CN103764670A - Polypeptides having glucoamylase activity and polynucleotides encoding same - Google Patents

Polypeptides having glucoamylase activity and polynucleotides encoding same Download PDF

Info

Publication number
CN103764670A
CN103764670A CN201280041671.1A CN201280041671A CN103764670A CN 103764670 A CN103764670 A CN 103764670A CN 201280041671 A CN201280041671 A CN 201280041671A CN 103764670 A CN103764670 A CN 103764670A
Authority
CN
China
Prior art keywords
polypeptide
seq
polynucleotide
sequence
glucoamylase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201280041671.1A
Other languages
Chinese (zh)
Inventor
孙天齐
李明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Priority to CN201280041671.1A priority Critical patent/CN103764670A/en
Priority claimed from PCT/CN2012/080553 external-priority patent/WO2013029496A1/en
Publication of CN103764670A publication Critical patent/CN103764670A/en
Pending legal-status Critical Current

Links

Classifications

    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Provided are isolated polypeptides having glucoamylase activity, catalytic domains, and polynucleotides encoding the polypeptides, catalytic domains. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains.

Description

There are polypeptide and its polynucleotide of coding of glucoamylase activity
The reference of sequence table
This application has comprised a sequence table that is computer-reader form, and this sequence table is combined in this by reference.
Background of invention
Invention field
The present invention relates to have the polypeptide of glucoamylase activity and the polynucleotide of these polypeptide of coding.The invention still further relates to the nucleic acid construct, carrier and the host cell that have comprised these polynucleotide, together with for generation of with use the method for these polypeptide, and the purposes that relates to glucoamylase of the present invention and transform to produce tunning (as ethanol) and syrup (as glucose) for starch.The invention still further relates to a kind of composition that comprises glucoamylase of the present invention.
Description of Related Art
Glucoamylase (Isosorbide-5-Nitrae-α-D-dextran glucose lytic enzyme, EC3.2.1.3) is catalysis discharges D-Glucose a kind of enzyme from the non-reducing end of starch or Related Oligosaccharides and polysaccharide molecule.Glucoamylase is produced by some filamentous funguss and yeast, from those of Aspergillus, is wherein commercially most important.
Commercially, glucoamylase is used to the starch material being partly hydrolyzed by α-amylase to change into glucose.Then glucose can be used a kind of fermentation organism to change into directly or indirectly tunning.The example of commercial tunning comprises alcohols (for example, ethanol, methyl alcohol, butanols, 1,3-PD); Organic acid (for example, citric acid, acetic acid, methylene-succinic acid, lactic acid, glyconic acid, gluconate, lactic acid, succsinic acid, 2,5-DKG); Ketone (for example acetone); Amino acids (for example L-glutamic acid); Gas (for example, H 2and CO 2), and more complicated compound, comprise for example antibiotics (for example, penicillin and tsiklomitsin); Enzyme; Vitamins (for example, riboflavin, B 12, β-carotene); Hormones; And other compounds that are difficult to produce synthetically.Fermenting process is for example also normally used for, in consumable alcohol (, beer and grape wine), milk preparation (for example,, for the manufacture of yoghourt and cheese) industry.
End product may be also syrup.For example, end product can be glucose, but also a kind of mixture that can for example change into fructose or be formed by almost equal ground glucose and fructose by glucose isomerase.This mixture, or other a kind of mixture of enrichment fructose are business-like the most frequently used high-fructose corn syrups (HFCS) worldwide.
An object of the present invention is to provide multiple the have polypeptide of glucoamylase activity and the polynucleotide of these polypeptide of coding, and they are in tunning production technique, as alcohol production technique, comprise by providing high yield in the step ethanol fermentation technique that raw (or uncooked) starch of gelationization does not carry out.According to one of glucoamylase of the present invention, SEQ ID NO:2, has 62.3% consistence with the GENESEQP:ASP18034 from Phoma herbarum (Phoma herbarum) included in WO2008080093-A2.Another kind of glucoamylase of the present invention, SEQ ID NO:4, with included from agglomerate capsule deer Cordyceps (Elaphocordyceps ophioglossoide) in WO2008080093-A2 gENESEOP:ASP18042have 73.0% consistence.
Summary of the invention
By fungi Talaromyces, produced and the polypeptide with glucoamylase activity has been identified and has been characterized.More particularly, Talaromyces is to be selected from Talaromyces emersonii (Talaromyces emersonii).
The present invention relates to have the isolated polypeptide of glucoamylase activity, this polypeptide is selected from lower group, and this group is comprised of the following:
(a) have at least 75%, at least 80%, at least 85%, at least 90%, a for example peptide species of 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 with the mature polypeptide of SEQ ID NO:2 or SEQ ID NO:4;
(b) by under medium-high stringency condition, the high stringency condition of high stringency conditioned disjunction with the mature polypeptide encoded sequence of (i) SEQ ID NO:1 or SEQ ID NO:3, (ii) its cDNA sequence or the (iii) coded peptide species of a kind of polynucleotide of total length complement hybridization (i) or (ii);
(c) for example, by having at least 75%, at least 80%, at least 85%, at least 90% with SEQ ID NO:1 or SEQ ID NO:3 mature polypeptide encoded sequence or its cDNA sequence, the coded peptide species of a kind of polynucleotide of 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;
(d) a kind of variant of the mature polypeptide of SEQ ID NO:2 or SEQ ID NO:4, this variant comprises a replacement, disappearance and/or inserts in one or more (several) position; And
(e) fragment with glucoamylase activity of (a) and (b), (c) or these polypeptide (d).
The invention still further relates to the isolated polypeptide that has comprised glucoamylase catalyst structure domain, this catalyst structure domain is selected from lower group, and this group is comprised of the following:
(a) there is a catalyst structure domain of at least 75% sequence identity with the amino acid 31 to 500 of SEQ ID NO:2 or the amino acid 29 to 499 of SEQ ID NO:4;
(b) by under high stringency condition with the (i) Nucleotide 91 to 1652 of SEQ ID NO:1 or the Nucleotide 85 to 1712 of SEQ ID NO:3, (ii) its cDNA sequence or the (iii) coded catalyst structure domain of a kind of polynucleotide of total length complement hybridization (i) or (ii);
(c) by with the (i) Nucleotide 91 to 1652 of SEQ ID NO:1 or the Nucleotide 85 to 1712 of SEQ ID NO:3 (ii) or its cDNA sequence there is a coded catalyst structure domain of a kind of polynucleotide of at least 90% sequence identity; And
(d) a kind of variant of the amino acid 31 to 500 of SEQ ID NO:2 or the amino acid 29 to 499 of SEQ ID NO:4, this variant for example, comprises a replacement, disappearance and/or inserts in one or more (several) position;
And wherein this catalyst structure domain has glucoamylase activity.
The invention still further relates to the polynucleotide of the separation of coding polypeptide of the present invention; Nucleic acid construct; Recombinant expression vector; The recombinant host cell that comprises these polynucleotide; And produce the method for these polypeptide.
The invention still further relates to the method by amyloid material produce tunning, and the composition that comprises glucoamylase of the present invention.
Definition
Glucoamylase: term glucoamylase (Isosorbide-5-Nitrae-α-D-dextran glucose lytic enzyme, EC3.2.1.3) is defined as a kind of enzyme of catalysis from the non-reducing end release D-Glucose of starch or Related Oligosaccharides and polysaccharide molecule.For purposes of the present invention, glucoamylase activity is the program determination of basis described in this ' materials and methods ' part.
Polypeptide of the present invention have SEQ ID NO:2 mature polypeptide at least 20%, preferably at least 40%, preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% and even most preferably at least 100% glucoamylase activity, or polypeptide of the present invention have SEQ ID NO:4 mature polypeptide at least 20%, preferably at least 40%, preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% and even most preferably at least 100% glucoamylase activity.
Allele variant: term " allele variant " meaning refers to any in two or more alternative forms of a gene that occupies same chromogene seat.Allelic variation is naturally to cause by sudden change, and may in colony, cause polymorphism.Transgenation can be reticent (in the polypeptide of coding, nothing changes), maybe can encode and provide the polypeptide of the aminoacid sequence changing.The allele variant of one peptide species is a peptide species coded by the allele variant of a gene.
Binding domains: term " carbohydrate binding domains " meaning refers to the region of this enzyme of mediation of a kind of enzyme and the combination of carbohydrate substrate.This carbohydrate binding domains (CBD) typically at the N-terminal place of glucoamylase or the end points place of C-terminal find.This CBD is called again starch binding domains, SBD sometimes.
Catalyst structure domain: term " catalyst structure domain " meaning refers to a kind of region of the catalysis machine that comprises this enzyme of enzyme.
CDNA: term " cDNA " meaning refers to can be by a kind of DNA molecular of preparing by reverse transcription from the ripe mRNA molecule through montage of an eucaryon or prokaryotic cell prokaryocyte acquisition.CDNA lacks intron sequences, and these sequences may reside in corresponding genomic dna.The precursor that initial elementary rna transcription thing is mRNA, this precursor, before the ripe mRNA being rendered as through montage, is processed by the series of steps including montage.
Encoding sequence: term " encoding sequence " meaning refers to so a kind of polynucleotide, and these polynucleotide have directly been specified the aminoacid sequence of polypeptide.The border of this encoding sequence is generally to be determined by open reading frame, and this open reading frame starts with an initiator codon (as ATG, GTG or TTG), and finishes with a terminator codon (as TAA, TAG or TGA).This encoding sequence can be a genomic dna, cDNA, synthetic DNA or its combination.
Control sequence: term " control sequence " meaning refers to the polynucleotide of the mature polypeptide of the present invention of encoding to express necessary nucleotide sequence.Each control sequence can be natural (that is, from same gene) or external (that is, from different genes) for the polynucleotide of this polypeptide of coding, or natural or external each other each other.This class control sequence includes but not limited to, leader sequence, polyadenylation sequence, propeptide sequence, promotor, signal peptide sequence and transcription terminator.On bottom line, these control sequences comprise a promotor, and transcribe and translation termination signal.These control sequences can possess multiple connexons, for the object that the specific limited site that contributes to these control sequences to be connected with the coding region of the polynucleotide of coded polypeptide is introduced.
Express: term " expression " has comprised and produces the related any step of a peptide species, includes but not limited to, transcribe, post transcriptional modificaiton, translation, posttranslational modification and secretion.
Expression vector: term " expression vector " meaning refers to the DNA molecular of a kind of linearity or annular, polynucleotide that this molecule has comprised coded polypeptide and be operably connected to the control sequence that its expression is provided.
Fragment: term " fragment " meaning refers to have one or more (for example several) the amino acid whose peptide species or catalysis or the carbohydrate binding domains that are not present in the amino of mature polypeptide or structural domain and/or C-terminal; Wherein this fragment has glucoamylase or carbohydrate in conjunction with activity.In one aspect, at least 85% the amino-acid residue that fragment has comprised SEQ ID NO:2 or SEQ ID NO:4, at least 90% amino-acid residue or at least 95% amino-acid residue.In a specific embodiment, the amino acid 31 to 500 that fragment has comprised SEQ ID NO:2 or the amino acid 29 to 499 of SEQ ID NO:4.
High stringency condition: term " the high stringency condition " meaning refers to the probe long at least 100 Nucleotide, follow standard Southern western blot procedure, at 42 ℃ in 5X SSPE, 0.3% SDS, 200 mcg/ml through shearing and the methane amide of the salmon sperm dna of sex change and 50% in carry out prehybridization and hybridization 12 to 24 hours.Solid support material is finally used 2X SSC, 0.2% SDS washing three times, each 15 minutes at 65 ℃.
Host cell: term " host cell " meaning refers to be subject to comprise any cell type of the nucleic acid construct of polynucleotide of the present invention or the conversion of expression vector, transfection, transduction etc.The sudden change and the inconsistent any filial generation of this parent cell because occurring between replicative phase of a parent cell contained in term " host cell ".
Separate: term " separation " meaning refers to a kind of material in the non-existent form of occurring in nature or environment.The limiting examples of the material separating comprises the material that (1) any non-natural exists; (2) any material of removing from one or more or all naturally occurring compositions that are connected with it at nature at least in part, includes but not limited to any enzyme, variant, nucleic acid, protein, peptide or cofactor; (3) this material of finding with respect to occurring in nature, by manually modified any material; Or (4) increase (multiple copies of the gene of this material of for example, encoding by making this amount of substance to some extent with respect to other components that are naturally connected with it; Use than and the stronger promotor of the promotor that is naturally connected of gene of this material of coding) any material of modifying.A kind of material of separation may reside in fermentation broth sample.
Low stringency condition: term " the low stringency condition " meaning refers to the probe long at least 100 Nucleotide, follow standard Southern western blot procedure, at 42 ℃ in 5X SSPE, 0.3% SDS, 200 mcg/ml through shearing and the methane amide of the salmon sperm dna of sex change and 25% in carry out prehybridization and hybridization 12 to 24 hours.Solid support material is finally used 2X SSC, 0.2% SDS washing three times, each 15 minutes at 50 ℃.
Mature polypeptide: term " mature polypeptide " meaning refers to be afterwards in translation and any posttranslational modification (as N-terminal processing, C-terminal brachymemma, glycosylation, phosphorylation etc.) peptide species of its final form.In one aspect, based on SignalP program (Nelson (Nielsen) etc., 1997, < < protein engineering > > (Protein Engineering) 10:1-6) predict that the amino acid/11 to 21 of SEQ ID NO:2 is signal peptides, this mature polypeptide is the amino acid 22 to 537 of SEQ ID NO:2.In yet another aspect, based on SignalP program (Nelson etc., 1997, < < protein engineering > > 10:1-6) predict that the amino acid/11 to 21 of SEQ ID NO:4 is signal peptides, this mature polypeptide is the amino acid 22 to 651 of SEQ ID NO:4.
As known in the art, a host cell can produce the mixture of two or more different mature polypeptides (that is, having different C-terminal and/or N-terminal amino acid) of being expressed by same polynucleotide.
Mature polypeptide encoded sequence: term " mature polypeptide encoded sequence " meaning refer to encode out a kind of polynucleotide of the mature polypeptide with glucoamylase activity.In one aspect, based on SignalP(Nelson etc., 1997, with above) signal peptide of Nucleotide 1 to 63 coding of prediction SEQ ID NO:1, this mature polypeptide encoded sequence is the Nucleotide 64 to 1763 of SEQ ID NO:1, or its cDNA sequence.In yet another aspect, this mature polypeptide encoded sequence is Nucleotide 64-226,294-1113 and the 1199-1763 of SEQ ID NO1.
In yet another aspect, based on SignalP(Nelson etc., 1997, with above) signal peptide of Nucleotide 1 to 63 coding of prediction SEQ ID NO:3, this mature polypeptide encoded sequence is the Nucleotide 64 to 2168 of SEQ ID NO:3, or its cDNA sequence.In yet another aspect, this mature polypeptide encoded sequence is Nucleotide 64-232,293-576,626-722,777-1414 and the 1467-2168 of SEQ ID NO3.
Medium stringency condition: term " the medium stringency condition " meaning refers to the probe long at least 100 Nucleotide, follow standard Southern western blot procedure, at 42 ℃ in 5X SSPE, 0.3% SDS, 200 mcg/ml through shearing and the methane amide of the salmon sperm dna of sex change and 35% in carry out prehybridization and hybridization 12 to 24 hours.Solid support material is finally used 2X SSC, 0.2% SDS washing three times, each 15 minutes at 55 ℃.
Medium-high stringency condition: term " medium-high stringency condition " meaning refers to the probe long at least 100 Nucleotide, follow standard Southern western blot procedure, at 42 ℃ in 5X SSPE, 0.3% SDS, 200 mcg/ml through shearing and the salmon sperm dna of sex change, and or 35% methane amide in carry out prehybridization and hybridization 12 to 24 hours.Solid support material is finally used 2XSSC, 0.2% SDS washing three times, each 15 minutes at 60 ℃.
Nucleic acid construct: term " nucleic acid construct " meaning refers to strand or double-stranded a kind of nucleic acid molecule, this nucleic acid molecule is from naturally occurring gene isolation, or modify in a certain way to comprise otherwise will not be present in the nucleic acid segment of occurring in nature, or for what synthesize, it comprises one or more control sequences.
Be operably connected: term " is operably connected " to look like and refers to a kind of like this configuration, in this configuration, a control sequence is placed on the position suitable with respect to the encoding sequence of polynucleotide, makes like this this control sequence guide the expression of this encoding sequence.
Sequence identity; Dependency between two aminoacid sequences or between two nucleotide sequences is to be described by parameter " sequence identity ".For purposes of the present invention, sequence identity between two aminoacid sequences be use as at EMBOSS routine package (EMBOSS: European molecular biology Freeware bag, Lai Si (Rice) etc., 2000, < < genetics trend > > (Trends Genet.) 16:276-277), the interior moral Leman-Wen Saiqi algorithm (Needleman-Wunsch algorithm) (interior moral Leman and the Wen Saiqi that preferably in the Needle program of 5.0.0 or version afterwards, implement, 1970, < < molecular biology magazine > > (J.Mol.Biol.) 48:443-453) measure.These parameters of using are that point penalty 0.5 is extended in the open point penalty 10 in room, room, and the EMBOSS version of EBLOSUM62(BLOSUM62) substitution matrix.The output (acquisition of use-nobrief option) of " the longest consistence " of using Needle mark is as consistence per-cent and be calculated as follows:
(consistent residue × 100)/(sum of comparison length-comparison Vacancy)
For purposes of the present invention, sequence identity between two deoxyribonucleotide sequences be use as at EMBOSS routine package (EMBOSS: European molecular biology Freeware bag, Lai Si etc., 2000, with above), interior moral Leman-Wen Saiqi algorithm of preferably implementing in the Needle program of 5.0.0 or version afterwards (interior moral Leman and Wen Saiqi, 1970, with above) measure.These parameters of using are that point penalty 0.5 is extended in the open point penalty 10 in room, room, and the EMBOSS version of EDNAFULL(NCBI NUC4.4) substitution matrix.The output (acquisition of use-nobrief option) of " the longest consistence " of using Needle mark is as consistence per-cent and be calculated as follows:
(consistent deoxyribonucleotide × 100)/(sum of comparison length-comparison Vacancy)
Subsequence: term " subsequence " meaning refers to have a kind of polynucleotide of one or more (for example several) Nucleotide that is not present in the 5' of mature polypeptide encoded sequence and/or 3' end; Wherein this subsequence is encoded out and has a fragment of glucoamylase activity.In one aspect, at least 85% the Nucleotide that subsequence has comprised SEQ ID NO:1 or SEQ ID NO:3, at least 90% Nucleotide or at least 95% Nucleotide.In a particular embodiment, the Nucleotide 91 to 1652 that subsequence has comprised SEQ ID NO:1, or the Nucleotide 85 to 1712 of SEQ ID NO:3.
Variant: term " variant " meaning refers to for example, comprise a kind of change in one or more (several) position, that is, and replacements, a peptide species with glucoamylase activity that inserts and/or lack.Replace the meaning and refer to that the amino acid that occupies a position is by a different amino acid replacement; The disappearance meaning refers to occupy the amino acid whose of a position and removes; And inserting the meaning refers to be adjacent to and add an amino acid afterwards followed by the amino acid that occupies a position.
High stringency condition: term " the high stringency condition " meaning refers to the probe long at least 100 Nucleotide, follow standard Southern western blot procedure, at 42 ℃ in 5X SSPE, 0.3% SDS, 200 mcg/ml through shearing and the methane amide of the salmon sperm dna of sex change and 50% in carry out prehybridization and hybridization 12 to 24 hours.Solid support material is finally used 2X SSC, 0.2% SDS washing three times, each 15 minutes at 70 ℃.
Extremely low stringency condition: term " the extremely low stringency condition " meaning refers to the probe long at least 100 Nucleotide, follow standard Southern western blot procedure, at 42 ℃ in 5X SSPE, 0.3% SDS, 200 mcg/ml through shearing and the methane amide of the salmon sperm dna of sex change and 25% in carry out prehybridization and hybridization 12 to 24 hours.Solid support material is finally used 2X SSC, 0.2% SDS washing three times, each 15 minutes at 45 ℃.
Detailed description of the invention
There is the polypeptide of glucoamylase activity
In one embodiment, the present invention relates to have at least 75%, at least 80%, at least 85%, at least 86% with the mature polypeptide of SEQ ID NO:2, the for example isolated polypeptide of 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% sequence identity, these isolated polypeptide have glucoamylase activity.Specifically, this glucoamylase have SEQ ID NO:2 mature polypeptide at least 20%, preferably at least 40%, preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% and even most preferably at least 100% glucoamylase activity.
In one aspect, the mature polypeptide of these polypeptide and SEQ ID NO:2 has and is no more than 10 (for example 1,2,3,4,5,6,7,8 or 9) amino acid whose differences.
Polypeptide of the present invention preferably includes or it consists of aminoacid sequence or its allele variant of SEQ ID NO:2; Or there is the fragment of glucoamylase activity for it.In yet another aspect, this polypeptide comprises or it consists of the mature polypeptide of SEQ ID NO:2.In yet another aspect, this polypeptide comprises or it consists of the amino acid 64 to 537 of SEQ ID NO:2.
In another embodiment, the present invention relates to have at least 75%, at least 80%, at least 85%, at least 86% with the mature polypeptide of SEQ ID NO:4, the for example isolated polypeptide of 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% sequence identity, these isolated polypeptide have glucoamylase activity.Specifically, this glucoamylase have SEQ ID NO:4 mature polypeptide at least 20%, preferably at least 40%, preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% and even most preferably at least 100% glucoamylase activity.In one aspect, the mature polypeptide of these polypeptide and SEQ ID NO:4 has and is no more than 10 (for example 1,4,3,4,5,6,7,8 or 9) amino acid whose differences.
Polypeptide of the present invention preferably includes or it consists of aminoacid sequence or its allele variant of SEQ ID NO:4; Or there is the fragment of glucoamylase activity for it.In yet another aspect, this polypeptide comprises or it consists of the mature polypeptide of SEQ ID NO:4.In yet another aspect, this polypeptide comprises or it consists of the amino acid 64 to 545 of SEQ ID NO:4.
In another embodiment, the present invention relates to a kind of isolated polypeptide with glucoamylase activity, this isolated polypeptide is by medium-high stringency condition, under the high stringency condition of high stringency conditioned disjunction with the (i) mature polypeptide encoded sequence of SEQ ID NO:1, (ii) its cDNA sequence, or (iii) coded (the Pehanorm Brooker (Sambrook) etc. of a kind of polynucleotide of total length complement (i) or (ii) hybridization, 1989, < < molecular cloning experiment guide > > (Molecular Cloning, A Laboratory Manual), the 2nd edition, cold spring port, New York (Cold Spring Harbor, New York)).
In another embodiment, the present invention relates to a kind of isolated polypeptide with glucoamylase activity, this isolated polypeptide is by medium-high stringency condition, under the high stringency condition of high stringency conditioned disjunction with the (i) mature polypeptide encoded sequence of SEQ ID NO:3, (ii) its cDNA sequence, or (iii) coded (the Pehanorm Brooker etc. of a kind of polynucleotide of total length complement (i) or (ii) hybridization, 1989, < < molecular cloning experiment guide > >, the 2nd edition, cold spring port, New York).
The polynucleotide of SEQ ID NO:1 or SEQ ID NO:3 or its subsequence, together with polypeptide or its fragment of SEQ ID NO:2 or SEQ ID NO:4, can be for designing nucleic acid probe, in order to according to method well known in the art, never belong to together or the bacterial strain of kind in differentiate and clone the DNA of the polypeptide with glucoamylase activity of having encoded.Specifically, can use this class probe, follow Southern western blot procedure, with genomic dna or the cDNA hybridization of cells of interest, to differentiate and isolate corresponding gene wherein.This class probe can be shorter than complete sequence significantly, but should be at least 15, for example at least 25, at least 35 or at least 70 Nucleotide long.Preferably, this nucleic acid probe is that at least 100 Nucleotide are long, and for example at least 200 Nucleotide, at least 300 Nucleotide, at least 400 Nucleotide, at least 500 Nucleotide, at least 600 Nucleotide, at least 700 Nucleotide, at least 800 Nucleotide or at least 900 Nucleotide are long.Two kinds of DNA and rna probes can be used.These probes are typically labeled for detection of corresponding gene and (for example, use 32p, 3h, 35s, vitamin H or avidin 9 white marker).This class probe is contained in the present invention.
Can for probe hybridization described above and the DNA of the polypeptide with glucoamylase activity of encoding out the genomic dna of being prepared by other bacterial strains of this class or cDNA storehouse are screened.Genome or other DNA from these other bacterial strains of class can pass through agarose or polyacrylamide gel electrophoresis, or other isolation technique separate.From the DNA in these storehouses or the DNA of separation, can transfer to and be fixed on Nitrocellulose or other applicable solid support materials.In order to differentiate and clone or the DNA of SEQ ID NO:1 or the hybridization of its subsequence, this solid support material is used for to Southern trace.
For purposes of the present invention, these polynucleotide of hybridization indication extremely low under high stringency condition with the nucleic acid probe hybridization through mark corresponding to the following: (i) SEQ ID NO:1 or SEQ ID NO:3; (ii) the mature polypeptide encoded sequence of SEQ ID NO:1 or SEQ ID NO:3; (iii) its cDNA sequence; (iv) its total length complement; Or (v) its subsequence.Can use for example X ray film or any other detection means as known in the art to detect with the molecule of this nucleic acid probe hybridization under these conditions.
In one aspect, this nucleic acid probe is Nucleotide 64-226,294-1113 and the 1199-1763 of SEQ ID NO1.In yet another aspect, this nucleic acid probe is the polypeptide of coding SEQ ID NO:2; Its mature polypeptide; Or the polynucleotide of its fragment.In yet another aspect, this nucleic acid probe is SEQ ID NO:1 or its cDNA sequence.
In one aspect, this nucleic acid probe is Nucleotide 64-232,293-576,626-722,777-1414 and the 1467-2168 of SEQ ID NO3.In yet another aspect, this nucleic acid probe is the polypeptide of coding SEQ ID NO:4; Its mature polypeptide; Or the polynucleotide of its fragment.In yet another aspect, this nucleic acid probe is SEQ ID NO:3 or its cDNA sequence.
In another embodiment, the present invention relates to a kind of isolated polypeptide with glucoamylase activity, this isolated polypeptide is that for example the polynucleotide of 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% sequence identity are coded by having at least 75%, at least 80%, at least 85%, at least 86% with mature polypeptide encoded sequence or its cDNA sequence of SEQ ID NO:1.
In another embodiment, the present invention relates to a kind of isolated polypeptide with glucoamylase activity, this isolated polypeptide is by have the polynucleotide of at least 75%, at least 80%, 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% sequence identity coded with the mature polypeptide encoded sequence of SEQ ID NO:3 or its cDNA sequence.
In another embodiment, the present invention relates to the variant of the mature polypeptide of SEQ ID NO:2 or SEQ ID NO:4, these variants for example, comprise a replacement, disappearance and/or insert in one or more (several) position.In one embodiment, the number of aminoacid replacement, disappearance and/or the insertion in the mature polypeptide of introducing SEQ ID NO:2 or SEQ ID NO:4 is no more than 10, for example 1,2,3,4,5,6,7,8 or 9.These amino acid change can have small character, that is, the folding and/or active conserved amino acid that can not affect significantly protein replaces or inserts; Typically 1-30 amino acid whose less disappearance; Less amino or C-terminal extend, as aminoterminal methionine residues; The less connexon peptide of 20-25 residue at the most; Or be convenient to come by changing net charge or another kind of function the less extension of purifying, as polyhistidyl sequence (tract), epitope or binding domains.
The conservative example replacing is in the scope of lower group: basic aminoacids (arginine, Methionin and Histidine), acidic amino acid (L-glutamic acid and aspartic acid), polare Aminosaeren (glutamine and l-asparagine), hydrophobic amino acid (leucine, Isoleucine and α-amino-isovaleric acid), die aromatischen Aminosaeuren (phenylalanine, tryptophane and tyrosine) and p1 amino acid (glycine, L-Ala, Serine, Threonine and methionine(Met)).The general aminoacid replacement that can not change specific activity is as known in the art and for example by H. knob Lars (H.Neurath) and R.L. Xi Er (R.L.Hill), 1979, < < protein G reatT.GreaT.GT > (The Proteins), new york academic press (Academic Press, New York) is described.Common replacement is Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.
As an alternative, these amino acid change a kind of character with the physics-chem characteristic change that makes polypeptide.For instance, amino acid changes the thermostability that can improve polypeptide, changes substrate specificity, changes optimum pH etc.
Primary amino acid in polypeptide can be according to program as known in the art, as site-directed mutagenesis or alanine scanning mutagenesis are differentiated (Cunningham (Cunningham) and Wei Ersi (Wells), 1989, < < science > > (Science) 244:1081-1085).In a rear technology, each the residue place in this molecule introduces single alanine mutation, and the glucoamylase activity of gained mutant molecule is tested to differentiate the vital amino-acid residue of activity for this molecule.Also referring to Hilton (Hilton) etc., 1996, < < journal of biological chemistry > > (J.Biol.Chem.) 271:4699-4708.The avtive spot of this enzyme or other biological interact and also can in conjunction with the amino acid whose sudden change in contact site of inferring, measure by structure physical analysis (as by being measured as technology such as nucleus magnetic resonance, crystallography, electron diffraction or photoaffinity labeling).Referring to for example, De Wosi (de Vos) etc., 1992, < < science > > 255:306-312; Smith (Smith) etc., 1992, < < molecular biology magazine > > 224:899-904; Wo Dawen (Wlodaver) etc., 1992, the < < Europe communication > > of biochemical society (FEBS Lett.) 309:59-64.The identity of primary amino acid also can be by comparing to know by inference with a kind of related polypeptide.
Single or multiple aminoacid replacement, disappearance and/or insertion can be used known mutagenesis, restructuring and/or Shuffling Method, the screening procedure of being correlated with subsequently, as Randt's Kazakhstan-Mancur Olson (Reidhaar-Olson) and Sol (Sauer), 1988, < < science > > 241:53-57; Bao Wei (Bowie) and Sol, periodical > > (Proc.Natl.Acad.Sci.USA) 86:2152-2156 of institute of 1989, < < NAS; WO95/17413; Or disclosed those of WO95/22625, produce and test.Operable additive method comprises that fallibility PCR, phage (for example present, Lao Man (Lowman) etc., 1991, < < biological chemistry > > (Biochemistry) 30:10832-10837; U.S. Patent number 5,223,409; WO92/06204) and determine region mutagenesis (Darbishire (Derbyshire) etc., 1986, < < gene > > (Gene) 46:145; Nail (unit of length) (Ner) etc., 1988, < < DNA > > 7:127).
Mutagenesis/reorganization method can combine with high throughput automated screening method the activity (Nai Si (Ness) etc. of the polypeptide of the mutagenesis that detects the clone expressed by host cell, 1999, < < nature-biotechnology > > (Nature Biotechnology) 17:893-896).The DNA molecular of the mutagenesis of coding active polypeptide can reclaim from these host cells, and uses the standard method in this area to check order rapidly.These methods allow to determine fast the importance of indivedual amino-acid residues in polypeptide.
This polypeptide can be a kind of hybrid polypeptide, and merge at N-terminal or the C-terminal place in a region of another polypeptide in a region of one of them polypeptide.
This polypeptide can be the fusion polypeptide of a kind of fusion polypeptide or cleavable, and wherein another polypeptide is to merge at N-terminal or the C-terminal place of polypeptide of the present invention.Fusion polypeptide is by polynucleotide and the polynucleotide of the present invention of another polypeptide of coding are merged to produce.For generation of the technology of fusion polypeptide, be as known in the art, and comprise the encoding sequence of coded polypeptide is connected, making thus them is that expression same frame and this fusion polypeptide is under the control in identical promoters and terminator.Fusion polypeptide can also be used intein technique construction, in this technology, fusion polypeptide is the (Ku Bai (Cooper) etc. that produce after translation, the magazine > > of 1993, < < European Molecular Bioglogy Organization (EMBO J.) 12:2575-2583; Road gloomy (Dawson) etc., 1994, < < science > > 266:776-779).
Fusion polypeptide can comprise in addition a cracking site between two polypeptide.After fusion rotein secretion, this site is cleaved, thereby discharges this two polypeptide.The example of cracking site includes but not limited to the site disclosed in the following: Martin (Martin) etc., 2003, < < industrial microbiology and biotechnology magazine > > (J.Ind.Microbiol.Biotechnol.) 3:568-576; Match civilian orange red Na (Svetina) etc., 2000, < < biotechnology magazine > > (J.Biotechnol.) 76:245-251; Hans Kjeld Rasmussen-Wei Ersen (Rasmussen-Wilson) etc., 1997, < < application and environmental microbiology > > (Appl.Environ.Microbiol.) 63:3488-3493; Ward (Ward) etc., 1995, < < biotechnology > > (Biotechnology) 13:498-503; And Kang Te Lars (Contreras) etc., 1991, < < biotechnology > > 9:378-381; Eton (Eaton) etc., 1986, < < biological chemistry > > 25:505-512; Collins-Lei Xi (Collins-Racie) etc., 1995, < < biotechnology > > 13:982-987; Ka Te (Carter) etc., 1989, < < protein: structure, function and genetics > > (Proteins:Structure, Function, and Genetics) 6:240-248; And Stevens (Stevens), 2003, < < world drug discovery > > (Drug Discovery World) 4:35-48.
There is the source of the polypeptide of glucoamylase activity
The polypeptide with glucoamylase activity of the present invention can obtain from the microorganism of any genus.For purposes of the present invention, as the term using in conjunction with given source at this " from ... obtain " should refer to, by the polypeptide of polynucleotide encoding, be to produce by this source or by a kind of bacterial strain having inserted from the polynucleotide in this source.In one aspect, the polypeptide obtaining from given source is cell exocrine.
This polypeptide can be a kind of fungi polypeptide.For instance, this polypeptide can be Penicillium or Talaromyces polypeptide.
In yet another aspect, this polypeptide is a kind of Ai Mosen mould (Penicillium emersonii) or a kind of Talaromyces emersonii polypeptide.
Will be appreciated that, for previously mentioned kind, two kinds of perfect and imperfect states are contained in the present invention, and the equivalent of other classification, and for example anamorph, regardless of the kind title of known they.Those of ordinary skill in the art will be easy to identify the identity of suitable equivalent.
The public is easy to obtain the bacterial strain of these kinds at many culture collections center, as American type culture collection (ATCC), German microbial strains preservation center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), Dutch DSMZ (Centraalbureau Voor Schimmelcultures, CBS) and american agriculture research research centre, DSMZ southern area (NRRL).
This polypeptide can be used above-mentioned probe differentiate and obtain from other sources, comprises the microorganism such as, separating from nature (soil, compost, water etc.) or the DNA sample directly such as, obtaining from natural materials (soil, compost, water etc.).Be used for is directly well known in the art from the technology of s natural element separate microorganism and DNA.Then, can pass through the genomic dna to another kind of microorganism or cDNA storehouse similarly, or the DNA sample mixing screens to obtain the polynucleotide of this polypeptide of coding.Once the polynucleotide to coded polypeptide by these one or more probe in detecting, just can be by utilizing technology known to persons of ordinary skill in the art that these polynucleotide are separated or cloned (referring to for example, Pehanorm Brooker etc., 1989, with above).
Catalyst structure domain
In one embodiment, the invention still further relates to the amino acid 31 to 500 of SEQ ID NO:2 and have at least 75%, at least 80%, at least 85%, at least 86%, for example catalyst structure domain of at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.In one aspect, the amino acid 31 to 500 of the aminoacid sequence that these catalyst structure domains comprise and SEQ ID NO:2 has and is no more than 10 (for example 1,2,3,4,5,6,7,8 or 9) amino acid whose differences.
This catalyst structure domain preferably includes or it consists of amino acid 31 to 500 or its allele variant of SEQ ID NO:2; Or there is the fragment of glucoamylase activity for it.
In another embodiment, the invention still further relates to by lower catalyst structure domain (the Pehanorm Brooker etc. coded with the Nucleotide 91 to 1652 of (i) SEQ ID NO:1, (ii) its cDNA sequence or polynucleotide that (iii) total length complement (i) or is (ii) hybridized of the high stringency condition of high stringency conditioned disjunction (as defined above), 1989, with above).
In another embodiment, for example the invention still further relates to, by having at least 75%, at least 80%, at least 85%, at least 86%, the coded catalyst structure domain of the polynucleotide of at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with amino acid 91 to 1652 or its cDNA sequence of SEQ ID NO:1.
In another embodiment, the invention still further relates to the catalyst structure domain variant of the amino acid 31 to 500 of SEQ ID NO:2, these variants for example, comprise a replacement, disappearance and/or insert in one or more (several) position.In one aspect, the number of aminoacid replacement, disappearance and/or insertion in the sequence of the amino acid 31 to 500 of introducing SEQ ID NO:2 is 10, for example 1,2,3,4,5,6,8 or 9.
In another embodiment, the invention still further relates to the amino acid 29 to 499 of SEQ ID NO:4 and have at least 75%, at least 80%, at least 85%, at least 86%, for example catalyst structure domain of at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.In one aspect, the amino acid 29 to 499 of the aminoacid sequence that these catalyst structure domains comprise and SEQ ID NO:4 has and is no more than 10 (for example 1,4,3,4,5,6,7,8 or 9) amino acid whose differences.
This catalyst structure domain preferably includes or it consists of amino acid 29 to 499 or its allele variant of SEQ ID NO:4; Or there is the fragment of glucoamylase activity for it.
In another embodiment, the invention still further relates to by lower catalyst structure domain (the Pehanorm Brooker etc. coded with the Nucleotide 85 to 1712 of (i) SEQ ID NO:3, (ii) its cDNA sequence or polynucleotide that (iii) total length complement (i) or is (ii) hybridized of the high stringency condition of high stringency conditioned disjunction (as defined above), 1989, with above).
In another embodiment, for example the invention still further relates to, by having at least 75%, at least 80%, at least 85%, at least 86%, the coded catalyst structure domain of the polynucleotide of at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with amino acid 85 to 1712 or its cDNA sequence of SEQ ID NO:3.
In another embodiment, the invention still further relates to the catalyst structure domain variant of the amino acid 29 to 499 of SEQ ID NO:4, these variants for example, comprise a replacement, disappearance and/or insert in one or more (several) position.In one aspect, the number of aminoacid replacement, disappearance and/or insertion in the sequence of the amino acid 29 to 499 of introducing SEQ ID NO:4 is 10, for example 1,2,3,4,5,6,8 or 9.
Binding domains
In one embodiment, the invention still further relates to the carbohydrate binding domains with the amino acid 536 to 651 of SEQ ID NO:4 with 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.In one aspect, the aminoacid sequence that this carbohydrate binding domains comprises and the amino acid 536 to 651 of SEQ ID NO:4 have and are no more than 10 (for example 1,2,3,4,5,6,7,8 or 9) amino acid whose differences.
This carbohydrate binding domains preferably includes or it consists of amino acid 536 to 651 or its allele variant of SEQ ID NO:4; Or there is carbohydrate in conjunction with active fragment for it.
In another embodiment, the invention still further relates to the carbohydrate binding domains variant of the amino acid 536 to 651 of SEQ ID NO:4, these variants for example, comprise a replacement, disappearance and/or insert in one or more (several) position.In one aspect, the number of aminoacid replacement, disappearance and/or insertion in the sequence of the amino acid 536 to 651 of introducing SEQ ID NO:4 is 10, for example 1,2,3,4,5,6,8 or 9.
The catalyst structure domain that is operably connected to this carbohydrate binding domains can be from lytic enzyme, isomerase, ligase enzyme, lyase, oxydo-reductase or transferring enzyme, for example aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, allosteric lytic enzyme, oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, zytase or xylobiase.The polynucleotide of coding catalyst structure domain can obtain from any protokaryon, eucaryon or other sources.
In a particular embodiment, this catalyst structure domain is included in the catalyst structure domain of the present invention in SEQ ID NO:2 or SEQ ID NO:4.
Heterozyme
The invention still further relates to heterozyme, these enzymes have comprised (for example starch degradation enzymic activity that has enzymic activity, as α-amylase, starch Pullulanase (amylopullulanase), beta-amylase, CGT enzyme, glucoamylase, isoamylase, raw wheat starch enzyme or Pullulanase activity) a catalyst structure domain, an and carbohydrate binding domains (CBD).This heterozyme can comprise a connexon in addition.
This heterocomplex can be by merging first DNA sequence dna of coding catalyst structure domain and second DNA sequence dna of encoding carbohydrate binding modules to produce, or this heterocomplex can be based on being applicable to CBD, connexon and catalyst structure domain the understanding of aminoacid sequence, as one intactly synthetic gene produce.
Term " heterozyme " (being called again " fusion rotein ", " heterocomplex ", " hybrid polypeptide " or " hybrid protein ") is used herein to and characterizes hybrid polypeptide of the present invention, these hybrid polypeptides have comprised one and have had enzymic activity (for example starch degradation enzymic activity, as α-amylase, starch Pullulanase, beta-amylase, CGT enzyme, glucoamylase, isoamylase, raw wheat starch enzyme or Pullulanase activity) catalytic module and a carbohydrate binding modules, wherein this catalyst structure domain and this carbohydrate binding modules are to derive from different sources.Term " source " includes but not limited to, female enzyme or its variant, for example amylase or glucoamylase, or other applicable catalytic module that comprises catalytic activity and/or an applicable CBD and/or applicable connexons.But this CBD derives from the polypeptide without catalytic activity.This catalyst structure domain and this carbohydrate binding modules can derive from same microorganism bacterial strain, derive from bacterial strain in identical type, derive from relevant nearly kind or not too relevant organism.Preferably, the catalyst structure domain of these heterocomplexs and carbohydrate binding modules are to derive from different sources, for example, derive from the different enzyme from same bacterial strain and/or kind, or for example derive from the bacterial strain in different sorts.
In one aspect, this heterozyme comprises according to CBD of the present invention and a catalyst structure domain.In a particular embodiment, this catalyst structure domain is a glucoamylase catalyst structure domain or a α-amylase catalyst structure domain.In yet another aspect, this heterozyme comprises a catalyst structure domain of the present invention and a glucoamylase CBD or a α-amylase CBD.The example that is applicable to CBD and connexon for example can see in WO06/069290.
Polynucleotide
The invention still further relates to the polynucleotide of the separation of coding polypeptide of the present invention as the described herein, catalyst structure domain or carbohydrate binding domains.
For separating of or the technology of clone's polynucleotide be as known in the art and comprise from genomic dna or cDNA, or its combination separates.Clone from the polynucleotide of genomic dna can be for example by using well-known polymerase chain reaction (PCR) or realizing in order to the expression library antibody screening that the DNA fragmentation of the clone with total constitutional features is detected.Referring to for example, Yi Nisi (Innis) etc., 1990, < < PCR method and application guide > > (PCR:A Guide to Methods and Application), new york academic press.Can use other nucleic acid amplification programs, as ligase chain reaction (LCR), connect activation transcribe (LAT) and the amplification based on polynucleotide (NASBA).These polynucleotide can be cloned by Talaromyces bacterial strain or relevant organism, and therefore, for example, can be allelotrope or the kind variants of the polypeptid coding area of these polynucleotide.
The modification of the polynucleotide of code book invention polypeptide can be essential for the synthetic polypeptide that is similar in fact this polypeptide.Term " similar in fact " refers in this polypeptide the form that the non-natural of this polypeptide exists.These polypeptide may be different from from its natural origin isolated polypeptide in certain through engineering approaches mode, for example, at different variants in aspect such as specific activity, thermostability, optimum pH.These variants can be based on SEQ ID NO:1 or SEQ ID NO:3 mature polypeptide encoded sequence, or the polynucleotide that present of its cDNA sequence (for example its subsequence) form, and/or by introducing the aminoacid sequence that can not change this polypeptide, but corresponding to the Nucleotide that is intended for the organic codon usage of host that produces this enzyme, replace, or replace to build by introducing the Nucleotide that may produce different aminoacids sequence.The general description that relevant Nucleotide replaces, referring to for example, Ford (Ford) etc., 1991, < < protein expression and purifying > > (Protein Expression and Purification) 2:95-107.
Nucleic acid construct
The invention still further relates to nucleic acid construct, these nucleic acid constructs have comprised the polynucleotide of the present invention that are operably connected to one or more control sequences, these one or more control sequences guide this encoding sequence with the compatible condition of these control sequences under express being applicable in host cell.
Polynucleotide can handle to provide by variety of way the expression of this polypeptide.Depend on expression vector, in insertion vector before to this Nucleotide handle can be make us wish or essential.It is well known in the art utilizing the technology that recombinant DNA method is modified polynucleotide.
This control sequence can be a promotor,, by host cell, is identified a kind of polynucleotide so that the polynucleotide of code book invention polypeptide are expressed that is.This promotor comprises transcriptional control sequence, and these sequences have mediated the expression of this polypeptide.This promotor can be any polynucleotide that demonstrate transcriptional activity in host cell, comprise mutant, brachymemma and hybrid promoter, and can be to be obtained by the gene of polypeptide in the extracellular of coding and this host cell homology or allos or cell.
For guiding nucleic acid construct of the present invention, at the example of the applicable promotor of bacterial host cell transcription, be by bacillus amyloliquefaciens (Bacillus amyloliquefaciens) alpha-amylase gene (amyQ), Bacillus licheniformis (Bacillus licheniformis) alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), the raw wheat starch enzyme gene of bacstearothermophilus (Bacillus stearothermophilus) (amyM), subtilis (Bacillus subtilis) type froctosan saccharase gene (sacB), subtilis xylA and xylB gene, bacillus thuringiensis (Bacillus thuringiensis) cryIIIA gene (A Gesi (Agaisse) and Le Leikusi (Lereclus), 1994, < < molecular biology > > (Molecular Microbiology) 13:97-107), intestinal bacteria lac operon, intestinal bacteria trc promotor (Ai Ge (Egon) etc., 1988, < < gene > > (Gene) 69:301-315), sky blue streptomycete (Streptomyces coelicolor) gelase gene (dagA) and protokaryon β-lactamase gene (Wella-Karma Lip river husband (Villa-Kamaroff) etc., 1978, the periodical > > 75:3727-3731 of institute of < < NAS), together with tac promotor (De Boer (DeBoer) etc., 1983, the periodical > > 80:21-25 of institute of < < NAS).Other promotors are described in " from the useful proteins matter (Useful proteins from recombinant bacteria) of recombinant bacteria ", gilbert (Gilbert) etc., 1980, < < Scientific Beauty compatriots > > (Scientific American) 242:74-94; And Pehanorm Brooker etc., 1989, together above.The example of Gene expression is disclosed in WO99/43835.
It for guiding nucleic acid construct of the present invention, at the example of the applicable promotor of filamentous fungal host cell transcription, is the promotor being obtained by the gene of following thing: Aspergillus nidulans (Aspergillus nidulans) acetamidase, aspergillus niger (Aspergillus niger) neutral alpha-amylase, aspergillus niger acid acceptance α-amylase, aspergillus niger or Aspergillus awamori (Aspergillus awamori) glucoamylase (glaA), aspergillus oryzae (Aspergillus oryzae) TAKA amylase, aspergillus oryzae Sumizyme MP, aspergillus oryzae triosephosphate isomerase, Fusarium oxysporum (Fusarium oxysporum) trypsin-like proteolytic enzyme (WO96/00787), fusarium (Fusarium venenatum) amyloglucosidase (WO00/56900), fusarium Daria(WO00/56900), fusarium Quinn(WO00/56900), rhizomucor miehei bacterium (Rhizomucor miehei) lipase, rhizomucor miehei bacterium aspartate protease, Trichodermareesei (Trichoderma reesei) beta-glucosidase enzyme, Trichodermareesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, trichoderma reesei endoglucanase I, trichoderma reesei endoglucanase II, trichoderma reesei endoglucanase III, trichoderma reesei endoglucanase IV, trichoderma reesei endoglucanase V, Xylanase from Trichoderma reesei I, Xylanase from Trichoderma reesei II, Trichodermareesei xylobiase, together with NA2-tpi promotor, (through the promotor from Aspergillus neutral alpha-amylase gene of modifying, wherein untranslated leader is substituted by the untranslated leader from Aspergillus phosphotriose isomerase gene, limiting examples comprises the promotor from aspergillus niger neutral alpha-amylase gene through modifying, and wherein untranslated leader is substituted by the untranslated leader from Aspergillus nidulans or aspergillus oryzae phosphotriose isomerase gene), with and mutant, brachymemma and hybrid promoter.
In yeast host, useful promotor is to be obtained by the gene of following thing: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) Hydratase, phosphoenolpyruvate (ENO-1), yeast saccharomyces cerevisiae galactokinase (GAL1), yeast saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), yeast saccharomyces cerevisiae triosephosphate isomerase (TPI), brewing yeast metallothionein (CUP1) and yeast saccharomyces cerevisiae glycerol 3-phosphate acid kinase.Other useful promotors of yeast host cell are by Luo Man Butterworth (Romanos) etc., and 1992, < < yeast > > (Yeast) 8:423-488 describes.
This control sequence can also be identified the transcription terminator that stops transcribing by host cell.This terminator is operably connected to the 3' end of the polynucleotide of this polypeptide of coding.Any terminator working in this host cell may be used in the present invention.
For the terminator of bacterial host cell, be preferably to be obtained by the gene of Bacillus clausii (Bacillus clausii) Sumizyme MP (aprH), bacillus licheniformis alpha-amylase (amyL) and intestinal bacteria ribosome-RNA(rRNA) (rrnB).
Preferred is to be obtained by the gene of Aspergillus nidulans anthranilate synthase, aspergillus niger glucoamylase, aspergillus niger alpha-glucosidase, aspergillus oryzae TAKA amylase and Fusarium oxysporum trypsin-like proteolytic enzyme for the terminator of filamentous fungal host cell.
Preferred is by yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate, brewing yeast cell pigment C(CYC1 for the terminator of yeast host cell) and the gene of yeast saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase obtain.For other useful terminators of yeast host cell by Luo Man Butterworth etc., 1992, with described above.
This control sequence can also be in promotor downstream and in the mRNA stable region of gene coded sequence upstream, and it increases the expression of this gene.
The example that is applicable to mRNA stable region is by bacillus thuringiensis cryIIIA gene (WO94/25612) and subtilis SP82 gene (mound (Hue) etc., 1995, < < bacteriology magazine > > (Journal of Bacteriology) 177:3465-3471) obtain.
This control sequence can also be a leader sequence, a kind of to the very important untranslated mRNA region of host cell translation.This leader sequence is operably connected to the 5' end of the polynucleotide of this polypeptide of coding.Any leader sequence working in host cell can be used.
Preferred is to be obtained by the gene of aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase for the leader sequence of filamentous fungal host cell.
For the applicable leader sequence of yeast host cell, be to be obtained by the gene of yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate (ENO-1), yeast saccharomyces cerevisiae glycerol 3-phosphate acid kinase, yeast saccharomyces cerevisiae α-factor and yeast saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate (ADH2/GAP).
This control sequence can also be a polyadenylation sequence, a kind of sequence of the 3' end that is operably connected to polynucleotide, and by host cell, be identified as a signal that polyadenylic acid residue is added to the mRNA transcribing when transcribing.Any polyadenylation sequence working in host cell can be used.
Preferred is to be obtained by the gene of Aspergillus nidulans anthranilate synthase, aspergillus niger glucoamylase, aspergillus niger alpha-glucosidase, aspergillus oryzae TAKA amylase and Fusarium oxysporum trypsin-like proteolytic enzyme for the polyadenylation sequence of filamentous fungal host cell.
Useful polyadenylation sequence for yeast host cell is by Guo (Guo) and Xia Erman (Sherman), 1995, < < molecule and cytobiology > > (Mol.Cellular Biol.) 15:5983-5990 describe.
This control sequence can be also a signal peptide coding region, this coding region encoded the N-terminal that is connected to polypeptide signal peptide and guide this polypeptide to enter in the secretion path of cell.The 5' end of the encoding sequence of polynucleotide can be included in translation reading frame the signal coding sequence being connected natively with the section of the encoding sequence of coded polypeptide inherently.As an alternative, can to comprise for this encoding sequence be an external signal coding sequence for the 5' of this encoding sequence end.In the situation that this encoding sequence not comprises a signal coding sequence natively, an external signal coding sequence may be required.As an alternative, external signal coding sequence can substitute natural signals peptide-coding sequence simply, to promote the secretion of this polypeptide.But any signal coding sequence that guides expressed polypeptide to enter in the secretion path of host cell can be used.
It for the useful signal peptide-coding sequence of bacterial host cell, is the signal coding sequence being obtained by the gene of the raw wheat starch enzyme of genus bacillus NCIB11837, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, bacillus stearothermophilus alpha-amylase, bacstearothermophilus neutral protease (nprT, nprS, nprM) and subtilis prsA.Other signal peptides are to cover (Simonen) and Pa Erwa (Palva) by plug, 1993, < < microbiology is commented > > (Microbiological Reviews) 57:109-137 and is described.
It for the useful signal peptide-coding sequence of filamentous fungal host cell, is the signal coding sequence being obtained by the gene of aspergillus niger neutral starch enzyme, aspergillus niger glucoamylase, aspergillus oryzae TAKA amylase, Humicola insolens (Humicola insolens) cellulase, Humicola insolens EGV, pubescence humicola lanuginosa (Humicola lanuginosa) lipase and rhizomucor miehei bacterium aspartate protease.
For the useful signal peptide of yeast host cell, be to be obtained by the gene of yeast saccharomyces cerevisiae α-factor and yeast saccharomyces cerevisiae saccharase.Other useful signal coding sequences are by Luo Man Butterworth etc., 1992, and with described above.
This control sequence can also be the propeptide code sequence that coding is positioned at the propetide at the N-terminal place of polypeptide.Gained polypeptide is called as preferment or front polypeptide (or being called in some cases proenzyme).Front polypeptide be generally non-activity and can be cracked into propetide by front polypeptide catalysis or autocatalysis and change into active polypeptide.This propeptide code sequence can be obtained by bacillus subtilis alkali proteinase (aprE), subtilis neutral protease (nprT), the thermophilic gene of ruining a bacterium (Myceliophthora thermophila) laccase (WO95/33836), rhizomucor miehei bacterium aspartate protease and yeast saccharomyces cerevisiae α-factor.
In the situation that signal peptide and propeptide sequence both exist, this propeptide sequence is immediately following location the N-terminal of polypeptide after, and this signal peptide sequence is immediately following locating after the N-terminal of this propeptide sequence.
Can also wish to add regulating and controlling sequence, these regulating and controlling sequences regulate and control the expression of polypeptide with respect to the growth of host cell.The example of regulator control system be in response to chemistry or physical stimulation (comprising the existence of regulating compound) and make that the expression of gene opens or close those.Regulator control system in prokaryotic system comprises lac, tac and trp operator gene system.In yeast, can use ADH2 system or GAL1 system.In filamentous fungus, can use aspergillus niger glucoamylase promotor, aspergillus oryzae TAKA α-amylase promotor and aspergillus oryzae glucoamylase promotor.Other examples of regulating and controlling sequence are those of permission gene amplification.In eukaryotic system, these regulating and controlling sequences have comprised the dihydrofolate reductase gene of amplification under methotrexate exists, and the metallothionein gene of amplification under heavy metal exists.In these cases, the encode polynucleotide of this polypeptide will be operably connected with this regulating and controlling sequence.
Expression vector
The invention still further relates to recombinant expression vector, these expression vectors comprise polynucleotide of the present invention, a promotor and transcribe and translation termination signal.Different IPs thuja acid and control sequence can be bonded together to produce a kind of recombinant expression vector, and this recombinant expression vector can comprise that one or more restriction sites are easily to allow in encode insertion or the replacement of polynucleotide of this polypeptide of these site.As an alternative, these polynucleotide can be by inserting these polynucleotide or the nucleic acid construct that comprises these polynucleotide for expressing in the suitable carrier of expressing.When producing expression vector, this encoding sequence is to be arranged in this carrier, and the suitable control sequence that makes thus this encoding sequence express with this confession is operably connected.
This recombinant expression vector can be any carrier (for example plasmid or virus) that can experience expediently recombinant DNA program and can cause the expression of these polynucleotide.The selection of carrier will typically be depended on the consistency of this carrier with the host cell of intending to introduce this carrier.This carrier can be the plasmid of a linearity or closed annular.
This carrier can be a kind of carrier of self-replicating, that is, a kind of carrier existing with the outer entity form of karyomit(e), it copies with chromosome duplication irrelevant, for example plasmid, extra-chromosomal element, minichromosome or artificial chromosome.This carrier can comprise any member for guaranteeing self replication.As an alternative, this carrier can be when being introduced in host cell, to be incorporated into the carrier copying in genome and together with it has been integrated into karyomit(e) wherein.In addition, can use single carrier or plasmid, or comprise together the total DNA intending in introducing host cell gene group, or two or more carriers or the plasmid of transposon.
This carrier preferably comprises one or more selectable markers, and these selectable markers make to be easy to select the cell through conversion, transfection, transduction etc.Selectable marker is so a kind of gene, and its product provides biocide or virus resistance, the resistance to heavy metal, to auxotrophic prototrophy etc.
The example of the selectable marker of bacterium is Bacillus licheniformis or subtilis dal gene, or gives the marker of antibiotics resistance (as Ampicillin Trihydrate (ampicillin), paraxin (chloramphenicol), kantlex (kanamycin), Liu Suanyan NEOMYCIN SULPHATE (neomycin), spectinomycin (spectinomycin) or tetracyclin resistance).Applicable marker for yeast host cell includes but not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.Selectable marker for filamentous fungal host cell includes but not limited to; amdS(acetamidase), argB(ornithine transcarbamylase), bar(glufosinates Transacetylase), hph(hygromix phosphotransferase), niaD(nitrate reductase), pyrG(orotidine-5'-phosphate decarboxylase), sC(sulfate adenylyl transferase) and trpC(anthranilate synthase), together with its equivalent.Being preferred in Aspergillus cell is Aspergillus nidulans or aspergillus oryzae amdS and pyrG gene, and streptomyces hygroscopicus (Streptomyces hygroscopicus) bar gene.
This carrier preferably comprises in the genome that makes this carrier can be incorporated into host cell or makes this carrier can in this cell, be independent of an element of this genome self-replicating.
For being incorporated in host cell gene group, this carrier can depend in the sequence of polynucleotide of coded polypeptide or this carrier and be incorporated into any other element in this genome by homology or non-homogeneous restructuring.As an alternative, this carrier can comprise the other polynucleotide that are guided through homologous recombination and be incorporated into (multiple) accurate location in karyomit(e) in host cell gene group.In order to increase the possibility that is incorporated into an accurate location, these integrated elements should comprise the nucleic acid of sufficient amount, as 100 to 10,000 base pair, 400 to 10,000 base pair and 800 to 10,000 base pair, these nucleic acid have the high sequence degree of consistency with corresponding target sequence, thereby have strengthened the probability of homologous recombination.These integrated elements can be with host cell gene group in any sequence of target sequence homology.In addition, these integrated elements can be non-coding or coded polynucleotide.On the other hand, this carrier can be incorporated in the genome of host cell by non-homogeneous restructuring.
For self-replicating, this carrier can comprise the replication orgin that this carrier can independently be copied in touched upon host cell in addition.This replication orgin can be any plasmid replicon of the mediation self-replicating that works in cell.Term " replication orgin " or " plasmid replicon " a kind of polynucleotide of instigating plasmid or carrier to copy in vivo that look like.
The example of the replication orgin of bacterium is the replication orgin of allowing plasmid pBR322, the pUC19, pACYC177 and the pACYC184 that copy in intestinal bacteria, and allows the replication orgin of pUB110, the pE194, pTA1060 and the p Α Μ β 1 that copy in genus bacillus.
The example that is used for the replication orgin of yeast host cell is 2 microns of replication orgin; ARS1; ARS4; The combination of ARS1 and CEN3; And the combination of ARS4 and CEN6.
The example that is applicable to the replication orgin in filamentous fungal cells is AMA1 and ANS1(Jim this (Gems) etc., 1991, < < gene > > 98:61-67; Coulomb (Cullen) etc., 1987, < < nucleic acids research > > (Nucleic Acids Res.) 15:9163-9175; WO00/24883).The structure of the separation of AMA1 gene and the plasmid that comprises this gene or carrier can be realized according to the method disclosed in WO00/24883.
Can will exceed in the polynucleotide Insertion Into Host Cell of the present invention of a copy to increase the output of polypeptide.The increase of this polynucleotide copies quantity can be by being incorporated at least one other sequence copy in host cell gene group, or by comprising that a selectable marker gene that can increase and this polynucleotide obtain, wherein comprise cell and the other polynucleotide copies by this of the selectable marker gene copy of amplification, can select by cultivate these cells under suitably can selective agent existing.
In order to connect these elements described above, to build the program of recombinant expression vector of the present invention, be those of ordinary skill in the art well-known (referring to for example, Pehanorm Brooker etc., 1989, with above).
Host cell
The invention still further relates to recombinant host cell, these host cells have comprised the polynucleotide of the present invention that are operably connected to one or more control sequences, and these one or more control sequences guide the generation of polypeptide of the present invention.The construct that comprises polynucleotide or carrier are introduced in host cell, make thus this construct or carrier be maintained the form of chromosomal integration body or the form of the outer carrier of self replication karyomit(e), as described in the early time.The sudden change and the inconsistent any filial generation of this parent cell because occurring between replicative phase of a parent cell contained in term " host cell ".The selection of host cell will be depended on gene and its source of this polypeptide of encoding to a great extent.
This host cell can be any cell that is applicable to restructuring generation polypeptide of the present invention, for example prokaryotic cell prokaryocyte or eukaryotic cell.
Prokaryotic host cell can be any Gram-positive or gram negative bacterium.Gram positive bacterium includes but not limited to, bacillus, fusobacterium, enterococcus spp, Geobacillus, lactobacillus genus, lactococcus, bacillus marinus genus, Staphylococcus, streptococcus and streptomyces.Gram negative bacterium includes but not limited to, Campylobacter, intestinal bacteria, Flavobacterium, Fusobacterium, Helicobacterium, molten Bacillaceae, eisseria, Rhodopseudomonas, salmonella and urine slurry Pseudomonas.
This bacterial host cell can be any bacillus cell, include but not limited to, Alkaliphilic bacillus (Bacillus alkalophilus), bacillus amyloliquefaciens, bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus clausii, Bacillus coagulans (Bacillus coagulans), bacillus firmus (Bacillus firmus), bacillus lautus (Bacillus lautus), bacillus lentus (Bacillus lentus), Bacillus licheniformis, bacillus megaterium (Bacillus megaterium), bacillus pumilus (Bacillus pumilus), bacstearothermophilus, subtilis and bacillus thuringiensis cell.
This bacterial host cell can be also any suis cell, include but not limited to streptococcus equisimilis (Streptococcus equisimilis), product Streptococcus pyrogenes (Streptococcus pyogenes), streptococcus uberis (Streptococcus uberis) and streptococcus equi beast pest subspecies (Streptococcus equi subsp.Zooepidemicus) cell.
This bacterial host cell can also be any streptomyces cell, include but not limited to, do not produce look streptomycete (Streptomyces achromogenes), Avid kyowamycin (Streptomyces avermitilis), sky blue streptomycete, streptomyces griseus (Streptomyces griseus) and muta lead mycillin (Streptomyces lividans) cell.
DNA is introduced in bacillus cell and can be realized in the following manner: protoplast transformation is (referring to for example, often (Chang) and Ke's sweat (Cohen), 1979, < < MGG > > (Mol.Gen.Genet.) 168:111-115), competent cell transforms (referring to for example, poplar (Young) and Spizien (Spizizen), 1961, < < bacteriology magazine > > 81:823-829, or Du Bunuo (Dubnau) and Davidoff-A Bosen (Davidoff-Abelson), 1971, < < molecular biology magazine > > 56:209-221), electroporation is (referring to for example, Mao Chuan (Shigekawa) and road dimension (Dower), 1988, < < biotechnology > > 6:742-751) or combined techniques (referring to for example, (Koehler) and Si Aomu (Thome) strangle in section, 1987, < < bacteriology magazine > > 169:5271-5278).DNA is introduced and in Bacillus coli cells, can pass through protoplast transformation (referring to for example, Hana rare (Hanahan), 1983, < < molecular biology magazine > > 166:557-580) or electroporation (referring to for example, road dimension etc., 1988, < < nucleic acids research > > 16:6127-6145) realize.DNA is introduced in streptomyces cell and can pass through protoplast transformation, electroporation is (referring to for example, Gong (Gong) etc., 2004, < < microorganism journal > > (Folia Microbiol.) (Prague) 49:399-405), combined techniques is (referring to for example, Ma Zuo Dell (Mazodier) etc., 1989, < < bacteriology magazine > > 171:3583-3585) or transduction (referring to for example, Bock (Burke) etc., 2001, the periodical > > 98:6289-6294 of institute of < < NAS) realize.DNA is introduced and in pseudomonad cells, can pass through electroporation (referring to for example, Cai (Choi) etc., 2006, < < micro-biological process magazine > > (J.Microbiol.Methods) 64:391-397) or combined techniques (referring to for example, send many (Pinedo) and Si Maite (Smets), 2005, < < application and environmental microbiology > > 71:51-57) realize.DNA is introduced and in suis cell, can pass through nature competence (referring to for example, send auspicious (Perry) and storehouse light (Kuramitsu), 1981, < < infects and immune > > (Infect.Immun.) 32:1295-1297), protoplast transformation is (referring to for example, Ka Te (Catt) and Qiao Like (Jollick), 1991, < < microbiology > > (Microbios) 68:189-207), electroporation is (referring to for example, cloth Cray (Buckley) etc., 1999, < < application and environmental microbiology > > 65:3800-3804) or combined techniques (referring to for example, Ke Laiwei (Clewell), 1981, < < microbiology is commented > > 45:409-436) realize.But, as known in the artly for any method of DNA being introduced to host cell, can use.
This host cell can also be eukaryotic cell, as Mammals, insect, plant or fungal cell.
This host cell can be a fungal cell." fungi " comprises ascomycetes as used in this, basidiomycetes, chytrid and zygomycetes together with oomycetes door and all mitospore fungi (as Huo Ke Butterworth (Hawksworth) etc., < < fungi dictionary > > (Ainsworth and Bisby's Dictionary of The Fungi), the 8th edition, 1995, CABI (CAB International), (the University Press of press of univ cambridge uk, Cambridge, UK)).
This fungal host cells can be a yeast cell." yeast " comprises ascosporogenous yeast (Saccharomycetes), produces load yeast and belong to the yeast (blastogenesis Zoopagales) of fungi impertecti as used in this.Because the classification of yeast may change future, therefore for purposes of the present invention, yeast should be as < < biology of yeast and active > > (Biology and Activities of Yeast) (Si Jinna (Skinner), Paasche is (Passmore) and Davenport (Davenport) editor not, the > > of < < SAB (Soc.App.Bacteriol.Symposium), the 9th volume, 1980) described in, define.
This yeast host cell can be Candida, Hansenula, genus kluyveromyces, pichia yeast belongs to, yeast belong, Schizosaccharomyces or Ye Shi yeast belong cell, as Kluyveromyces lactis (Kluyveromyces lactis), Ka Ersibai yeast (Saccharomyces carisbergensis), yeast saccharomyces cerevisiae, saccharomyces diastaticus (Saccharomyces diastaticus), Doug Laplace yeast (Saccharomyces douglasii), kluyveromyces (Saccharomyces kluyveri), promise ground yeast (Saccharomyces norbensis), saccharomyces ellipsoideus (Saccharomyces oviformis) or Yarrowia lipolytica (Yarrowia lipolytica) cell.
This fungal host cells can be a filamentous fungal cells." filamentous fungus " comprises all thread form (as Huo Ke Butterworth etc., 1995, with defining) of Mycophyta and oomycetes door subgroup above.The feature of these filamentous funguss is generally the mycelia wall consisting of chitin, Mierocrystalline cellulose, dextran, chitosan, mannosans and other complicated polysaccharide.Nourish and grow and extend and carry out and carbon katabolism is inevitably aerobic by mycelia.By contrast, by nourishing and growing of carrying out as yeast such as yeast saccharomyces cerevisiaes, by unicellular prothallus, sprout and carry out and carbon katabolism can be fermentation.
This filamentous fungal host cell can be top spore Pseudomonas, Aspergillus, aureobasidium genus, smoke pipe Pseudomonas, intend wax Pseudomonas, Chrysosporium, Coprinus, white rot Pseudomonas, genera cryptococcus, net spore Pseudomonas, fusarium, Humicola, huge seat shell belongs to, Mucor, myceliophthora, new U.S. whip Pseudomonas, the mould genus of arteries and veins spore, paecilomyces, Penicillium, flat lead fungi belongs to, white rot Pseudomonas, pears capsule whip Pseudomonas, pleurotus, Schizophyllum, Talaromyces, thermophilic ascomycete belongs to, Thielavia, Tolypocladium, trametes or Trichoderma cell.
For instance, this filamentous fungal host cell can be Aspergillus awamori, smelly aspergillus (Aspergillus foetidus), fumigation look aspergillus (Aspergillus fumigatus), aspergillus japonicus (Aspergillus japonicus), Aspergillus nidulans, aspergillus niger, aspergillus oryzae, smoke pipe bacterium (Bjerkandera adusta), dry plan wax bacterium (Ceriporiopsis aneirina), a kind of fungi (Ceriporiopsis caregiea), pale yellow plan wax bacterium (Ceriporiopsis gilvescens), a kind of fungi (Ceriporiopsis pannocinta), a kind of fungi (Ceriporiopsis rivulosa), a kind of fungi (Ceriporiopsis subrufa), worm is intended wax bacterium (Ceriporiopsis subvermispora), a kind of fungi (Chrysosporium inops), chrysosporium keratinophilum (Chrysosporium keratinophilum), a kind of fungi (Chrysosporium lucknowense), excrement gold pityrosporion ovale (Chrysosporium merdarium), the golden pityrosporion ovale of hair (Chrysosporium pannicola), a kind of fungi (Chrysosporium queenslandicum), chrysosporium tropicum (Chrysosporium tropicum), a kind of fungi (Chrysosporium zonatum), Coprinus cinereus (Coprinus cinereus), hair Coriolous Dersicolor (Fr.) Quel fungus (Coriolus hirsutus), bar spore shape Fusariumsp (Fusarium bactridioides), chinese sorghum Fusariumsp (Fusarium cerealis), gram ground Fusariumsp (Fusarium crookwellense), yellow Fusariumsp (Fusarium culmorum), Fusarium graminearum (Fusarium graminearum), the red Fusariumsp of standing grain (Fusarium graminum), different spore Fusariumsp (Fusarium heterosporum), albizzia Fusariumsp (Fusarium negundi), Fusarium oxysporum, net Fusariumsp (Fusarium reticulatum), pink Fusariumsp (Fusarium roseum), Williams Elder Twig Fusariumsp (Fusarium sambucinum), colour of skin Fusariumsp (Fusanum sarcochroum), intend branch spore Fusariumsp (Fusanum sporotrichioides), sulphur look Fusariumsp (Fusanum sulphureum), beads Fusariumsp (Fusarium torulosum), intend silk spore Fusariumsp (Fusarium trichothecioide), fusarium, Humicola insolens, pubescence humicola lanuginosa, Mucor (Mucor miehei), thermophilicly ruin a bacterium, Neuraspora crassa (Neurospora crassa), penicillium purpurogenum (Penicillium purpurogenum), the flat lead fungi of raw wool (Phanerochaete chrysosporium), penetrate arteries and veins hedgehog fungus (Phlebia radiata), Pleurotus eryngii (Pleurotus eryngii), Thielavia terrestris (Thielavia terrestris), long wool hair bolt bacterium (Trametes villosa), variable color bolt bacterium (Trametes versicolor), trichoderma harziarum (Tnchoderma harzianum), koning trichoderma (Tnchoderma koningii), long shoot wood mould (Tnchoderma longibrachiatum), Trichodermareesei or viride (Tnchoderma viride) cell.
Fungal cell can transform by relating to a kind of method that protoplastis formation, protoplast transformation and cell walls regenerate in a manner known way.The applicable program description that is used for transforming Aspergillus and Trichoderma host cell is in EP238023, Ye Dun (Yelton) etc., 1984, the periodical > > 81:1470-1474 of institute of < < NAS, and Christon gloomy (Christensen) etc., in 1988, < < biotechnology > > (Bio/Technology) 6:1419-1422.For the appropriate methodology that transforms Fusarium kind, be by horse traction Dell (Malardier) etc., 1989, < < gene > > 78:147-156, and WO96/00787 describes.Yeast can be used the program described in following document to transform: Bake that (Becker) and Gu Lunte (Guarente), this Ademilson J.N.(Abelson ends, J.N.) and plug cover M.I.(Simon, M.I.) editor, < < yeast genetics and molecular biology guide > > (Guide to Yeast Genetics and Molecular Biology), < < Enzymology method > > (Methods in Enzymology), the 194th volume, 182-187 page, company limited of new york academic press, Ai Tuo (Ito) etc., 1983, < < bacteriology magazine > > 153:163, and pungent human relations (Hinnen) etc., the periodical > > 75:1920 of institute of 1978, < < NAS.
Manufacture method
The invention still further relates to and produce the method for polypeptide of the present invention, comprise that (a) cultivates a cell being of value under the condition that produces this polypeptide, this cell produces this polypeptide with its wild-type form; And (b) reclaim this polypeptide.One preferred aspect, this cell is Penicillium or Talaromyces cell.One preferred aspect, this cell is Ai Mosen Penicillium notatum or Talaromyces emersonii cell.The invention still further relates to and produce the method for polypeptide of the present invention, comprise that (a) cultivates a recombinant host cell of the present invention being of value under the condition that produces this polypeptide; And (b) reclaim this polypeptide.
These host cells are to cultivate in a kind of nutritional medium that is suitable for using method as known in the art to produce this polypeptide.For instance; can pass through shake-flask culture, or in laboratory or industrial fermentation tank in be applicable in substratum and allowing the small-scale of carrying out under the condition of this expression of polypeptides and/or separation or large scale fermentation (comprise continuously, in batches, charging in batches or solid state fermentation) cultivate this cell.This cultivation is to use program as known in the art, in one, is applicable to occurring in nutritional medium, and this substratum comprises carbon and nitrogen source and inorganic salt.Applicable substratum is obtainable from commercial supplier, or can for example, according to disclosed composition (, in the catalogue of American type culture collection), prepare.If this polypeptide is secreted in nutritional medium, this polypeptide can directly reclaim from this substratum so.If this polypeptide is not secreted, it can reclaim from cell lysates so.
This polypeptide can be used the specific method for these polypeptide as known in the art to detect.These detection methods include but not limited to, use, the formation of enzyme product or the disappearance of enzyme substrates of specific antibody.For instance, can check to measure with a kind of enzyme the activity of this polypeptide.
This polypeptide can be used method as known in the art to reclaim.For instance, this polypeptide can pass through conventional procedure, includes but not limited to, collect, centrifugal, filter, extraction, spraying are dry, evaporation or precipitation, from this nutritional medium, reclaim.
This polypeptide can carry out purifying by multiple programs as known in the art, include but not limited to, chromatography (for example, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromatofocusing method and size exclusion chromatography), electrophoretic procedures (for example, preparative isoelectric focusing method), difference solubleness (for example ammonium sulfate precipitation method), SDS-PAGE or extraction process are (referring to for example, < < protein purification > > (Protein Purification), outstanding gloomy (Janson) and Leyton (Ryden) editor, (the VCH Publishers of New York VCH press, New York), 1989), obtain thus pure in fact polypeptide.
One substituting aspect, this polypeptide is not recovered, but uses the host cell of the present invention of expressing this polypeptide as this polypeptide source.
Plant
The invention still further relates to the plant of separation, for example transgenic plant, plant part or vegetable cell, these plants comprise polypeptide of the present invention, thereby express and produce polypeptide or the structural domain of callable amount.This polypeptide or structural domain can reclaim from this plant or plant part.As an alternative, the plant that comprises this polypeptide or structural domain or plant part can in statu quo for improving food or quality of the fodder, for example, improve nutritive value, palatability and rheological properties, or destroy anti-nutritional factors.
These transgenic plant can be dicots (dicotyledonss) or monocotyledonous (monocotyledons).Monocotyledonous example is grass, if English grass (bluegrass, Poa L .), herbage are as festuca, lolium, cold ground type herbage, as cuts Gu Ying and cereal, for example wheat, oat, rye, barley, rice, Chinese sorghum and Zea mays (corn).
The example of dicotyledons is tobacco, beans legumen, as lupine, potato, sugar beet, pea, Phaseolus and soybean, and cress (Cruciferae), mustard as Arabic in Cauliflower, Semen Brassicae campestris and correlation model organism.
The example of plant part is stem, callus, leaf, root, fruit, seed and stem tuber, for example, together with the indivedual tissues that comprise these parts, epidermis, mesophyll, essence, vascular tissue, meristematic tissue.Specified plant cell cell, as chloroplast(id), apoplast, plastosome, vacuole, peroxysome and tenuigenin, is also considered to a kind of plant part.In addition, which kind of tissue origin no matter any vegetable cell, be, is all considered to a kind of plant part.Equally, separated to contribute to the plant part of utilization of the present invention, as particular organization and cell, be also considered to plant part, for example plumule, endosperm, aleuron and seed coat.
In scope of the present invention, also comprise the filial generation of these plants, plant part and vegetable cell.
Transgenic plant or the vegetable cell of expressing this polypeptide or structural domain can build according to procedures known in the art.In brief, by the expression construct of this polypeptide of one or more codings or structural domain is incorporated in this plant host genome or chloroplast gene group, and make gained breed and in transgenic plant or vegetable cell, build this plant or vegetable cell through the plant of modifying or vegetable cell.
This expression construct is a kind of nucleic acid construct expediently, this nucleic acid construct comprised to the polynucleotide of expressing a peptide species that the required appropriate regulation sequence of polynucleotide is operably connected or structural domain encoding in the plant of selecting or plant part.In addition, this expression construct can comprise the selectable marker that a kind of vegetable cell being applicable to being integrated into this expression construct is differentiated, and this construct is introduced to DNA sequence dna essential in the plant of touching upon (the latter is depended on the DNA introducing method of intending use).
The selection of regulating and controlling sequence (as promotor and terminator sequence and optionally signal or transit sequence) be for example based on when, wherein and how to express desirable polypeptide or structural domain decides.For example, the expression of the gene of coded polypeptide or structural domain can be composition or derivable, can be maybe growth, stage or tissue-specific, and can make gene product target particular organization or plant part, as seed or leaf.Regulating and controlling sequence is such as tower lattice (Tague) etc., and 1988, < < plant physiology > > (Plant Physiology) 86:506 is described.
For constructive expression, can use 35S-CaMV, Zea mays ubiquitin 1 or other rice Actin muscle 1 promotors (Frank (Franck) etc., 1980, < < cell > > (Cell) 21:285-294; Christon is gloomy etc., 1992, < < molecular biology of plants > > (Plant Mol.Biol.) 18:675-689; (Zhang) etc., 1991, < < vegetable cell > > (Plant Cell) 3:1155-1165).Organ specific promoters can be for example from storage vault tissue (storage sink tissue), as seed, potato tuber and fruit (Edward (Edwards) and Cruz (Coruzzi), 1990, > > (Ann.Rev.Genet.) 24:275-303 is commented in < < heredity academic year), or from metabolic pool tissue, as meristematic tissue (dust holder etc., 1994, < < molecular biology of plants > > 24:863-878) promotor, seed specific promoters, as the gluten from rice, prolamine, sphaeroprotein or albumin promoter (Wu (Wu) etc., 1998, < < plant cell physiology > > (Plant Cell Physiol.) 39:885-889), from legumin B4 with from the broad bean promotor (Joseph Conrad (Conrad) etc. of the unknown seed protein gene of broad bean, 1998, < < plant physiology magazine > > (J.Plant Physiol.) 152:708-711), from the promotor (old (Chen) etc., 1998, < < plant cell physiology > > 39:935-941) of seed oil body protein, from the storage protein napA promotor of Semen Brassicae campestris, or any other seed specific promoters as known in the art, for example, described in WO91/14772.In addition, this promotor can be a kind of leaf specificity promoter, as (the Jing Zhong (Kyozuka) etc. of the rbcs promotor from rice or tomato, 1993, < < plant physiology > > (Plant Physiol.) 102:991-1000); Chlorella virus VITAMIN B4 methyl transferase gene promotor (meter Te La (Mitra) and Huo Jinsi (Higgins), 1994, < < molecular biology of plants > > 26:85-93); From the aldP gene promoter (Ka Jiaya (Kagaya) etc., 1995, < < MGG > > 248:668-674) of rice; Or wound inducible promoter, as potato pin2 promotor (permitted (Xu) etc., 1993, < < molecular biology of plants > > 22:573-588).Equally, this promotor can be by inducing as abiotic processing such as temperature, arid or salinity variations, or apply by outer seedbed material (for example ethanol that makes this promotor activation; Oestrogenic hormon; Plant hormone, as ethene, dormin and gibberic acid; And heavy metal) induce.
Can also reach polypeptide or the more high expression level of structural domain in this plant with promotor enhancer element.For example, this promotor enhancer element can be the intron being placed between this promotor and the polynucleotide of coded polypeptide or structural domain.For example, permitted etc., 1993, with above, disclosed and strengthened expression with the First Intron of rice Actin muscle 1 gene.
Any other part of this selectable marker gene and this expression construct can be selected from available those in this area.
This nucleic acid construct is to be incorporated in this Plant Genome according to routine techniques as known in the art, comprise agrobacterium-mediated conversion, virus-mediated conversion, microinjection, particle bombardment, via Particle Bombardment Transformation and electroporation (Gai Se (Gasser) etc., 1990, < < science > > 244:1293; Po Tekusi (Potrykus), 1990, < < biotechnology > > 8:535; Island this (Shimamoto) etc., 1989, < < nature > > 338:274).
The transgenosis of Agrobacterium tumefaciens mediation is a kind of for generation of transgenosis dicotyledons (relevant commentary, referring to Huo Yika (Hooykas) and Si Chapote (Schilperoort), 1992, < < molecular biology of plants > > 19:15-38) and for the method for transforming monocots, but other method for transformation also can be for these plants.For generation of the monocotyledonous method of transgenosis, be particle bombardment (scribbling micro-gold or tungsten particle of transfering DNA) (Chris Austria (Christou) of the plumule of embryo callus or growth, 1992, < < plant magazine > > (Plant J.) 2:275-281; Originally, 1994, < < biotechnology is newly shown in > > (Curr.Opin.Biotechnol.) 5:158-162 on island; Wa Xier (Vasil) etc., 1992, < < biotechnology > > 10:667-674).A kind of alternative method for monocotyledonous conversion is based on protoplast transformation, as Ao meter Lu Le (Omirulleh) etc., 1993, < < molecular biology of plants > > 21:415-428 describes.Other method for transformation comprises U.S. Patent number 6,395,966 and 7,151,204(both with it, be in full incorporated into this by reference) described in those.
After transforming, according to method well known in the art, to being incorporated to the transformant of this expression construct, select and make its regeneration to become complete plant.Conventionally, this Transformation Program is to be designed at regeneration period or afterwards in generation, by the selectivity of for example using the Select gene carrying out with two independent T-DNA construct cotransformations, eliminate, or the excision of the locus specificity of the Select gene being undertaken by specific recombinase.
Except directly transforming specified plant genotype with construct of the present invention, can also be by making the plant with this construct hybridize to produce transgenic plant with the second plant that lacks this construct.For instance, can the construct of coded polypeptide or structural domain be introduced in specified plant kind, without the plant that always directly transforms this given kind by hybridization.Therefore, the plant directly being regenerated by the cell transforming according to the present invention is not only contained in the present invention, but also contains the filial generation of these plants.As used herein, filial generation can refer to the offspring in arbitrary generation of female plant prepared in accordance with the present invention.This class filial generation can comprise DNA construct prepared in accordance with the present invention.Hybridization makes by initial plant being carried out to cross-pollination by the strain of transgenosis introduced plant with donor plant strain.The limiting examples of these steps is described in U.S. Patent number 7,151, in 204.
Plant can produce by the conversion method that backcrosses.For instance, plant has comprised the plant of genotype, plant strain, selfing plant or hybrid plant that being called backcrosses transforms.
Can use genetic marker to help one or more transgenosiss of the present invention infiltrates another kind from a kind of genetic background gene.With respect to conventional herd breeding, help the benefit that provides of marker of selecting to be, it can be for the mistake of avoiding being caused by phenotypic variation.In addition, genetic marker can provide the data about the relative extent of excellent germplasm in indivedual filial generations of a specific cross.For instance, when by proterties likely tool and there is in addition the plant of the genetic background of wishing on non-agronomy and during the hybridization of a kind of excellent maternal plant, can select not only there is interested proterties with genetic marker, but also there is the filial generation of the germplasm of the hope of relatively large ratio.In such a way, making one or more character genes infiltrate required number of passages in a kind of specific genetic background minimizes.
The invention still further relates to the method that produces polypeptide of the present invention or structural domain, these methods comprise that (a) cultivates transgenic plant or vegetable cell, the polynucleotide that these transgenic plant or vegetable cell have comprised encode this polypeptide or structural domain under the condition that is of value to this polypeptide of generation or structural domain; And (b) reclaim this polypeptide or structural domain.
Composition
The invention still further relates to the composition that has comprised polypeptide of the present invention.Preferably, these these type of polypeptide of composition enrichment.Term " enrichment " indication, the glucoamylase activity of said composition increases, and for example enrichment factor is at least 1.1.
Said composition for example can comprise polypeptide of the present invention, as Major Enzymes component, single-component composition.As an alternative, said composition can comprise plurality of enzymes activity, as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, haloperoxidase, saccharase, laccase, lipase, mannosidase, oxydase, pectin decomposing enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases or zytase.Other enzyme can be for example by following microorganisms: belong to Aspergillus, preferably backshank aspergillus, Aspergillus awamori, Aspergillus fumigatus, smelly aspergillus, aspergillus japonicus, Aspergillus nidulans, aspergillus niger or aspergillus oryzae; Fusarium, preferably the thin end of the scroll spore shape Fusariumsp, chinese sorghum Fusariumsp, gram ground Fusariumsp, yellow Fusariumsp, Fusarium graminearum, the red Fusariumsp of standing grain, different spore Fusariumsp, albizzia Fusariumsp, Fusarium oxysporum, net Fusariumsp, pink Fusariumsp, Williams Elder Twig Fusariumsp, colour of skin Fusariumsp, sulphur look Fusariumsp, beads Fusariumsp, plan silk spore Fusariumsp or fusarium; Humicola, preferably Humicola insolens or pubescence humicola lanuginosa; Or Trichoderma, preferably wooden mould, the Trichodermareesei of mould, the long shoot of trichoderma harziarum, healthy and free from worry wood or viride.
These peptide compositions can be prepared according to procedures known in the art, and can be the form of liquid or drying composition.For example, this peptide composition can be the form of particle or particulate.The polypeptide being included in said composition can carry out stabilization according to procedures known in the art.
Below provided the example of the advantageous applications of peptide composition of the present invention.Other conditions of the dosage of peptide composition of the present invention and use said composition can be determined based on method as known in the art.
the combination of glucoamylase and acid alpha-amylase
According to this aspect of the invention, glucoamylase of the present invention can with α-amylase, preferably acid alpha-amylase with between 0.3 and 5.0AFAU/AGU between ratio combination.More preferably, the ratio between acid alpha-amylase activity and glucoamylase activity is at least 0.35, at least 0.40, at least 0.50, at least 0.60, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.85 or or even 1.9AFAU/AGU at least.But the ratio between acid alpha-amylase activity and glucoamylase activity should preferably be less than 4.5, be less than 4.0, be less than 3.5, be less than 3.0, be less than 2.5 or be even less than 2.25AFAU/AGU.In AUU/AGI, the activity of acid alpha-amylase and glucoamylase preferably with between 0.4 and 6.5AUU/AGI between ratio exist.More preferably, the ratio between acid alpha-amylase activity and glucoamylase activity is at least 0.45, at least 0.50, at least 0.60, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4 or or even 2.5AUU/AGI at least.But the ratio between acid alpha-amylase activity and glucoamylase activity is preferably less than 6.0, be less than 5.5, be less than 4.5, be less than 4.0, be less than 3.5 or be even less than 3.0AUU/AGI.
Above composition is suitable in the following starch conversion process for the production of syrup and tunning (as ethanol) of mentioning.
Below provided the example of the advantageous applications of peptide composition of the present invention.Other conditions of the dosage of peptide composition of the present invention and use said composition can be determined based on method as known in the art.
Application
The present invention is also for the process/method about using the polypeptide with glucoamylase activity of the present invention.
Application according to the present invention has comprised that starch becomes the starch conversion of for example syrup and tunning (comprising ethanol and beverage).Can use the example of the technique of glucoamylase of the present invention to comprise that WO92/20777, WO03/066816, WO03/066826, WO2004/080923 and WO2004/081193(are all incorporated into this to quote) described in method.
the production of tunning
by the method for the material produce tunning containing gelation starch
At this on the one hand, the present invention relates to a kind of method by amyloid material produce tunning, especially ethanol, the method comprises liquefaction step and the saccharification and the fermentation step that carry out in order or side by side.
The present invention relates to a kind of method by amyloid material produce tunning, comprise the following steps:
(a) use α-amylase to liquefy to amyloid material,
(b) use glucoamylase of the present invention to carry out saccharification to the liquefied material obtaining in step (a); And
(c) use fermentation organism to ferment to the material of saccharification.
This tunning, as ethanol especially, can be optionally after fermentation, for example reclaim by distilling.The starch-containing parent material being applicable to is listed in following " amyloid material " part.The enzyme of containing is listed in following " enzyme " part.This liquefaction is preferably carried out under α-amylase exists.This fermentation is preferably at yeast, and preferably yeast belong bacterial strain carries out under existing.Being applicable to fermentation organism lists in following " fermentation organism " part.In a preferred embodiment, step (b) and (c) be (that is, as the SSF method) of carrying out in order or side by side.
In a particular embodiment, method of the present invention comprises following steps before in addition in step (a):
X) preferably by grinding, reduce the granularity of this amyloid material; And
Y) slurries that comprise this amyloid material and water have been formed.
This water-soluble serous can comprising from 10wt.% to 40wt.%, preferably 25wt.% is to the amyloid material of 35wt.%.These slurries are heated to above gelatinization temperature, and can add α-amylase, and preferably bacterium and/or acid fungal alpha-amylase are to start liquefaction (dilution).In one embodiment, these slurries can spray culinary art (jet-cooked) so that the before further gelationization of the α-amylase of this slurries in experience step of the present invention (a).
More particularly, liquefaction can be used as a hot slurry method of three steps and carries out.These slurries are heated between 60 ℃ to 95 ℃, preferably between 80 ℃ to 85 ℃, and add α-amylase to start liquefaction (dilution).Then, these slurries can, between 95 ℃ to 140 ℃, preferably spray culinary art 1-15 minute, preferably 3-10 minute, especially approximately 5 minutes at the temperature between 105 ℃ to 125 ℃.These slurries are cooled to 60 ℃ to 95 ℃, and add α-amylase to finish hydrolysis (secondary liquefaction) again.This liquefaction process is often at pH4.5-6.5, particularly under the pH between between 5 and 6, carries out.Through the complete particulate grinding and liquefy, be called mashed prod (mash).
Saccharification in step (b) can be used condition well known in the art to carry out.For example, all saccharifying can continue from approximately 24 to approximately 72 hours, but, conventionally only at the temperature between between 30 ℃ to 65 ℃, typically carry out the typically premashing of 40-90 minute at approximately 60 ℃, at the same time in saccharification and zymotechnique (SSF technique), during fermentation carry out complete saccharification subsequently.Saccharification is typically from 30 ℃ to 65 ℃, typically at the temperature of approximately 60 ℃, and at the pH between 4 and 5, conventionally under about pH4.5, carries out.
At tunning, especially in the production of ethanol, the most widely used method is synchronous glycosylation and fermentation (SSF) method, and in the method, this saccharification does not exist holding stage, means that fermentation organism (as yeast) and enzyme can add together.SSF can, typically between 25 ℃ and 40 ℃, as between 29 ℃ and 35 ℃, as between 30 ℃ and 34 ℃, carry out at the temperature of 32 ℃ according to appointment.According to the present invention, this temperature can during fermentation be adjusted up and down.
According to the present invention, this fermentation step (c) comprises and being not limited to, for example, for the production of alcohols (ethanol, methyl alcohol, butanols); Organic acid (for example citric acid, acetic acid, methylene-succinic acid, lactic acid, glyconic acid); Ketone (for example acetone); Amino acids (for example L-glutamic acid); Gas (for example H 2and CO 2); Antibiotics (for example penicillin and tsiklomitsin); Enzyme; The fermentation process of vitamins (for example riboflavin, B12, β-carotene).Preferred fermentation process comprises alcohol fermentation process, as known in the art.Preferred fermentation process comprises anaerobic fermentation method, as known in the art.
by containing the not method of the material produce tunning of gelation starch
At this on the one hand, the present invention relates to, in the case of not carrying out the gelationization of amyloid material, by this amyloid material (that is, uncooked amyloid material), be produced the method for tunning.In one embodiment, only between saccharification and yeast phase, use glucoamylase of the present invention.According to the present invention, can, in the case of not to water-soluble serous liquefaction that has comprised this amyloid material, produce the tunning of wishing, as ethanol.In one embodiment, method of the present invention is included in lower than under gelatinization temperature, under glucoamylase of the present invention exists, (grinding) amyloid material (for example pearl starch) is carried out to saccharification, to produce sugar, these sugar can be fermented into by being applicable to fermentation organism the tunning of hope.
Therefore, at this on the one hand, the present invention relates to a kind of method by amyloid material produce tunning, comprising:
(a) at the temperature of the initial gelatinization temperature lower than amyloid material, with preferably have sequence as shown in the amino acid 22 to 651 of the amino acid 22 to 537 of SEQ ID NO:2 or SEQ ID NO:4 according to ripe glucoamylase of the present invention, or there is at least 75% conforming glucoamylase with it described amyloid material is carried out to saccharification, and preferably also add α-amylase
(b) use a kind of fermentation organism to ferment.
The step (a) of the inventive method and (b) can carry out in order or side by side.In one embodiment, the slurries that comprise water and amyloid material are prepared before in step (a).
This fermentation process can carry out one section 1 to 250 hours, preferably from 25 to 190 hours, more preferably from 30 to 180 hours, more preferably from 40 to 170 hours, even more preferably from 50 to 160 hours, more preferably from 60 to 150 hours again, more preferably from 70 to 140 hours and time of from 80 to 130 hours most preferably even again.
Term " initial gelatinization temperature " meaning refers to that this starch gelling melts the minimum temperature of beginning.The starch heating in water is starting gelationization between 50 ℃ and 75 ℃; The exact temperature of gelationization depends on concrete starch, and can easily by skilled people in the industry, be determined.Therefore, this initial gelatinization temperature can change together with growth conditions according to floristics, this floristic certain species.In the context of the present invention, a kind of initial gelatinization temperature of given amyloid material is to use Gu Linsitan (Gorinstein) and Lee (Lii), 1992, < < starch > >
Figure BDA0000469949290000371
44(12): 461-466(1992) described method, makes 5% starch granules lose birefringent temperature.
In step (a) before, can prepare and there is the drying solid of 10wt.% to the amyloid material of 55wt.%, preferably 25wt.% is to the drying solid of 40wt.%, and more preferably 30wt.% is to the slurries of the amyloid material (as pearl starch) of the drying solid of 35wt.%.These slurries can comprise water and/or process water, as stillage (adverse current), washing water, evaporator condensate or distilled water, the side stripper water that obtained by distillation, or other tunning factory technics waters.Due to method of the present invention be under this gelatinization temperature, carry out and therefore there is not significant viscosity and increase, therefore if desired, can use the stillage of higher level.In one embodiment, this water-soluble serous stillage comprising from about 1vol.% to about 70vol.%, preferably 15vol.% is to the stillage of 60vol.%, the especially stillage from about 30vol.% to 50vol.%.
This amyloid material can be by by particle size reduction (preferably by dry type or wet grinding), to 0.05 to 3.0mm, preferably prepared by 0.1-0.5mm.After experience method of the present invention, the drying solid of this amyloid material of 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% or preferably at least 99% is changed into Zulkovsky starch hydrolysate.
Method of the present invention is to carry out at the temperature lower than this initial gelatinization temperature.Preferably, the temperature that step (a) is carried out is between 30 ℃ to 75 ℃, preferably between 45 ℃ to 60 ℃.
In a preferred embodiment, step (a) and step (b) are that saccharification and fermentation process sequentially or simultaneously to carry out carries out.In this preferred embodiment, the method, typically between 25 ℃ and 40 ℃, as between 29 ℃ and 35 ℃, as between 30 ℃ and 34 ℃, is carried out at the temperature of 32 ℃ according to appointment.According to the present invention, this temperature can during fermentation be adjusted up and down.
In one embodiment, synchronous glycosylation and fermentation make sugar level, as glucose level remains on lower level, as lower than 6wt.%, be preferably lower than about 3wt.%, be preferably lower than about 2wt.%, preferred lower than about 1wt.%, even preferred lower than about 0.5wt.% or even preferred 0.25%wt.%, as lower than about 0.1wt.%.This lower sugar level can by adopt simply adjustment amount enzyme and fermentation organism realize.Those of ordinary skill in the art can easily determine the enzyme and the fermentation organism that use how many amounts.The enzyme adopting and the organic scale of construction of fermentation can also be through selecting to maintain lower maltose concentration in fermented liquid.For example, this maltose level can keep below about 0.5wt.% or lower than about 0.2wt.%.
Method of the present invention can, in scope between 3 and 7, preferably from pH3.5 to 6, or more preferably be carried out from the pH of pH4 to 5.
amyloid material
Any applicable amyloid material (comprising pearl starch) can be used according to the invention.This parent material is generally that the tunning based on hope is selected.The example that is suitable for the amyloid material in method of the present invention comprises stem tuber, root, stem, whole grain, corn, cob, wheat, barley, rye, milo (milo), sago, cassava, Tapioca Starch, Chinese sorghum, rice, beans, Kidney bean or sweet potato, or its mixture, or cereal, sugary raw material, as molasses, fruit material, sugar-cane or sugar beet, potato, and cellulose-containing material, as timber or plant residue, or its mixture.Contain corn and the barley of two types of wax and non-waxs.
Term " pearl starch " meaning refers to uncooked raw starch, that is, and and the starch that is its natural form of finding in cereal, stem tuber or grain.Starch is as the molecule form being insoluble in water, to form in vegetable cell.When putting into cold water, these starch granuless can absorb a small amount of liquid, and expand.At the temperature of maximum 50 ℃ to 75 ℃, this expansion can be reversible.But, under higher temperature, start irreversible expansion, be called " gelationization ".Having pearl starch to be processed can be highly refined starch quality, preferably at least 90%, at least 95%, at least 97% or at least 99.5% is pure, or it can be the material of the starch containing more coarse, this material comprises the whole grain through grinding, including non-starch part, as plumule residue and fiber.These starting material (as whole grain) are processed so that deployed configuration and permission are further through grinding.According to the present invention, below two kinds of Ginding process be preferred: wet type and dry grinding.In dry grinding, whole kernel is polished and uses.Wet grinding makes plumule and meal (starch granules and protein) good separation, and is often applied to the place of using starch hydrolysate to produce syrup.Dry type and wet grinding are starch processing methods well known in the art and are contained comparably for method of the present invention.
Preferably by dry type or wet grinding, reduce the granularity of this amyloid material, to expose larger surface-area.In one embodiment, this granularity is between 0.05 to 3.0mm, preferably between 0.1-0.5mm, or make at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably this amyloid material of at least 90% is applicable to having 0.05 to 3.0mm screen cloth by one, preferably 0.1 sieve to 0.5mm screen cloth.
tunning
Term " tunning " meaning refers to the product of producing by the method including the fermentation step that uses fermentation organism to carry out.The tunning of containing according to the present invention comprises alcohols (for example ethanol, methyl alcohol, butanols); Organic acid (for example citric acid, acetic acid, methylene-succinic acid, lactic acid, glyconic acid); Ketone (for example acetone); Amino acids (for example L-glutamic acid); Gas (for example H 2and CO 2); Antibiotics (for example penicillin and tsiklomitsin); Enzyme; Vitamins (for example riboflavin, B 12, β-carotene); And hormones.In a preferred embodiment, this tunning is ethanol, for example alcohol fuel; Drinking alcohol, that is, and drinkable alcohol; Or industrial alcohol, or the product for example, for example, using for consuming alcohol industry (beer and grape wine), milk preparation industry (milk preparation of fermentation), leather industry and tobacco industry.Preferred beer type comprises that pale beer, stout, Britain produce strong stout (porter), Lager, bitter, malt liquor, low malt beer (happoushu), high alcohol beer, lab, low-heat beer or light beer.The preferred fermentation process using comprises alcohol fermentation process, as known in the art.Preferred fermentation process comprises alcohol fermentation process, as known in the art.
fermentation organism
" fermentation organism " refers to any organism that is suitable in fermenting process and can produces the tunning of hope, comprises bacterium and fungi organism.Especially the tunning that applicable fermentation organism can directly or indirectly become to wish by sugar (as glucose or maltose) fermentation (that is, transforming).The organic example that ferments comprises fungi organism, as yeast.Preferred yeast comprises yeast belong bacterial strain, particularly yeast saccharomyces cerevisiae.Commercially available yeast comprises for example Red Star tM/ Lesaffre Ethanol Red(is from (the Red Star/Lesaffre of U.S. Red Star company, USA) obtainable), FALI(is from (the Fleischmann's Yeast of Fei Laiximan yeast company of branch office of U.S. Berne Philips food company, a division of Burns Philp Food Inc., USA) obtainable), SUPERSTART(is obtainable from Ao Taike company (Alltech)), GERT STRAND(is from the bent moral AB(Gert Strand of Sweden Ztel Si AB, Sweden) obtainable) and FERMIOL(obtainable from the extraordinary product companies of DSM (DSM Specialties)).
enzyme
glucoamylase
This glucoamylase is glucoamylase of the present invention preferably.But as mentioned above, glucoamylase of the present invention also can combine with other glucoamylases.Term " glucoamylase " (Isosorbide-5-Nitrae-α-D-dextran glucose lytic enzyme, EC3.2.1.3) is catalysis discharges D-Glucose a kind of enzyme from the non-reducing end of starch or Related Oligosaccharides and polysaccharide molecule.
The addition of this glucoamylase can be 0.001 to 10AGU/g DS, preferably from 0.01 to 5AGU/g DS, as be approximately 0.1,0.3,0.5,1 or 2AGU/g DS, especially 0.1 to 0.5AGU/g DS or 0.02 to 20AGU/g DS, preferably 0.1 to 10AGU/g DS.
α-amylase
This α-amylase can any source.The preferably α-amylase of fungi or bacterial origin.
In a preferred embodiment, this α-amylase is a kind of acid alpha-amylase, for example fungi acid alpha-amylase or bacterium acid alpha-amylase.Term " acid alpha-amylase " meaning refers to add with significant quantity, has a kind of α-amylase (EC3.2.1.1) of optimum activity the pH in 3 to 7, preferably from 3.5 to 6 or more preferably from 4 to 5 scope.
bacterialα-amylase
Bacterialα-amylase can preferably derive from bacillus.
In a preferred embodiment, this bacillus α-amylase is to derive from Bacillus licheniformis, bacillus amyloliquefaciens, subtilis or bacstearothermophilus bacterial strain, but also can derive from other bacillus.The specific examples of the α-amylase containing comprises the bacillus licheniformis alpha-amylase (BLA) that the SEQ ID NO:4 in WO99/19467 is shown; The shown bacillus amyloliquefaciens α-amylase (BAN) of SEQ ID NO:5 in WO99/19467; And the shown bacillus stearothermophilus alpha-amylase (BSG) of the SEQ ID NO:3 in WO99/19467.In one embodiment of the invention, this α-amylase be respectively with WO99/19467 in arbitrary sequence as shown in SEQ ID NO:1,2,3,4 or 5 there is at least 60% the degree of consistency, preferably at least 70%, preferred at least 80%, even preferred at least 90%, as conforming a kind of enzyme of at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
This bacillus α-amylase can also be the combinations by reference hereby of all documents of a kind of variant and/or heterocomplex, especially WO96/23873, WO96/23874, WO97/41213, WO99/19467, WO00/60059 and WO02/10355() the middle described variant of any one and/or heterocomplex.Specifically, the alpha-amylase variants of containing is disclosed in U.S. Patent number 6, 093, 562, 6, 187, 576 and 6, 297, 038(is combination by reference hereby) in, and comprise bacillus stearothermophilus alpha-amylase (BSG α-amylase) variant, these variants have the disappearance of one or two Amino acid in position 179 to 182, two disappearances of preferably disclosing in WO96/23873-referring to for example the 20th page, 1-10 capable (combination by reference hereby), preferably compare with the wild-type BSG α-amylase aminoacid sequence of stating in the SEQ ID NO:3 disclosing in WO99/19467 and corresponding to δ (181-182), or use WO99/19467(this reference hereby by reference in conjunction with) in the disappearance of amino acid/11 79 and 180 of numbering of SEQ ID NO:3.Even more preferably genus bacillus α-amylase, especially bacillus stearothermophilus alpha-amylase, these α-amylase have had the two disappearances corresponding to δ (181-182), and compare with the wild-type BSG α-amylase aminoacid sequence of stating in the SEQ ID NO:3 disclosing in WO99/19467, comprise in addition N193F and replace (being expressed as again I181*+G182*+N193F).
This α-amylase can also be a kind of raw wheat α-amylase." raw wheat α-amylase " (dextran-Isosorbide-5-Nitrae-α-Fructus Hordei Germinatus lytic enzyme, EC3.2.1.133) can be hydrolyzed into amylose starch and amylopectin the maltose of α-configuration.Raw wheat α-amylase from bacstearothermophilus bacterial strain NCIB11837 is commercially available from Denmark Novozymes Company (Novozymes A/S, Denmark).This raw wheat α-amylase is described in U.S. Patent number 4,598, and in 048,4,604,355 and 6,162,628, these patents are combination by reference hereby.
bacterium hybrid alpha-amylases
445 C-terminal amino-acid residues (as shown in the SEQ ID NO:4 in WO99/19467) that the hybrid alpha-amylases of containing especially comprises bacillus licheniformis alpha-amylase and derive from 37 N-terminal amino-acid residues (as shown in the SEQ ID NO:3 as in WO99/19467) of the α-amylase of bacillus amyloliquefaciens, and following one or more, especially whole replacements: G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S(is used Bacillus licheniformis numbering).Further preferably there is the variant of following one or more sudden change (or sudden change accordingly in other genus bacillus α-amylase main chains): H154Y, A181T, N190F, A209V and Q264S; And/or the disappearance of two residues between position 176 and 179, the preferably disappearance of E178 and G179 (using the SEQ ID NO:5 numbering of WO99/19467).
Can adding by amount well known in the art of this bacterialα-amylase.When (being described in following " materials and methods " part) take KNU as metric unit, this alpha-amylase activity is preferably with 0.5-5, the amount of 000NU/g DS, with the amount of 1-500NU/g DS, or more preferably with 5-1,000NU/g DS, as the amount of 10-100NU/g DS exists.
fungal alpha-amylase
Fungal alpha-amylase has comprised the acid alpha-amylase that derives from Aspergillus bacterial strain, as Aspergillus albicans, aspergillus niger or aspergillus oryzae α-amylase.
Preferred acid fungal alpha-amylase is a kind of α-amylase of Fungamyl sample, and this α-amylase preferably derives from aspergillus oryzae strain.In this disclosure, term " α-amylase of Fungamyl sample " indicated with WO96/23874 in SEQ ID NO:10 in the maturing part of shown aminoacid sequence show high consistence,, exceed 70%, exceed 75%, exceed 80%, exceed 85%, exceed 90%, exceed 95%, exceed 96%, exceed 97%, exceed 98%, exceed conforming a kind of α-amylase of 99% or even 100%.
Another kind of preferred acid alpha-amylase is to derive from Aspergillus niger strain.In a preferred embodiment, this acidity fungal alpha-amylase be from aspergillus niger in Swiss-prot/TeEMBL database, take " AMYA_ASPNG ", disclose enter to hide the α-amylase of registration number as P56271, and this α-amylase is described in greater detail in WO89/01969(example 3) in.This erie black aspergillus acid alpha-amylase is also in WO2004/080923(Novozymes Company) in SEQ ID NO:1 show, this patent is combination by reference hereby.The SEQ ID NO:1 with WO2004/080923 of also containing described acid fungal amylase has at least 70% consistence, as at least 80% or or even at least 90% consistence, as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% conforming variant.The commercially available acid fungal alpha-amylase being applicable to that derives from aspergillus niger is that SP288(is obtainable from Denmark Novozymes Company).
In a preferred embodiment, this α-amylase is to derive from Aspergillus albicans and by gold (Kaneko) etc., 1996, < < fermentation and biotechnology magazine > > (J.Ferment.Bioeng.) 81:292-298, " molecular cloning of the nucleotide sequence of the gene of the acid acceptance α-amylase of encoding in Aspergillus albicans and mensuration (Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid-stable alpha-amylase from Aspergillus kawachii) ", and with EMBL:#AB008370, disclose in addition.
This fungi acid alpha-amylase can also be a kind of wild-type enzyme (that is, non-heterozygosis) that comprises a carbohydrate binding modules (CBM) and a α-amylase catalyst structure domain, or its variant.In one embodiment, this wild-type α-amylase is to derive from Aspergillus albicans bacterial strain.
fungi hybrid alpha-amylases
In a preferred embodiment, this fungi acid alpha-amylase is a kind of hybrid alpha-amylases.
The preferred example of fungi hybrid alpha-amylases comprises the open case 2005/0054071(of WO2005/003311 or U. S. application Novozymes Company) or U. S. application number 60/638,614(Novozymes Company) α-amylase disclosed in (hereby by reference in conjunction with).Hybrid alpha-amylases can comprise a α-amylase catalyst structure domain (CD) and a carbohydrate binding domains/module (CBM) and an optional connexon.
The specific examples of the hybrid alpha-amylases of containing comprises U. S. application number 60/638, those disclosed in 614, comprised the Fungamyl variant (U. S. application number 60/638 with catalyst structure domain JA118 and Roche Ah too bacterium SBD, SEQ ID NO:100 in 614), there is the Rhizomucor pusillus α-amylase (U. S. application number 60/638 of Roche Ah too bacterium AMG connexon and SBD, SEQ ID NO:101 in 614) and there is the large-scale sub-Grifolas frondosa germ α-amylase (U. S. application number 60/638 of Roche Ah too bacterium glucoamylase connexon and SBD, SEQ ID NO:102 in 614).
Other specific exampless of the hybrid alpha-amylases of containing comprise those disclosed in U. S. application publication number 2005/0054071, comprise the 15th page of above those disclosed in table 3, as there is the aspergillus niger α-amylase of Aspergillus albicans connexon and starch binding domains.
commercial α-amylase product
The commercial composition that preferably comprises α-amylase comprises from DSM(Ji Site Brocades Co., Ltd (Gist Brocades)) MYCOLASE; BAN tM, TERMAMYL tmsC, FUNGAMYL tM, LIQUOZYME tMx and SAN tMsUPER, SAN tMeXTRA L(Novozymes Company); And CLARASE tMl-40,000, DEX-LO tM, SPEZYME tMfRED, SPEZYME tMaA, SPEZYME tMethyl, GC358, GC980, SPEZYME tMrSL and SPEZYME tMindustrial of DELTA AA(Jie Neng section (Genencor Int.)); And obtainable from Denmark Novozymes Company with trade(brand)name SP288() sell acid fungal alpha-amylase.
Acid alpha-amylase can, according to the present invention with 0.1 to 10AFAU/g DS, preferably 0.10 arrive 5AFAU/g DS, and especially 0.3 to 2AFAU/g DS amount is added.
the production of syrup
The present invention also provides a kind of glucoamylase of the present invention that uses, by the method for amyloid material produce syrup (as glucose etc.).In part that applicable parent material is illustrated in above " amyloid material ".In general, the method comprises the following steps: under α-amylase exists, make amyloid material partly be hydrolyzed (liquefaction), and then further under glucoamylase of the present invention exists, carry out saccharification, from the non-reducing end of this starch or Related Oligosaccharides and polysaccharide molecule, discharge glucose.
Liquefaction and saccharification can be as produced described carrying out about tunning above.
The glucoamylase of the present invention using can be to be also the form being fixed.This be applicable to and often for the production of superfine syrup, as maltose syrups, and in addition for example, for the stream and produce fructose syrups, high fructose syrups (HFS) at residual night of oligosaccharides.
Therefore, of the present invention this relates in one aspect to a kind of method by amyloid material produce syrup, comprising:
(a), under α-amylase exists, make amyloid material liquefaction; And
(b) use glucoamylase of the present invention to carry out saccharification to the material obtaining in step (a).
The saccharification salvage material that syrup can obtain from step (b).
About the details that are applicable to condition can see above.
Brewage
Glucoamylase of the present invention can also be used for making method.Glucoamylase of the present invention is to add with significant quantity, and these significant quantities can easily be determined by those of ordinary skill in the art.
In the invention of this description and requirement, be not limited to the scope of specific embodiment disclosed here, because these embodiment intentions are as the explanation of the some aspects of the present invention.Any equivalent embodiment is intended within the scope of the invention.In fact, those of ordinary skills from above stated specification by apparent to revising except of the present invention different those of this demonstration description.These are revised also and are intended within the scope of the appended claims.In conflicting situation, will disclose with this (comprise be defined in) to be as the criterion.
At this, quoted different reference, its disclosure content is by reference with its combination in full.Following instance further describes the present invention, and these examples should not be construed as limiting the scope of the invention.
Signal peptide and propetide
The invention still further relates to a kind of polynucleotide of separation of coded signal peptide, this signal peptide comprises or it consists of the amino acid/11 to 21 of SEQ ID NO:2 or SEQ ID NO:4.These polynucleotide can comprise the gene of a coded protein in addition, and this protein is operably connected to this signal peptide.This protein is preferably external for this signal peptide and/or propetide.In one aspect, the encode polynucleotide of this signal peptide are the Nucleotide 1 to 63 of SEQ ID NO:1 or SEQ ID NO:3.
The invention still further relates to the nucleic acid construct, expression vector and the recombinant host cell that comprise these polynucleotide.
The invention still further relates to generation method of protein, comprise that (a) cultivates the recombinant host cell that comprises this polynucleotide; And (b) reclaim this protein.
This protein is natural or allos for host cell.Term " protein " is not intended to refer to a kind of coded product of length-specific at this, and therefore contains peptide, oligopeptides and polypeptide.Two or more polypeptide of the product that is combined to form coding also contained in term " protein ".These protein also comprise hybrid polypeptide and fusion polypeptide.
Preferably, this protein is a kind of hormone, enzyme, acceptor or its part, antibody or its part, or report.For instance, this protein can be a kind of lytic enzyme, isomerase, ligase enzyme, lyase, oxydo-reductase or transferring enzyme, for example aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, allosteric lytic enzyme, oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, zytase or xylobiase.
This gene can obtain from any protokaryon, eucaryon or other sources.
Following instance further describes the present invention, and these examples should not be construed as limiting the scope of the invention.
Example
Glucoamylase activity
Glucoamylase activity can be unit or according to glucose starch unit of enzyme (AGU) according to AGI, or according to other applicable checks, as for example from Kikkoman (Kikkoman) or and commercial test test kit (the LabAssay glucose of light (Wako), with light Co., Ltd., catalog number (Cat.No.) 298-65701) measure.
glucoamylase activity (AGI)
Glucoamylase (being equivalent to amyloglucosidase) changes into glucose by starch.The amount of glucose is to measure by the glucose oxidase method for determination of activity at this.The method is described in from association of U.S. cereal chemistry man (American Association of Cereal Chemists), (2000); 76-11 partial starch-glucose starch enzyme process in " U.S. cereal chemistry man association approval method (Approved methods of the American Association of Cereal Chemists) " 1-2 volume AACC of ISBN:1-891127-12-8 and measuring subsequently in glucose (Starch-Glucoamylase Method with Subsequent Measurement of Glucose with Glucose Oxidase) with glucose oxidase.
A glucose starch unit of enzyme (AGI) is under the standard conditions of the method, and per minute will form the enzyme amount of 1 micromole's glucose.
standard conditions/reaction conditions:
Substrate: Zulkovsky starch, concentration is about 16g dry-matter/L.
Damping fluid: acetate, about 0.04M, pH=4.3
pH：4.3
Incubation temperature: | 60 ℃
Reaction times: 15 minutes
Reaction terminating: add the concentration (pH～9) of NaOH to about 0.2g/L
Enzyme concn: 0.15-0.55AAU/mL.
This starch should be lintner starch (Lintner starch), and this starch is the one that is used as colorimetric indicator in the laboratory starch that gently boils.Lintner starch is to obtain by the dilute hydrochloric acid processing of native starch, and therefore it has kept being blue ability under iodine exists.
glucoamylase activity (AGU)
Novo glucose starch unit of enzyme (AGU) is defined as under following standard conditions per minute and is hydrolyzed the enzyme amount of 1 micromole's maltose: 37 ℃, and pH4.3, substrate: maltose 23.2mM, damping fluid: acetate 0.1M, 5 minutes reaction times.
Can use automatic analysis instrument system.Mutarotase is added in Hexose phosphate dehydrogenase reagent, make thus any alpha-D-glucose existing become β-D-Glucose.Hexose phosphate dehydrogenase in above-mentioned reaction with β-D-Glucose specific reaction, form NADH, under 340nm, use this NADH of photometric determination measuring as raw glucose concentration.
AMG is hatched： ?
Substrate: Maltose 23.2mM
Damping fluid: Acetate 0.1M
pH： 4.30±0.05
Incubation temperature: 37℃±1℃
Reaction times: 5 minutes
Enzyme working range: 0.5-4.0AGU/mL
Color reaction： ?
GlucDH： 430U/L
Mutarotase: 9U/L
NAD： 0.21mM
Damping fluid: Phosphoric acid salt 0.12M; 0.15M NaCl
pH： 7.60±0.05
Incubation temperature: 37℃±1℃
Reaction times: 5 minutes
Wavelength: 340nm
glucoamylase activity check (Kikkoman Co., Ltd)
Product code: 60211
This test kit forms active for measuring meter Qu (rice koji) glucose.
Inspection principle:
Substrate 4-nitrophenyl-beta-maltose glycosides (G2-β-pNP) is degraded into 4-nitrophenyl-β-glucoside (G1-β-pNP) by glucoamylase or alpha-glucosidase.G1-β-pNP is further degraded into 4-nitrophenols (pNP) by the beta-glucosidase enzyme in this test kit.Reaction is at room temperature carried out under about pH4.By adding sodium carbonate, stop this reaction, and simultaneously, this solution becomes alkaline pH so that the absorbancy maximum of pNP.It is by this pNP quantitatively being measured under 400nm that this glucose forms activity.
1) reaction and display of measuring goes out G2-β-pNP degrading activity of glucoamylase and alpha-glucosidase in sample.This glucose being considered in sample forms activity.
2) this test can be for the bent extract of rice of not dialysis.
3) this check is not affected by the α-amylase in sample.
Reagent constituents
Reagent Main ingredient Amount
Substrate solution G2-β-pNP 60ml
Enzyme solution Beta-glucosidase enzyme 60ml
Stop bath Sodium carbonate 120ml
1) with 1:1, mix " substrate solution " and " enzyme solution " of this test kit.
2) draw 20 these samples of μ l (or as blank water) and transferred in the hole of microtiter plates.(in duplicate)
3) this substrate-enzyme mixture of 60 μ l is added in this hole.
4) at room temperature hatch 20 minutes.
5) this stop bath of 120 μ l is added in this hole.
6) read OD400nm.The clean OD of # 400amount=OD 400(sample)-OD 400(blank)
1. blank: conventionally, this blank absorbancy is less than 0.200.
2. specificity: this reaction is not subject to glucose (nearly 100g/l) or α-amylase (725U/ml) impact.
3. reproducibility: when same sample is analyzed to 10 times, the CV of absorbancy is less than 1%.
4. linearity range: maximum 1.6 clean OD 400should be proportional with this enzyme concn.
5. colour stability: this absorbancy does not change for 2 hours at 25 ℃.
With light glucose test test kit (LabAssay glucose, and light Co., Ltd., catalog number (Cat.No.) 298-65701).
LabAssay tMglucose is the test kit that carries out enzyme method and check a kind of reagent of glucose based on the combination with mutarotase and glucose oxidase.
Alpha-D-glucose and β-D-Glucose maintain balance with constant ratio in solution.Glucose oxidase only reacts with β-D-Glucose and does not react with alpha-D-glucose.Therefore, use mutarotase that alpha-D-glucose is changed into β-D-Glucose.
When by a duplicate samples and chromogen reagent mix, in sample, the glucose of alpha-form is changed into beta form by mutarotase.β-D-Glucose is oxidized and obtains hydrogen peroxide by glucose oxidase (GOD).Under peroxidase (POD) exists, the hydrogen peroxide of formation obtains red pigment by carrying out quantitative oxidative condensation with phenol and 4-AA.Glucose concn is that the absorbancy by measuring this red pigment obtains.
This check is to have used with the microplate reader of 505nm wavelength filter to carry out in 96 hole microplates.Check is carried out 5 minutes at 37 ℃.
It is beautiful and (Miwa) etc. that this inspection party's ratio juris is described in addition, and 1972, < < clinical chemistry journal > > (Clin.Chim.Acta.), in 37,538-540.
In the scope aspect concrete disclosed here that the invention is not restricted to of this description and requirement, because these aspects intentions are as the explanation of the some aspects of the present invention.Any equivalent aspect is all intended within the scope of the invention.In fact, those of ordinary skills from above stated specification by apparent to revising except of the present invention different those of this demonstration description.These are revised also and are intended within the scope of the appended claims.In conflicting situation, will disclose with this (comprise be defined in) to be as the criterion.
Material
As the chemical of damping fluid and substrate, it is the commercial product of SILVER REAGENT at least.
Bacterial strain
Ai Mosen Penicillium notatum (NN051602) has been used as the source of the polypeptide with glucoamylase activity.NN051602 separates from the compost of economizing from Chinese yunnan for 2009.Sequence information shows, this strain isolated is relevant extremely nearly to Talaromyces emersonii, and can be in fact identical type or its anamorph.In these embodiments, therefore this bacterial strain is called as Talaromyces emersonii.Aspergillus niger strain HowB112 is used to the Talaromyces emersonii gene of the polypeptide that has had glucoamylase activity to having encoded and expresses.Aspergillus niger HowB112 is the amdS(acetamidase of aspergillus niger BO1) the destroyed derivative of gene.Bacterial strain HowB112 is with following genotype: AMG -(glucoamylase gene of destruction), ASA -(the acid acceptance amylase gene of destruction) and tgs -(α-1 of destruction, 6-transglucosidase gene).
Substratum and solution
YMD substratum is to consist of 0.3% yeast extract, 0.5% peptone, 0.3% malt extract and 5% maltodextrin.
PDA agar plate by potato leach liquor (potato leach liquor is by the potato of 300g section (through washing but do not peel) is boiled to 30 minutes in water, and then by this nutrient solution decant or filter cheese cloth and make) form.Then add distilled water, until the cumulative volume of suspension is one liter, add subsequently the dextrose of 20g and the agar powder of 20g.This substratum carries out sterilizing (< < bacteriological analysis handbook > > (Bacteriological Analytical Manual) for 15 minutes by high pressure sterilization under 15psi, the 8th edition, revision A, 1998).
LB plate be by the bacterium agar of the sodium-chlor of the yeast extract of the bacto-tryptone of 10g (Bacto-Tryptone), 5g, 10g, 15g and supply 1 liter deionized water form.
LB substratum is by the yeast extract of the bacto-tryptone of 10g, 5g and the sodium-chlor of 10g, and supply 1 liter deionized water form.
YPG substratum has comprised 0.4% yeast extract, 0.1% KH2PO4,0.05% MgSO47H2O, 1.5% glucose in deionized water.
COVE-N-gly swash plate is by the agar powder of the COVE salts solution of the KNO3 of the glycerine of the Sorbitol Powder of 218g, 10g, 2.02g, 50ml, 25g and supplies the deionized water of 1 liter and form.
For the COVE plate of protoplast regeneration, be by the COVE salts solution of the agar powder of the sucrose of 342g, 20g, 20ml and supply the deionized water of 1 liter and form.This substratum carries out sterilizing (< < bacteriological analysis handbook > >, the 8th edition, revision A, 1998) for 15 minutes by high pressure sterilization under 15psi.This substratum is cooled to 60 ℃ and add the ethanamide, the CsCl of 15mM of 10mM.
COVE top agarose be by the COVE salts solution of the sucrose of 342.3g, 20ml, the GTG agarose of 6g (SeaKem, catalog number (Cat.No.) 50070) and supply 1 liter deionized water form.This substratum carries out sterilizing (< < bacteriological analysis handbook > >, the 8th edition, revision A, 1998) for 15 minutes by high pressure sterilization under 15psi.This substratum is cooled to 60 ℃ and the interpolation ethanamide of 10mM and the CsCl of 15mM.For separating of COVE-2 plate be by the agar powder of the COVE salts solution of the sucrose of 30g, 20ml, 30g and supply the deionized water of 1 liter and form.This substratum carries out sterilizing (< < bacteriological analysis handbook > >, the 8th edition, revision A, 1998) for 15 minutes by high pressure sterilization under 15psi.This substratum is cooled to 60 ℃ and add the ethanamide of 10mM.
COVE salts solution is the MgSO by 26g 47H 2the KCL of O, 26g, the KH of 26g 2pO 4, 50ml COVE trace-metal solution and supply 1 liter deionized water form.
COVE trace-metal solution is the Na by 0.04g 2b 4o 710H 2the CuSO of O, 0.4g 45H 2the FeSO of O, 1.2g 47H 2the MnSO of O, 0.7g 4h 2the Na of O, 0.8g 2moO 42H 2the ZnSO of O, 10g 47H 2o and supply 1 liter deionized water form.
MD substratum is by 1.34% YNB, 4 × 10 -5the vitamin H of % and 2% dextrose form.For plate, the agar of 7.5g is added in the ability of swimming pressure sterilizer of 200ml, be cooled to 60 ℃, and then add the 10X D-Glucose of 10X YNB, 25ml and the 500X vitamin H of 400 μ l of 25ml.
BMSY is by 1% yeast extract, 2% peptone (bacterium is used), the potassium phosphate buffer (pH6.0) of 100mM, 1.34%YNB, 4 × 10 -5the vitamin H of % and 1.82% Sorbitol Powder form.
The Sorbitol Powder of the peptone of the yeast extract of 10g, 20g (bacterium with) and 18.2g is dissolved in 800ml water and in liquid circulation to high pressure sterilization 20 minutes.When by the substratum cool to room temperature of high pressure sterilization, add 1M potassium phosphate buffer (pH6.0) and the 10X YNB of 100ml and the 500X vitamin H of 2ml of 100ml.
Example 1: the extraction of Talaromyces emersonii genomic dna
The bacterial strain NN051602 of Talaromyces emersonii is inoculated on a PDA plate, and at 45 ℃, in dark place, hatches 3 days.Several mycelia-PDA plug is inoculated in the 500ml shaking flask of the YPG substratum that has comprised 100ml.Under with 160rpm vibration, these flasks are hatched 3 days at 45 ℃.By filtration, pass through MIRACLOTH
Figure BDA0000469949290000512
(the Kang Biquan company (Calbiochem, La Jolla, CA, USA) of California, USA La Jolla) collects these mycelia, and freezing in liquid nitrogen.Freezing mycelia is milled to fine powder with mortar and pestle, and uses DNeasy
Figure BDA0000469949290000513
plant Maxi test kit (the Kai Jie company limited (QIAGEN Inc., Valencia, CA, USA) of California, USA Valencia) is followed the explanation of manufacturers and is isolated genomic dna.
Example 2: by 2 GH15 glucoamylase genes of Talaromyces emersonii genomic dna cloning
In following table 2, shown Oligonucleolide primers is designed to the genomic dna amplification GH15 glucoamylase gene (SEQ ID:1 and 3) by Talaromyces emersonii NN051602.Primer is synthetic by hero company (the hero company (Invitrogen, Beijing, China) of BeiJing, China).
Table 1: from the GH15 glucoamylase gene of Talaromyces emersonii
Gene title DNA sequence dna
AMG51602-1 SEQ?ID:1
AMG51602-2 SEQ?ID:3
Table 2: in order to the primer by Talaromyces emersonii genomic dna amplification total length glucoamylase gene
Figure BDA0000469949290000511
Capitalization indicates 5'-and the 3'-region of gene to be amplified, and lowercase is at insertion point place and the carrier sequence homology of pCaHj505 carrier.This expression vector pCaHj505 has comprised the TAKA-amylase promotor and the aspergillus niger glucoamylase terminator element that derive from aspergillus oryzae.In addition, pCaHj505 has had the sequence in pUC18 for selecting intestinal bacteria and breed source, and one derive from Aspergillus nidulans for selecting amds +the amdS gene of Aspergillus transformant, this genes encoding acetamidase gene.
For each gene, in PCR reaction, used the primer pair (forward and oppositely in each) of 20pmol, this PCR reaction is to consist of the following: in dATP, dTTP, dGTP and the dCTP of DMSO, the 2.5mM of the Talaromyces emersonii NN051602 genomic dna of 2 μ l, the 5X GC damping fluid of 10 μ l, 1.5 μ l each, and the Phusion of 0.6 unit tMhigh frequency high fidelity archaeal dna polymerase (the Fei Zemu company (Finnzymes Oy, Espoo, Finland) of Espoo, Finland), final volume is 50 μ l.This amplification is to use Pei Erteer thermal cycler (Peltier Thermal Cycler) (M J research (the M J Research Inc. of company limited in San Francisco, California, USA south, South San Francisco, CA, USA)) carry out, this thermal cycler program setting was for sex change at 98 ℃ 1 minute; The sex change at 98 ℃ of 10 circulations 15 seconds, at 65 ℃, anneal 30 seconds (and each circulation reduces by 1 ℃), and at 72 ℃, extend 90 seconds; And respectively at 98 ℃ at 15 seconds, 60 ℃ at 30 seconds and 72 ℃ 90 seconds of other 26 circulations; Final extension 10 minutes at 72 ℃.Then, make heat block forward the infusion of 4 ℃ to.
By 0.7% agarose gel electrophoresis, use 90mM Tris-borate and 1mM EDTA(TBE) damping fluid separates these PCR products, wherein under UV light observation at the product band at each PCR product size place of expection.Then, according to the explanation of manufacturers, by using GFX PCR DNA and gel band purification kit (Gel Band Purification Kit) (the GE Medical Group (GE Healthcare, Buckinghamshire, UK) of Britain's Buckinghamshire) to be purified into these PCR products from solution.
Table 3: the size of PCR product in example 2
Gene title The size of PCR product
AMG51602-1 1.9kb
AMG51602-2 2.3kb
BamHI and XhoI digestion for plasmid pCaHj505, separate by 0.7% agarose gel electrophoresis carrying out with tbe buffer liquid, and according to the explanation of manufacturers, use illustra PCR DNA and gel band purification kit to carry out purifying.
Use In-Fusion CF dry clone test kit (Dry-down Cloning Kit) (Tyke, Crow experiment (the Clontech Laboratories of company limited in California, USA mountain scene city, Inc., Mountain View, CA, USA)) this fragment is directly cloned in this expression vector pCaHj505.
Use IN-FUSION tMthe dry clone of CF test kit (Tyke, the Crow experiment company limited in California, USA mountain scene city) links together the carrier of PCR product and digestion, produce respectively the plasmid in table 4, wherein Humicola insolens GH15 glucoamylase gene transcribes under the control in the TAKA-amylase promotor from aspergillus oryzae.Clone operations is to carry out according to the explanation of manufacturers.Briefly, for each ligation, the PCR product with BamHI and the pCaHj505 of XhoI digestion and the purifying of 60ng of 30ng is added in reaction bottle, and by adding deionized water, this powder is suspended again, final volume is 10 μ l.These reactions are hatched 15 minutes at 37 ℃, and then at 50 ℃, hatch 15 minutes.According to the scheme of manufacturers, the reaction of 3 μ l is transformed into intestinal bacteria TOP10 competent cell (sky (TIANGEN Biotech (Beijing) Co.Ltd. of root biotechnology (Beijing) company limited of BeiJing, China, Beijing, China)) in, and be coated onto on the LB plate that is supplemented with 0.1mg/ml Ampicillin Trihydrate.At 37 ℃ after overnight incubation, see that bacterium colony grows on LB Ampicillin Trihydrate plate.By bacterium colony PCR, detect the intestinal bacteria transformant that has comprised expression construct, and confirmed (by (the SinoGenoMax Company Limited of Nuo Sai gene company limited of BeiJing, China by carry out DNA sequencing with carrier primer, Beijing, China) carry out).For the plasmid DNA pAMG51602-1_C505 and the pAMG51602-2_C505 that express at aspergillus niger, be by using QIAprep Spin Miniprep test kit (the Kai Jie company limited of California, USA Valencia) to extract from correct intestinal bacteria transformant.
Table 4: the plasmid (expression construct) in example 2
Gene title Plasmid
AMG51602-1 pAMG51602-1_C505
AMG51602-2 pAMG51602-2_C505
Example 3: the expression of Talaromyces emersonii GH15 glucoamylase gene in aspergillus niger
With the spore inoculating agar swash plate (COVE-N-gly) of aspergillus niger HowB112 and it is grown at 32 ℃, until its complete real estate spore.These spores are suspended in the aseptic 0.05%tween20 water of 5-10ml again.By approximately 10 8individual spore is transferred to the NaNO that has comprised 100ml YPG substratum and 10mM 3the shaking flask of 500ml with baffle plate in, and at 32 ℃, in Innova vibrator, with 99rpm, hatch 16 hours.Then, gather the preparation of mycelia for protoplastis.The preparation of aspergillus niger HowB112 protoplastis and conversion are to carry out according to the method described in patent WO2004/111218 or EP238023.With pAMG51602-1_C505 and the individual aspergillus niger HowB112 that transforms individually of the each 10 μ g of pAMG51602-2_C505.
On COVE plate, the aspergillus niger HowB112 transformant with pAMG51602-1_C505 and pAMG51602-2_C505 is selected, for protoplast regeneration (being described in substratum and solution part).For each conversion, on option board, observe approximately 15 transformants.On COVE-2 plate, at 32 ℃, from each conversion, isolate six transformants, keep 3-4 days.
After separating, is inoculated into those six transformants that at every turn transform in the 3ml YMD substratum in 24 orifice plates individually, and hatches under 30 ℃, 220rpm.After 3 days hatch, according to the explanation of manufacturers, at ((the Invitrogen Corporation of hero company of California, USA Carlsbad of the NuPAGE Novex4%-12%Bis-Tris gel with MES, Carlsbad, CA, USA)) above the 20 μ l supernatant liquors from each culture are analyzed.The gel obtaining dyes by instant indigo plant (Instant Blue) (the Ai Bende company limited (Expedeon Ltd., Babraham Cambridge, UK) of Britain Camb Ba Bulahan).The SDS-PAGE spectrogram of these cultures shows, they have the protein belt of the expection of pAMG51602-1_C505 and pAMG51602-2_C505 expression product.Expression product numbering and the expression strain numbering of those six genes are shown in table 5.
Table 5: expression strain
Expression construct Expression product Expression strain
pAMG51602-1_C505 P245A6 O5MXC
pAMG51602-2_C505 P245A5 O5MXB
Example 4: the fermentation of aspergillus niger expression strain
Wash with the swash plate of each expression strain in the YMD his-and-hers watches 5 of 10ml, and be inoculated in 2 liters of flasks that comprised 400ml YMD substratum to produce the sign of nutrient solution for this enzyme.This culture is hatched with 150rpm at 30 ℃ on vibrator.In the time of the 3rd day, gather this culture, and use 0.45 μ m DURAPORE film (Millipore Corp. (Millipore, Bedford, MA, USA) of Massachusetts, United States Bedford) to filter.The culture nutrient solution filtering characterizes for enzyme.
Example 5: the sign of glucoamylase
Substrate: containing the deionized water of 1% Zulkovsky starch (S-9765 of Sigma (Sigma))
Reaction buffer: 0.1M acetate buffer, pH4.3
Glucose concentration determination test kit: and light glucose test test kit (LabAssay glucose, and light Co., Ltd., catalog number (Cat.No.) 298-65701).
reaction conditions:
The Zulkovsky starch of 20 μ l is mixed with the acetate buffer (pH5.3) of 50 μ l.The enzyme solution (50 μ g zymoprotein/ml) that adds 30 μ l reaches 100 μ l final volumes, hatches 15 minutes subsequently at 37 ℃.
Glucose concn be by with light kit measurement.
All these all parallel carrying out of experiment.
optimal temperature.
For evaluate temperature curve, reaction conditions check described above is carried out at 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃.These the results are shown in table 6.
Table 6: optimal temperature
From this result, can find out, under specified criteria, the optimum temps of P245A6 and P245A5 is that approximately 60 ℃ and glucoamylase maintain the activity that approaches 50% at 90 ℃.
thermostability:
In order to assess the thermostability of these glucoamylases, the modification part of this reaction conditions check is, by this enzyme solution and acetate buffer preincubate 0,10,30,60 and 120 minutes at 60 ℃.After hatching, 20 μ l starch are added in this solution, and carry out this check as described above.
These the results are shown in table 7.
Table 7: thermostability
Figure BDA0000469949290000552
Optimum pH:
In order to assess the optimum pH of glucoamylase, reaction conditions described above is checked at pH2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0, is carried out for 10.0 and 11.0 times.Replace the acetate buffer described in the check of this reaction conditions with following damping fluid: the succsinic acid of 100mM, HEPES, CHES, CAPSO, 1mM CaCl 2, 150mM KCl, 0.01%Triton X-100, pH adjusts to 2.0,3.0,3.5,4.0,4.5,5.0,6.0,7.0,8.0,9.0,10.0 or 11.0 with HCl or NaOH.
These the results are shown in table 8.
Table 8: optimum pH
Figure BDA0000469949290000561
From this result, can find out, P245A6 and P245A5 are respectively that best pH is 4 and 5 acidicenzym.
pH stability:
30 μ l enzyme solution (50 μ g/ml) and 50 μ l damping fluids are mixed and preincubate 0,10,30,60,120 minutes.After preincubate, by 20 μ l Zulkovsky starches, (final volume is that 100 μ l) are hatched 15 minutes at 37 ℃.
Finally, use and light kit measurement glucose concn.These the results are shown in table 9.
Table 9:pH stability
Figure BDA0000469949290000562
From this result, can find out, two kinds of glucoamylases are stable under tested condition under acidic conditions.
Figure IDA0000469949320000011
Figure IDA0000469949320000021
Figure IDA0000469949320000031
Figure IDA0000469949320000051
Figure IDA0000469949320000061
Figure IDA0000469949320000071
Figure IDA0000469949320000081

Claims (25)

1. have an isolated polypeptide for glucoamylase activity, this polypeptide is selected from lower group, and this group is comprised of the following:
(a) have at least 75%, at least 80%, at least 85%, at least 90%, a for example peptide species of 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 with the mature polypeptide of SEQ ID NO:2 or SEQ ID NO:4;
(b) by under medium-high stringency condition, the high stringency condition of high stringency conditioned disjunction with the mature polypeptide encoded sequence of (i) SEQ ID NO:1 or SEQ ID NO:3, (ii) its cDNA sequence or the (iii) coded peptide species of a kind of polynucleotide of total length complement hybridization (i) or (ii);
(c) for example, by having at least 75%, at least 80%, at least 85%, at least 90% with SEQ ID NO:1 or SEQ ID NO:3 mature polypeptide encoded sequence or its cDNA sequence, the coded peptide species of a kind of polynucleotide of 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;
(d) a kind of variant of the mature polypeptide of SEQ ID NO:2 or SEQ ID NO:4, this variant comprises a replacement, disappearance and/or inserts in one or more (several) position; And
(e) fragment with glucoamylase activity of (a) and (b), (c) or these polypeptide (d).
2. polypeptide as claimed in claim 1, the mature polypeptide of this polypeptide and SEQ ID NO:2 or SEQ ID NO:4 has 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.
3. polypeptide as claimed in claim 1 or 2, this polypeptide be by under medium-high stringency condition, the high stringency condition of high stringency conditioned disjunction with the mature polypeptide encoded sequence of (i) SEQ ID NO:1 or SEQ ID NO:3, (ii) its cDNA sequence or (iii) a kind of polynucleotide of total length complement hybridization (i) or are (ii) coded.
4. the polypeptide as described in any one in claim 1-3, this polypeptide is by have a kind of polynucleotide of 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 coded with the mature polypeptide encoded sequence of SEQ ID NO:1 or SEQ ID NO:3 or its cDNA sequence.
5. the polypeptide as described in any one in claim 1-4, this polypeptide comprises or it consists of SEQ ID NO:2 or SEQ ID NO:4, or the mature polypeptide of SEQ ID NO:2 or SEQ ID NO:4.
6. polypeptide as claimed in claim 5, wherein this mature polypeptide is the amino acid 22 to 537 of SEQ ID NO:2, or the amino acid 22 to 651 of SEQ ID NO:4.
7. the polypeptide as described in any one in claim 1-4, this polypeptide is a kind of variant of the mature polypeptide of SEQ ID NO:2 or SEQ ID NO:4, this variant comprises a replacement, disappearance and/or inserts in one or more (several) position.
8. polypeptide as claimed in claim 1, this polypeptide is a fragment of SEQ ID NO:2 or SEQ ID NO:4, wherein this fragment has glucoamylase activity.
9. comprise an isolated polypeptide for glucoamylase catalyst structure domain, this catalyst structure domain is selected from lower group, and this group is comprised of the following:
(a) there is a catalyst structure domain of at least 75% sequence identity with the amino acid 31 to 500 of SEQ ID NO:2 or the amino acid 29 to 499 of SEQ ID NO:4;
(b) by under high stringency condition with the (i) Nucleotide 91 to 1652 of SEQ ID NO:1 or the Nucleotide 85 to 1712 of SEQ ID NO:3, (ii) its cDNA sequence or the (iii) coded catalyst structure domain of a kind of polynucleotide of total length complement hybridization (i) or (ii);
(c) by with the (i) Nucleotide 91 to 1652 of SEQ ID NO:1 or the Nucleotide 85 to 1712 of SEQ ID NO:3 (ii) or its cDNA sequence there is a coded catalyst structure domain of a kind of polynucleotide of at least 75% sequence identity; And
(d) a kind of variant of the amino acid 31 to 500 of SEQ ID NO:2 or the amino acid 29 to 499 of SEQ ID NO:4, this variant for example, comprises a replacement, disappearance and/or inserts in one or more (several) position;
And wherein this catalyst structure domain has glucoamylase activity.
10. polypeptide as claimed in claim 9, this polypeptide comprises a connexon and a carbohydrate binding domains in addition.
11. polypeptide as claimed in claim 10, the amino acid 500 to 535 that wherein this connexon comprises SEQ ID NO:4, and this carbohydrate binding domains amino acid 536 to 651 of comprising SEQ ID NO:4.
12. 1 kinds of compositions, comprise the polypeptide as described in any one in claim 1-11.
13. composition according to claim 12, comprises a kind of α-amylase and a kind of polypeptide as described in any one in claim 1-11.
The purposes of 14. 1 kinds of polypeptide as described in any one in claim 1-11, this polypeptide is for the production of syrup and/or a kind of tunning.
15. purposes according to claim 14, wherein parent material is gelationization or the amyloid material of gelationization not.
The purposes of 16. 1 kinds of polypeptide as described in any one in claim 1-11, this polypeptide is used for brewageing.
17. 1 kinds of methods by amyloid material produce tunning, comprise the following steps:
(a) make amyloid material liquefaction;
(b) material of this liquefaction is carried out to saccharification; And
(c) with a kind of fermentation organism, ferment;
Wherein step (b) is to use at least one glucoamylase as described in any one in claim 1-11 to carry out.
18. 1 kinds of methods by amyloid material produce tunning, comprise the following steps:
(a) with a kind of α-amylase with a kind ofly make amyloid material carry out saccharification at a temperature of the initial gelatinization temperature lower than described amyloid material according to the glucoamylase described in any one in claim 1-11; And
(b) with a kind of fermentation organism, ferment,
Wherein step (a) is to use at least one glucoamylase as described in any one in claim 1-11 to carry out.
19. polynucleotide for separation, the polypeptide of this polynucleotide encoding as described in any one in claim 1-11.
20. 1 kinds of nucleic acid constructs or expression vector, comprised the polynucleotide as claimed in claim 19 that are operably connected to one or more control sequences, the generation of these one or more control sequences guiding this polypeptide in an expressive host.
21. 1 kinds of recombinant host cells, have comprised the polynucleotide as claimed in claim 19 that are operably connected to one or more control sequences, and these one or more control sequences guide the generation of this polypeptide.
22. a method for the polypeptide of generation as described in any one in claim 1-11, comprising:
(a) be of value under the condition that produces this polypeptide, cultivating a cell, this cell produces this polypeptide with its wild-type form; And
(b) reclaim this polypeptide.
23. a method for the polypeptide of generation as described in any one in claim 1-11, comprising:
(a) be of value under the condition that produces this polypeptide, cultivating host cell as claimed in claim 21; And
(b) reclaim this polypeptide.
Transgenic plant, plant part or the vegetable cell of 24. 1 kinds of polynucleotide conversions of the polypeptide as described in any one in claim 1-11 with coding.
25. a method for the polypeptide of generation as described in any one in claim 1-11, comprising:
(a) be of value under the condition that produces this polypeptide, cultivating transgenic plant as claimed in claim 26 or vegetable cell; And
(b) reclaim this polypeptide.
CN201280041671.1A 2011-08-26 2012-08-24 Polypeptides having glucoamylase activity and polynucleotides encoding same Pending CN103764670A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201280041671.1A CN103764670A (en) 2011-08-26 2012-08-24 Polypeptides having glucoamylase activity and polynucleotides encoding same

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN2011078999 2011-08-26
CNPCT/CN2011/078999 2011-08-26
CN201280041671.1A CN103764670A (en) 2011-08-26 2012-08-24 Polypeptides having glucoamylase activity and polynucleotides encoding same
PCT/CN2012/080553 WO2013029496A1 (en) 2011-08-26 2012-08-24 Polypeptides having glucoamylase activity and polynucleotides encoding same

Publications (1)

Publication Number Publication Date
CN103764670A true CN103764670A (en) 2014-04-30

Family

ID=50531049

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201280041671.1A Pending CN103764670A (en) 2011-08-26 2012-08-24 Polypeptides having glucoamylase activity and polynucleotides encoding same

Country Status (1)

Country Link
CN (1) CN103764670A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108977422A (en) * 2018-08-13 2018-12-11 天津科技大学 A kind of tool glucoamylase activity lactic acid monomer production bacterium and its application
CN110628648A (en) * 2019-08-07 2019-12-31 中国科学院成都生物研究所 Rhizomucor fungi producing liquefied amylase and application
CN112725302A (en) * 2021-01-15 2021-04-30 江南大学 Construction of fusion protein, method for degrading polymer by using fusion protein and application of fusion protein

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1284129A (en) * 1997-11-26 2001-02-14 诺沃挪第克公司 Thermostable glucoamylase
CN1875097A (en) * 2003-10-28 2006-12-06 诺维信北美公司 Hybrid enzymes
US20110078829A1 (en) * 2009-09-29 2011-03-31 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1284129A (en) * 1997-11-26 2001-02-14 诺沃挪第克公司 Thermostable glucoamylase
CN1875097A (en) * 2003-10-28 2006-12-06 诺维信北美公司 Hybrid enzymes
US20110078829A1 (en) * 2009-09-29 2011-03-31 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108977422A (en) * 2018-08-13 2018-12-11 天津科技大学 A kind of tool glucoamylase activity lactic acid monomer production bacterium and its application
CN108977422B (en) * 2018-08-13 2022-02-22 天津科技大学 Lactic acid monomer producing strain with glucoamylase activity and application thereof
CN110628648A (en) * 2019-08-07 2019-12-31 中国科学院成都生物研究所 Rhizomucor fungi producing liquefied amylase and application
CN110628648B (en) * 2019-08-07 2022-04-22 中国科学院成都生物研究所 Rhizomucor fungi producing liquefied amylase and application
CN112725302A (en) * 2021-01-15 2021-04-30 江南大学 Construction of fusion protein, method for degrading polymer by using fusion protein and application of fusion protein

Similar Documents

Publication Publication Date Title
CN102869771B (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
CN103781910B (en) Alpha-amylase variants and its coded polynucleotide
US11180746B2 (en) Polypeptides having alpha-amylase activity and polynucleotides encoding same
US8530216B2 (en) Polypeptides having alpha-amylase activity and polynucleotides encoding same
CN103298932A (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
EP2884852B1 (en) Thermostable asparaginase variants and polynucleotides encoding same
CN104169417A (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
EP2751131B1 (en) Polypeptides having alpha-amylase activity and polynucleotides encoding same
CN101443447A (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
CN105339494A (en) Pullulanase chimeras and polynucleotides encoding same
EP2748188A1 (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
CN102869770A (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
CN102482658B (en) Polypeptides having isoamylase activity and polynucleotides encoding same
US8697412B2 (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
CN103764670A (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
US9909112B2 (en) Polypeptides having alpha-amylase activity and polynucleotides encoding same
CN103298359A (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
US9650620B2 (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
CN102933596B (en) There are the polypeptide of glucoamylase activity and the polynucleotide of this polypeptide of coding
US9567574B2 (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
WO2013034097A1 (en) Polypeptides having glucoamylase activity and polynucleotides encoding same
WO2013029494A1 (en) Polypeptides having glucoamylase activity and polynucleotides encoding same

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
AD01 Patent right deemed abandoned
AD01 Patent right deemed abandoned

Effective date of abandoning: 20180907