Cellulase composition, protease and flocculent were same with used in Example 9 Method
1. 50 mL sludge was weight and put into a 100ml_ beaker.
2. Enzyme were added into the sludge sample. Polypeptide with CHAP domain was added on top of protease+celluase composition.
3. Sealed the reaction beaker and put into a shaker, incubated overnight at 35°C in a shaker with slow speed at 90 rpm.
4. The sludge was taken out from shaker and a known volume of polymer solution was added, and stirred immediately using magnetic stirring at a 150 rpm for 15s. and then stirred very slowly for 60s.
5. Took photo and observed the flocculation status. Calculated the optimum dosage according to the floe size and clarity of bulk water. Poured the flocculated sludge onto a 7um filter cloth, and measured the absorbance of the filtrate at 450 nm.
Results Each condition was carried out in triplicated. The polypeptide was used in amount of 50 ug protein/t-DS, protease was used in amount of 0.5 kg/t-DS, and cellulase composition was used in amount of 0.5 kg/t-DS. Table 10 shown that the addition of SEQ ID NOs: 22 or 30 on top of protease and cellulase composition brought in a better flocculation and clearer bulk water, and could achieve 9% reductions of optimum polymer dosage compared to the sludge only, and 7% reduction compared to protease + cellulase composition.
Table 10. Optimum polymer dosage for two difference polypeptides with CHAP domain
Figure imgf000097_0001
Example 11 : CST test for the polypeptides with CHAP domain. Materials
Digested sludge from a municipal wastewater plant in China. This sludge was generated by mesothermal digestion of wasted activated sludge.
Cellulase composition, protease and flocculent were same with used in Example 9
Method
1. 50 ml_ sludge was weight and put into a 100ml_ beaker.
2. Enzyme were added into the sludge sample. Polypeptide with CHAP domain was added on top of protease+celluase composition.
3. Sealed the reaction beaker and put into a shaker, incubated overnight at 35°C in a shaker with slow speed at 90 rpm.
4. The sludge was taken out from shaker and a known volume of polymer solution was added, and stirred immediately using magnetic stirring at a 150 rpm for 15s. and then stirred very slowly for 60s. 5. 5ml mixture obtained from step 4 were collected for CST (Capillary Suction Time) test with the standard Type 304 CST apparatus produced by Triton Electronics Ltd. The dosage of polymer used to reach the lowest CST was recorded.
Capillary suction time (CST) was a simple and precise measure of the rate at which water was released from a sludge matrix. This measure of sludge dewaterability was used to optimize the performance and operation of sludge dewatering processes. Sludges that released water quickly had a low CST (see Progress in Filtration and Separation, Edition: 1 , Chapter: 17. Capillary Suction Time (CST), Publisher: Academic Press, Editors: E.S. Tarleton, pp.659-670).
Each condition was carried out in triplicated. The polypeptide was used in amount of 50 ug protein/t-DS, protease was used in amount of 0.5 kg/t-DS, and cellulase composition was used in amount of 0.5 kg/t-DS. The result shown in Table 11. The addition of SEQ ID Nos:35, 38, 41. 42 and 45 on top of protease and cellulase composition significantly improved the dewaterability of the digested sludge by 22%, 12%, 22%, 12% and 12% respectively compared with protease + cellulase composition.
Table 11. Optimum polymer dose calculated by the CST.
Figure imgf000098_0001
*The optimum polymer dose represented the dosage of polymer used to reach the lowest CST.
The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

CLAIMS What is claimed is:
1. A method of treating sludge comprising i) adding an effective amount of one or more amidase enzyme having a CHAP domain, and one or more flocculants to the sludge to form a mixture of water and coagulated and flocculated solids and ii) separating the coagulated and flocculated solids from the water.
2. A method of treating sludge according to claim 1 , wherein the amidase enzyme having a CHAP domain comprises a polypeptide amino acids 126 to 212 of SEQ ID NO: 22, amino acid 128 to 214 of SEQ ID NO:23, amino acids 129 to 215 of SEQ ID NO: 24, amino acid 125 to 211 of SEQ ID NO:25, amino acids 131 to 213 of SEQ ID NO: 26, amino acid 127 to 213 of SEQ ID NO:27, amino acids 125 to 211 of SEQ ID NO: 28, amino acid 126 to 212 of SEQ ID NO:29, amino acids 129 to 213 of SEQ ID NO: 30, amino acid 132 to 217 of SEQ ID NO:31 , amino acids 121 to 207 of SEQ ID NO: 32, amino acid 129 to 214 of SEQ ID NO:33, amino acids 127 to 213 of SEQ ID NO: 34, amino acid 128 to 211 of SEQ ID NO:35, amino acids 54 to 138 of SEQ ID NO: 36, amino acid 56 to 139 of SEQ ID NO:37, amino acids 136 to 218 of SEQ ID NO: 38, amino acid 141 to 227 of SEQ ID NO:39, amino acids 126 to 211 of SEQ ID NO: 40, amino acid 122 to 208 of SEQ ID NO:41 , or amino acids 131 to 218 of SEQ ID NO: 42.
3. A method according to claim 1 , wherein the amidase enzyme having a CHAP domain is an isolated or purified polypeptide having amidase activity, selected from the group consisting of:
(a) a polypeptide having at least 60% sequence identity to mature polypeptide of any of SEQ ID NOs: 22-42;
(b) a polypeptide encoded by a polynucleotide that hybridizes under medium, medium-high, high or very high stringency conditions with the full-length complement of the mature
polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA sequence thereof;
(e) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1-21 ;
(f) a fragment of the polypeptide of (a), (b), or (e) that has amidase activity.
4. An isolated or purified polypeptide having amidase activity, selected from the group consisting of:
(a) a polypeptide having at least 60% sequence identity to mature polypeptide of any of SEQ ID NOs: 22-42; (b) a polypeptide encoded by a polynucleotide that hybridizes under medium, medium-high, high or very high stringency conditions with the full-length complement of the mature
polypeptide coding sequence of any of SEQ ID NOs: 1-21 or the cDNA sequence thereof;
(e) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1-21 ;
(f) a fragment of the polypeptide of (a), (b), or (e) that has amidase activity.
5. The polypeptide of claim 1 , which is a fragment of any of SEQ ID NOs: 22-42 or the mature polypeptide thereof, wherein the fragment preferably contains amino acids 21-236 of SEQ ID NO:22, amino acids 21-238 of SEQ ID NO:23, amino acids 21-239 of SEQ ID NO:24, amino acids 21-235 of SEQ ID NO:25, amino acids 21-237 of SEQ ID NO:26, amino acids 21- 237 of SEQ ID NO:27, amino acids 21-235 of SEQ ID NO:28, amino acids 21-236 of SEQ ID NO:29, amino acids 21-236 of SEQ ID NO:30, amino acids 22-239 of SEQ ID NO:31 , amino acids 21-230 of SEQ ID NO:32, amino acids 21-238 of SEQ ID NO:33, amino acid 21-239 of SEQ ID NO:34, amino acids 21-235 of SEQ ID NO:35, amino acid 17-161 of SEQ ID NO:36, amino acids 19-163 of SEQ ID NO:37, amino acid 21-242 of SEQ ID NO:38, amino acids 22- 251 of SEQ ID NO:39, amino acid 21-235 of SEQ ID NO:40, amino acids 20-230 of SEQ ID NO:41 , or amino acid 20-242 of SEQ ID NO:42, and wherein the fragment has amidase activity.
6. An isolated or purified polynucleotide encoding the polypeptide of claim 4 or 5.
7. The polynucleotide of claim 6, which comprises SEQ ID NO: 1 or nucleotides 61 to 711 of SEQ ID NO: 1 , SEQ ID NO: 2 or nucleotides 61 to 400, 452 to 768 of SEQ ID NO: 2, SEQ ID NO: 3 or nucleotides 61 to 403, 477-793 of SEQ ID NO: 3, SEQ ID NO: 4 or nucleotides 61 to 391 , 456 to 772 of SEQ ID NO: 4, SEQ ID NO: 5 or nucleotides 61 to 397, 470 to 786 of SEQ ID NO: 5, SEQ ID NO: 6 or nucleotides 61 to 391 , 455 to 771 of SEQ ID NO: 6, SEQ ID NO: 7 or nucleotides 61 to 391 , 452-768 of SEQ ID NO: 7, SEQ ID NO: 8 or nucleotides 61 to 394,
455 to 771 of SEQ ID NO: 8, SEQ ID NO: 9 or nucleotides 61 to 397, 475 to 788 of SEQ ID NO: 9, SEQ ID NO: 10 or nucleotides 64 to 411 , 492 to 800 of SEQ ID NO: 10, SEQ ID NO: 11 or nucleotides 61 to 379, 482-795 of SEQ ID NO: 1 1 , SEQ ID NO: 12 or nucleotides 61 to 717 of SEQ ID NO: 12, SEQ ID NO: 13 or nucleotides 61 to 720 of SEQ ID NO: 13, SEQ ID NO: 14 or nucleotides 61 to 708 of SEQ ID NO: 14, SEQ ID NO: 15 or nucleotides 49 to 486 of SEQ ID NO: 15, SEQ ID NO: 16 or nucleotides 55 to 492 of SEQ ID NO: 16, SEQ ID NO: 17 or nucleotides 61 to 412, 463-779 of SEQ ID NO: 17, SEQ ID NO: 18 or nucleotides 64 to 756 of SEQ ID NO: 18, SEQ ID NO: 19 or nucleotides 61 to 708 of SEQ ID NO: 19, SEQ ID NO: 20 or nucleotides 58 to 693 of SEQ ID NO: 20, SEQ ID NO: 21 or nucleotides 58 to 729 of SEQ ID NO: 21.
8. A nucleic acid construct or expression vector comprising the polynucleotide of claim 6 or 7, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.
9. A recombinant host cell comprising the polynucleotide of claim 6 or 7 operably linked to one or more control sequences that direct the production of the polypeptide.
10. A method of producing a protein, comprising cultivating the recombinant host cell of claim 6 under conditions conducive for production of the protein.
11. An enzyme composition comprising a protease and the polypeptide of claim 4 or 5.
12. The use of the enzyme composition of claim 8 in enhancing the dewaterability of sludge.
13. A method of treating sludge, comprising:
(c) Contacting the sludge with the enzyme composition of claim 8, and
(d) Removing water from the sludge.
PCT/CN2020/076877 2019-02-28 2020-02-27 Polypeptides with chap domain and their use for treating sludge WO2020173473A1 (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
US20030141256A1 (en) * 2002-01-29 2003-07-31 Jawed Sarkar Method of dewatering sludge using enzymes
WO2005073132A1 (en) * 2004-01-23 2005-08-11 Nalco Company Enzyme-assisted clarification and dewatering of wastewater
WO2013096707A1 (en) * 2011-12-21 2013-06-27 General Electric Company Microwave processing of wastewater sludge

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030141256A1 (en) * 2002-01-29 2003-07-31 Jawed Sarkar Method of dewatering sludge using enzymes
WO2005073132A1 (en) * 2004-01-23 2005-08-11 Nalco Company Enzyme-assisted clarification and dewatering of wastewater
WO2013096707A1 (en) * 2011-12-21 2013-06-27 General Electric Company Microwave processing of wastewater sludge

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* Cited by examiner, † Cited by third party
Title
BATEMAN, A. ET AL.: "The CHAP domain:a large family of amidases including GSP amidase and peptidoglycan hydrolases", TRENDS IN BIOCHEMICAL SCIENCES, vol. 28, no. 5, 31 May 2003 (2003-05-31), pages 234 - 237, XP004425561, DOI: 10.1016/S0968-0004(03)00061-6 *
DATABASE GenBank 14 August 2010 (2010-08-14), Database accession no. XM_003039550.1 *
DATABASE GenBank 14 August 2010 (2010-08-14), Database accession no. XP_003039596 .1 *
DATABASE GenBank 21 March 2015 (2015-03-21), Database accession no. ENH71971.1 *
DATABASE GenBank 24 June 2016 (2016-06-24), Database accession no. OBT58177.1 *
SHARMA, M. ET AL.: "Amidases:versatile enzymes in nature", REV ENVIRON SCI BIOTECHNOL, vol. 8, 24 October 2009 (2009-10-24), pages 343 - 366, XP019774338, DOI: 10.1007/s11157-009-9175-x *
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