WO2006088325A1 - Bacillus sp. i-52, permettant d'isoler une protease haloalcalophile stable vis-a-vis des detergents et des oxydants, gene assurant son codage et utilisation - Google Patents

Bacillus sp. i-52, permettant d'isoler une protease haloalcalophile stable vis-a-vis des detergents et des oxydants, gene assurant son codage et utilisation Download PDF

Info

Publication number
WO2006088325A1
WO2006088325A1 PCT/KR2006/000545 KR2006000545W WO2006088325A1 WO 2006088325 A1 WO2006088325 A1 WO 2006088325A1 KR 2006000545 W KR2006000545 W KR 2006000545W WO 2006088325 A1 WO2006088325 A1 WO 2006088325A1
Authority
WO
WIPO (PCT)
Prior art keywords
protease
transformant
culture supernatant
strain
activity
Prior art date
Application number
PCT/KR2006/000545
Other languages
English (en)
Inventor
Chung Soon Chang
Han Seung Joo
Jang Won Choi
Original Assignee
Inha-Industry Partnership Institute
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 Inha-Industry Partnership Institute filed Critical Inha-Industry Partnership Institute
Publication of WO2006088325A1 publication Critical patent/WO2006088325A1/fr

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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F24—HEATING; RANGES; VENTILATING
    • F24C—DOMESTIC STOVES OR RANGES ; DETAILS OF DOMESTIC STOVES OR RANGES, OF GENERAL APPLICATION
    • F24C7/00—Stoves or ranges heated by electric energy
    • F24C7/04—Stoves or ranges heated by electric energy with heat radiated directly from the heating element
    • F24C7/046—Ranges
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F24—HEATING; RANGES; VENTILATING
    • F24C—DOMESTIC STOVES OR RANGES ; DETAILS OF DOMESTIC STOVES OR RANGES, OF GENERAL APPLICATION
    • F24C7/00—Stoves or ranges heated by electric energy
    • F24C7/06—Arrangement or mounting of electric heating elements
    • F24C7/067—Arrangement or mounting of electric heating elements on ranges
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the present invention relates to a Bacillus sp. strain, Bacillus clausii 1-52, producing a protease, a protease mass-produced therefrom, a gene encoding the same and a use thereof, more precisely, Bacillus clausii 1-52 producing a protease which was isolated from west coast mudflat of Korea, a protease therefrom which retains an enzyme activity under severe conditions, a gene encoding the same and a use thereof.
  • protease has been widely used in variety of industrial fields, for example, for the treatment of human disease such as thrombosis, as a laundry detergent component, as a contact lens detergent component, for the modification of milk protein, for silk degumming, for improving feed, for leather soaking, for dehairing, for recovering silver from x-ray film and for regeneration of ultrafiltration membrane used for the concentration of protein solution.
  • human disease such as thrombosis
  • a laundry detergent component as a contact lens detergent component
  • for the modification of milk protein for silk degumming
  • for improving feed for leather soaking, for dehairing
  • recovering silver from x-ray film and for regeneration of ultrafiltration membrane used for the concentration of protein solution The availabilty of enzymes produced from a microorganism has been well known and the industrial enzyme market is now over a billion dollar.
  • proteases take more than 60%, which are the biggest parts of all, and thus the marketability of the protease is growing more and more.
  • Proteases have a long history of industrial
  • proteases As detergent additives, proteases have been added to such detergents as laundry detergent, contact lens detergent, denture detergent, etc, and approximately 25% of industrial proteases are used as detergent additives. Since a bacterial protease was first used as a detergent additive in the name of BIO-40 in 1956, 13 billion tons of proteases have been annually produced as of today as detergent additives. Proteases for detergent have to retain wide substrate specificity enabling the elimination of food and blood or body fluid components and stability not to lose its enzyme activity under severe conditions such as alkali or surfactant environment and high temperature. In the past, proteases isolated from plants had been generally used. But, recently, proteases isolated from microorganisms, in particular Bacillus sp., have been popular .
  • Proteases are divided into exoproteases and endoproteases according to operating properties thereof; serine proteases, aspartic proteases and cysteine proteases according to the functional group of the active site; alkaline, neutral and acidic proteases according to the optimum pH for the enzyme activity; or intracellular proteases and extracellular proteases according to their location.
  • alkaline proteases are mainly used for detergent industry, which are generally isolated from a Bacillus sp. microorganism such as Bacillus subtilis and B. licheniformis, and fungi such as Aspergillus oryzae but preferably isolated from a Bacillus sp. microorganism.
  • a protease For industrial use, a protease has to be very stable so as to keep its enzyme activity under severe external environmental conditions which are extremely variable, compared with those of internal conditions. For example, under the presence of surfactants, most proteases lose much or the entire of their activities, so proteases for the use as a detergent component are very limited. Under severe external environmental conditions such as exposure on severe pH, heavy metal or the level of oxidation-reduction, which are highly variable and strongly affect the enzyme activity of a protease, the enzyme loses its activity as soon as those conditions are out of an acceptable range. Therefore, to use protease industrially, it is important to keep the enzyme activity of a protease under extremely unstable physical and chemical conditions.
  • the protease activity should not be affected by surfactants; second, the protease activity should not be affected by an oxygen-bleaching agent; third, the protease has to be an alkaline protease to keep its activity under high pH condition; forth, the protease activity should not be affected by low temperature (20-30 ° Q; fifth, the protease activity should not be affected by a heavy metal; and sixth, the protease activity does not have to require metal ions such as calcium.
  • a halophilic protease is more preferred since the protease can keep its enzyme activity under the high concentration of salt (more than 10% salt) , which is because salt has to be added as a core material during the process for praparing the protease in the form of granules.
  • a halophilic protease is especially welcomed by the high salinity food industry.
  • the unique taste and flavor of Geotgals (pickled fish) and fermented sauces which are Korean traditional foods prepared by preserving with high salt and depends on the amino acids produced from proteins therein by the action of a protease.
  • the fermented food includes salt more than 15% for long-term storage, thus enzyme activity of a protease has to be kept under the high concentration of salt.
  • U.S. Patent Nos. 534075 and 6136553 and Korean Patent No. 258740 describe that a novel protease having an excellent activity and stability has been produced to fulfill the above conditions from a conventional protease by inducing a specific mutation.
  • the present inventors screened various strains from the west coast mudflat of Korea, and among them, isolated a specific strain which can produce high amounts of a protease that retains its enzyme activity in the presence of heavy metals, anionic surfactants and oxidants used for oxygen-bleaching agents (ex; hydrogen peroxide, sodium perborate, etc.) as well as under the condition of a wide range of pH have been identified. Further, the inventors isolated a protease having those characteristics therefrom, examined conditions for mass-production of the protease, and finally completed the present invention by establishing a simple and easy method for mass-production of an alkaline protease from the strain.
  • surfactants more than 0.5%; ex; sodium dodecyl sulfate, etc.
  • the present invention provides a Bacillus clausii 1-52 strain (KCTC 10277BP) producing a protease retaining its enzyme activity under severe physical and chemical conditions.
  • the present invention also provides a culture supernatant of the strain harboring the protease activity.
  • the present invention further provides a protease isolated from the strain, a gene encoding the same, an expression vector containing the gene and a transformant transfected with the expression vector.
  • the present invention also provides an expression vector for the insertion into Bacillus genome containing the above gene and a transformant transfected with the expression vector.
  • the present invention provides a method for isolation of the protease from the strain.
  • the present invention provides a method for mass- production of the protease from the strain.
  • the present invention provides a detergent composition containing a culture supernatant of the strain and/or the protease of the invention.
  • the present invention provides a method for processing foods and animal feeds by using the Bacillus clausii 1-52 strain, the transformant , the culture supernatant and/or the protease isolated therefrom.
  • the present invention provides a food or feed additive containing the Bacillus clausii 1-52 strain, the transformant , the culture supernatant and/or the protease as an effective ingredient.
  • the present invention provides a method for treating textiles, fibers and leather by using the Bacillus clausii 1-52 strain, the transformant , the culture supernatant and/or the protease.
  • the present invention provides a method for treating paper and pulp by using the culture supernatant of the strain and/or the protease.
  • the present invention provides a method for decomposing organic waste by using the culture supernatant of the strain and/or the protease.
  • protease activity includes all polypeptides having a protease activity, including a peptidase and/or a proteinase activity.
  • expression cassette refers to a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as a protease of the invention) in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional elements necessary or helpful in effecting expression may also be used, e.g., enhancers.
  • expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like.
  • a "vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
  • the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
  • Vectors include, but are not limited to, replicons (e.g., RNA replicons, bacteriophages) to which, fragments of DNA may be attached and become replicated.
  • Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Patent No. 5,217,879), and include both the expression and non-expression plasmids.
  • a recombinant microorganism or a cultured cell is described as a host of the "expression vector”
  • this includes both extra-chromosomal circular and linear DNA and a DNA that has been incorporated into the host chromosome (s) .
  • the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
  • promoter includes all sequences capable of driving transcription of a coding sequence in a cell, e.g., a bacterial cell.
  • promoters used in the constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in temporal or spatial transcriptional regulation and/or regulation or variation of transcription rate of a gene.
  • a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation.
  • cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.
  • Constutive promoters are those that drive expression continuously under most environmental conditions and states of development or cell differentiation.
  • Inducible or “regulatable” promoters direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.
  • “Plasmid” herein indicates a circular single stranded or double stranded polynucleotide nucleic acid molecule which is capable of self-replication in prokaryotes like bacteria or in lower eukaryotes like yeast.
  • “Plasmids” can be commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. Equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.
  • gene includes a nucleic acid sequence comprising a segment of DNA involved in producing a transcription product (e.g., mRNA) , which in turn is translated to produce a polypeptide chain, or regulates gene transcription, reproduction or stability.
  • Genes can include regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).
  • nucleic acid or “nucleic acid molecule” includes oligonucleotide, nucleotide, polynucleotide, or a fragment of any of these, DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, peptide nucleic acid (PNA) , or any DNA-like or RNA-like material, natural or synthetic in origin, including, e. g., iRNA, ribonucleoproteins (e.g., double stranded iRNAs, e.g., iRNPs) .
  • DNA or RNA e.g., mRNA, rRNA, tRNA, iRNA
  • PNA peptide nucleic acid
  • DNA-like or RNA-like material natural or synthetic in origin, including, e. g., iRNA
  • nucleic acids i.e., oligonucleotides, containing known analogues of natural nucleotides.
  • the term also encompasses nucleic acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol . 144: 189-197; Strauss-Soukup (1997) Biochemistry 36: 8692- 8698; Straussense Nucleic Acid Drug Dev. 6: 153-156.
  • amino acid or “amino acid molecule” includes oligopeptide, peptide, polypeptide, or protein sequence, or a fragment, portion, or subunit of any of these, and naturally occurring or synthetic molecules.
  • polypeptide and “protein” include amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids.
  • polypeptide also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides.
  • the peptides and polypeptides of the invention also include all “mimetic” and “peptidomimetic” forms, as described in further detail, below.
  • isolated includes a material removed from its original environment, e.g., the natural environment if it is naturally occurring.
  • a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition.
  • Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity.
  • the invention provides nucleic acids which have been purified from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five or more orders of magnitude.
  • transgenic cell or organism indicates a transgenic cell or organism with the insertion of a foreign gene.
  • the cell or organism can be either prokaryotes or eukaryotes .
  • a foreign gene can be inserted episomally or integrated into a host genome by a vector such as plasmid, phagemid, cosmid, BAC and YAC.
  • a vector such as plasmid, phagemid, cosmid, BAC and YAC.
  • various methods including the method of Sambrook, known in the art can be used.
  • heat shock and electroporation can be used.
  • nucleic acids can include nucleic acids adjacent to a "backbone” nucleic acid to which it is not adjacent in its natural environment.
  • nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid "backbone molecules".
  • Backbone molecules include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest.
  • the enriched nucleic acids represent 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules.
  • Recombinant polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein.
  • synthetic polypeptides or proteins are those prepared by chemical synthesis, as described in further detail, below.
  • Olionucleotide includes either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized.
  • Variant includes polynucleotides or polypeptides of the invention modified at one or more base pairs, codons, introns, exons, or amino acid residues (respectively) yet still retain the biological activity of a protease of the invention (which can be assayed by, e.g., the hydrolysis of casein in zymograms, the release of fluorescence from gelatin, or the release of p-nitroanalide from various small peptide substrates) .
  • Variants can be produced by any number of means included methods such as, for example, error-prone PCR, DNA shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site- directed mutagenesis, gene reassembly, GSSM (Gene Site Saturation Mutagenesis) and any combination thereof.
  • Techniques for producing variant protease having activity at a pH or temperature for example, that is different from a wild-type protease, are included herein.
  • “Culture supernatant” or “microorganism culture supernatant” herein indicates a liquid component obtained by eliminating solid components including cells from centrifugation after liquid culture of Bacillus clausii I- 52 of the invention or cells transfected with an expression vector containing an expression cassette harboring a gene encoding the protease of the invention or cells having a genome with an integrated expression cassette. The fractions partially purified from the culture supernatant containing the protease of the invention are also included.
  • RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly .
  • Recombinant polypeptides e.g., proteases
  • Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acids Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth . Enzymol. 68: 90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Patent No. 4,458,066.
  • nucleic acids such as, e.g., subcloning, labeling probes (e.g., random- primer labeling using Klenow polymerase, nick translation, amplification) , sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL
  • Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones.
  • a nucleic acid encoding a polypeptide of the invention is assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof.
  • the invention provides fusion proteins and nucleic acids encoding them.
  • a polypeptide of the invention can be fused to a heterologous peptide or polypeptide, such as N- terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification.
  • Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like.
  • Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, WA) .
  • an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams (1995) Biochemistry 34: 1787-1797; Dobeli (1998) Protein Expr. Purif. 12: 404-414) .
  • histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein.
  • Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll (1993) DNA CeIl. Biol., 12: 441-53.
  • the invention provides nucleic acid (e.g., DNA) sequences operably linked to expression (e.g., transcriptional or translational) control sequence (s) , e.g., promoters or enhancers, to direct or modulate RNA synthesis/expression.
  • expression control sequence can be in an expression vector.
  • Exemplary bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR, PL, trp and aprE.
  • Exemplary eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein.
  • Promoters suitable for expressing a polypeptide in bacteria include the E. coli lac or trp promoters, the lad promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), the acid phosphatase promoter and promoter of the alkaline protease from Bacillus sp. ⁇ aprE) .
  • PGK 3-phosphoglycerate kinase
  • Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, heat shock promoters, the early and late SV40 promoter, LTRs from retroviruses, and the mouse metallothionein-I promoter. Other promoters known to control expressions of genes in prokaryotic or eukaryotic cells or their viruses may also be used.
  • the invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the proteases of the invention.
  • Expression vectors and cloning vehicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes (BAC), viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), Pl- based artificial chromosomes, yeast plasmids, yeast artificial chromosomes (YAC) , and any other vectors specific for specific hosts of interest (such as Bacillus, Aspergillus and yeast) .
  • Vectors of the invention can include chromosomal, non-chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available.
  • Exemplary vectors are: bacterial: pQE vectors (Qiagen) , pBluescript plasmids, pNH vectors, lambda-ZAP vectors (Stratagene) ; pET vectors (Novagen) ; ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXTl, pSG5
  • any other plasmids or other vectors may be used so long as they are replicable and viable in the host.
  • Low copy number or high copy number vectors may be employed with the present invention.
  • the expression vector can comprise a promoter, a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • Mammalian expression vectors can comprise an origin of replication, any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5 '-flanking non-transcribed sequences.
  • DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • the expression vectors contain one or more selectable marker genes to permit selection of host cells containing the vector.
  • selectable markers include genes encoding dihydrofolate reductase or genes conferring neomycin resistance for eukaryotic cell culture, genes conferring chloramphenicol, gentamycin, kanamycin, tetracycline, erythromycin or ampicillin resistance in E. coli, and the S. cerevisiae TRPl gene.
  • Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers .
  • CAT chloramphenicol transferase
  • Enhancers are cis-acting elements of DNA, usually from about 10 to about 300 bp in length that act on a promoter to increase its transcription, Examples include the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer and the adenovirus enhancers.
  • a nucleic acid sequence can be inserted into a vector by a variety of procedures.
  • the sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases .
  • blunt ends in both the insert and the vector may be ligated.
  • a variety of cloning techniques are known in the art, e.g., as described in Ausubel and Sambrook. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the vector can be in the form of a plasmid, a viral particle, or a phage.
  • Other vectors include chromosomal, non-chromosomal and synthetic DNA sequences, derivatives of SV40/ bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies .
  • a variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by, e.g., Sambrook.
  • Particular bacterial vectors which can be used include the commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEMl (Promega Biotec, Madison, WI, USA), pQE70, pQE ⁇ O, pQE-9 (Qiagen) , pD 10, psiX174 pBluescript II KS, pNH8A, pNHl ⁇ a, pNH18A, pNH46A (Stratagene) , ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia), pKK232-8 and pCM7, pET vectors such as pET-3, pET-11 and pET-22 (Novagen) .
  • eukaryotic vectors include pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia) .
  • any other vector may be used as long as it is replicable and viable in the host cell.
  • the nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses and transiently or stably expressed in plant cells and seeds.
  • One exemplary transient expression system uses episomal expression systems, e.g., cauliflower mosaic virus (CaMV) viral RNA generated in the nucleus by transcription of an episomal mini-chromosome containing supercoiled DNA, see, e.g., Covey (1990) Proc. Natl. Acad. Sci. USA 87: 1633-1637, Alternatively, coding sequences, i.e., all or sub-fragments of sequences of the invention can be inserted into a plant host cell genome becoming an integral part of the host chromosomal DNA. Sense or antisense transcripts can be expressed in this manner.
  • CaMV cauliflower mosaic virus
  • a vector comprising the sequences (e.g., promoters or coding regions) from nucleic acids of the invention can comprise a marker gene that confers a selectable phenotype on a plant cell or a seed.
  • the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta.
  • Expression vectors capable of expressing nucleic acids and proteins in plants are well known in the art, and can include, e.g., vectors from Agrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J. 16: 3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene 173: 69-73), tomato bushy stunt virus (see, e.g., Hillman (1989)
  • the expression vector can have two replication systems to allow it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression vector can contain at least one sequence homologous to the host cell genome. It can contain two homologous sequences which flank the expression construct.
  • the integrating vector can be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
  • Expression vectors of the invention may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed, e.g., genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline.
  • Selectable markers can also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. The biosynthetic genes can be used for the selection of a transformant with the insertion of a foreign gene from a nutrient medium deficient in the essential nutrient which is the product of the gene.
  • the invention also provides a transformed cell comprising a nucleic acid sequence of the invention, e.g., a sequence encoding a protease of the invention, or a vector of the invention.
  • the host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
  • Exemplary bacterial cells include E. coli, Streptomyces, Bacillus subtilis, Salmonella typhlmurlum and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • Exemplary insect cells include Drosophila S2 and Spodoptera Sf9.
  • Exemplary animal cells include, CHO, COS or Bowes melanoma or any mouse or human cell line.
  • the selection of an appropriate host is within the abilities of those skilled in the art. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Weising (1988) Ann. Rev. Genet. 22: 421-477; U.S. Patent No. 5,750,870.
  • the vector can be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer.
  • nucleic acids or vectors of the invention are introduced into the cells for screening, thus, the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid.
  • the method of introduction is largely dictated by the targeted cell type.
  • Exemplary methods include CaPO 4 precipitation, liposome fusion, lipofection (e.g., LIPOFECTINTM) , electroporation, viral infection, etc.
  • the candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction) or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.). As many pharmaceutically important screens require human or model mammalian cell targets, retroviral vectors capable of transfecting such targets are preferred.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the invention.
  • the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof,
  • Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.
  • Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art.
  • the expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate, ethanol or acetone precipitation, acid extraction, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts and other cell lines capable of expressing proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines.
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides produced by host cells containing the vector may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may or may not also include an initial methionine amino acid residue.
  • Cell-free translation systems can also be employed to produce a polypeptide of the invention.
  • Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof.
  • the DNA construct may be linearized prior to conducting an in vitro transcription reaction.
  • the transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.
  • the expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • nucleic acids of the invention and nucleic acids encoding the proteases of the invention, or modified nucleic acids of the invention can be reproduced by amplification.
  • Amplification can also be used to clone or modify the nucleic acids of the invention.
  • the invention provides amplification primer sequence pairs for amplifying nucleic acids of the invention.
  • One of skills in the art can design amplification primer sequence pairs for any part of or the full length of these sequences .
  • forward primers F2 and F3 represented by SEQ. ID. NO: 6 and SEQ. ID. NO: 7 and reverse primers Rl, R2 and R3 represented by SEQ. ID. NO: 8, No 9 and No 10 were used for PCR but not always limited thereto .
  • Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such as the amount of message in a cell sample), label the nucleic acid (e.g., to apply it to an array or a blot) , detect the nucleic acid, or quantify the amount of a specific nucleic acid in a sample.
  • message isolated from a cell or a cDNA library is amplified.
  • the skilled artisan can select and design suitable oligonucleotide amplification primers.
  • Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N. Y. (1990) and PCR STRATEGIES (1995), ed. Innis,
  • Probes 10: 257- 271) and other RNA polymerase mediated techniques e.g., NASBA, Cangene, Mississauga, Ontario
  • RNA polymerase mediated techniques e.g., NASBA, Cangene, Mississauga, Ontario
  • the present inventors deposited the Bacillus clausii 1-52 strain isolated by the present inventors at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB, #52, Oun- dong, Yusong-ku, Taejon, Korea) , an international depository authority designated by the Budapest Treaty, on June 14, 2002 (Accession No: KCTC 10277BP) , and deposited the transformant Escherichia coli JMlO9/pHPS-BCAP on January 26, 2005 (Accession No: KCTC 10772BP) .
  • the deposit meets all the conditions asked by the Budafest Treaty in relation to the usages of the deposited microorganisms.
  • the present inventors certify that (a) all restriction on the availability to the public of the deposited microorganisms will be irrevocably removed upon issuance of a United States patent of which any of such deposited microoranisms are subject matters; (b) the deposited microorganisms will be maintained for the longest period among the followings; i) at least for 5 years after the most recent request for the furnishing of a sample of any of the deposited microorganisms were received by the KCTC, ii) at least for 30 years from the date of deposit and iii) for the effective life of such patent; (c) vitality test for the microorganisms has to be performed on the date of deposit; (d) the original materials will be redeposited in case the microorganisms cannot survive any longer; and (e) access to the deposited microorganisms will be available during the pendency of the patent application to
  • Fig. 1 is an illustration of results of SDS-PAGE examining the protease of the present invention: M: standard protein, 1: Phenyl-Sepharose phase,
  • Fig. 2 is a graph showing the pH-dependent enzyme activity of a protease of the invention.
  • Fig. 3 is a graph showing the temperature-dependent enzyme activity of a protease of the invention.
  • Fig. 4 is a graph showing the enzyme activity of the protease of the invention according to the concentration of anionic surfactant, sodium dodecyl sulfate.
  • Fig. 5 is a graph showing the enzyme activity of the protease of the invention according to the concentration of a hydrogen peroxide solution.
  • Fig. 6 is a graph showing the enzyme activity of the protease of the invention according to the concentration of sodium perborate.
  • Fig. 7 is a graph showing the relative activity of the protease of the invention according to the concentration of salt.
  • Fig. 8 is a graph showing the result of the experiment examining whether a protease of the invention could retain the enzyme stability in the presence of a commercial detergent.
  • Fig. 9 is a graph showing the detergency of the protease of the invention produced by Bacillus clausii in the presence of the detergent composition linear alkylbenzene sulfonates (LAS, 15%) .
  • Fig. 10 is a photograph showing the soybean cake decomposing capacity of a protease of the invention produced by Bacillus clausii:
  • Lane M protein marker
  • Lane 1 before treatment
  • Fig. 11 is a graph showing the decomposition of soybean trypsin inhibitor (SBTI) by a protease of the invention produced by Bacillus clausii.
  • Fig. 12 is a schematic diagram showing the locations of primers and amplified areas by PCR using the chromosomal DNA of Bacillus clausii 1-52 as a template.
  • SBTI soybean trypsin inhibitor
  • Fig. 13 is a photograph of gel electrophoresis showing the result of PCR using different combinations of primers :
  • Lane M 100 bp DNA marker
  • Lane 1 combination of primers F2/R1
  • Lane 2 combination of primers F2/R2
  • Lane 3 combination of primers F2/R3
  • Lane 4 combination of primers F3/R1
  • Lane 5 combination of primers F3/R2
  • Lane 6 combination of primers F3/R3.
  • Fig. 14 is a photograph of gel electrophoresis illustrating that the PCR product obtained from PCR using primers F3/R2 was introduced into pGEM T easy plasmid, which was digested with restriction enzyme to confirm the insertion of a target DNA:
  • Lane M lambda DNA marker
  • Fig. 15 is a schematic diagram illustrating the plasmid pT7-BCAP for the expression of a mature protease (BCAP) .
  • Fig. 16 is a photograph of gel electrophoresis illustrating that a gene encoding the mature protease (BCAP) was introduced into the plasmid pT7-7, which was then digested with restriction enzyme to confirm the insertion of a target DNA, Lane M: lambda DNA marker, and
  • Lane 1, 2 and 3 inserted DNA fragments after treated with Nco I and Hind III, clone 1, 2 and 3, respectively
  • Fig. 17 is a photograph showing the result of SDS- PAGE examining the mature protease induced by the culture of a transformant expressing the mature protease in the presence of IPTG:
  • Lane 1 before induction with IPTG
  • Lane 2 1 hour after induction with IPTG
  • FIG. 18 is a schematic diagram illustrating the plasmid pHPS-BCAP harboring a gene represented by SEQ. ID. NO: 1 for the integration into Bacillus clausii genome.
  • the present invention provides a Bacillus clausii I- 52 strain producing a protease.
  • the Bacillus clausii of the invention was isolated from the west coast mudflat of Korea, which is known to have bad growth conditions and produced a protease confirmed to keep its enzyme activity under severe physical and chemical conditions and in the presence of a heavy metal, and was proved to be stable in the wide range of pH and in the presence of an anionic surfactant and an oxidant (ex; hydrogen peroxide, sodium perborate, etc.) used as an additive to an oxygen-bleaching agent.
  • the Bacillus clausii of the invention was confirmed to be a Gram- positive, to form a spore, to have catalase and oxidase activities but not to have urease activity.
  • the Bacillus clausii uses sugar such as glucose, fructose, galactose, mannose, maltose, mannitol, sorbitol and starch, and proteins such as casein and gelatin to produce acid and gas, and other morphological and biochemical properties of the Bacillus clausii are presented in Table 1. And also, the Bacillus clausii contains 16S ribosomal RNA represented by SEQ. ID. NO: 1.
  • Table 1 Bacterial property of Bacillus clausii: morphological and biochemical characteristics
  • the present inventors named the strain producing the protease of the invention as 'Bacillus clausii 1-52' and deposited at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB, #52, Oun-dong, Yusong-ku, Taejon, Korea), on Jun 14, 2002 (Accession No: KCTC 10277BP) .
  • KCTC Korean Collection for Type Cultures
  • the present invention provides a protease produced by Bacillus clausii and a purification method of the protease.
  • the purification method of the protease produced by Bacillus clausii of the invention comprises the following steps :
  • the synthetic absorbent can be any of aromatic synthetic absorbents, and Diaion HP absorbents are preferred.
  • an absorbent is recovered by filtering and then washed briefly with tap water. Then, an active fraction is eluted.
  • the chromatography is either hydrophobic chromatography or ion exchange chromatography, and/or ion exchange chromatography and hydrophobic chromatography are used serially in that order.
  • the eluent was loaded to Phenyl- Sepharose column, and then an active fraction was eluted.
  • a protease produced by Bacillus clausii of the invention has the titer of 120,000 U per 1 mi of culture supernatant, indicating that the Bacillus clausii produces a protease much more than any other strain (see Korean Patent No. 258740) .
  • the protease of the invention consists of 381 amino acids, and 32 amino acids of N-terminal are a signal sequence and the 107 th amino acid alanine (Ala) is the N- terminal amino acid of an active protein.
  • the active protease region consists of 275 amino acid residues represented by SEQ. ID.
  • protease of the invention is a very stable enzyme retaining its activity under various physical and chemical conditions including high pH and the presence of a detergent, an organic solvent and a heavy metal.
  • the protease of the invention shows its protease activity in the wide range of pH (pH 7.0 - pH 12.0), but preferable pH range to show the maximum activity is 9.0 - 12.0, more preferable pH range is 10.0 - 11.0, and the most preferable pH is 11.0 (see Fig. 2).
  • protease of the invention retains its activity in the temperature range of 10 - 70 ° C but to show the maximum protease activity, 40 - 65 ° C is preferred, 55 - 65 ° Cis more preferred and 60 - 65 ° C is most preferred (see Fig. 3) .
  • the protease of the invention can keep its enzyme activity in the presence of not only non-ionic surfactants such as Tween 20 and Triton X-100 but also strong anionic surfactants such as sodium dodecyl sulfate (SDS) and linear alkylbenzene sulfonates (LAS) , in particular the protease retained its activity for three days even by the treatment of 5% SDS (see Fig.
  • protease of the invention shows the maximum activity in the presence of 2 - 5% salt, and keeps its activity further in the high concentration of salt (20%) , confirming that the protease of the invention is a halophilic protease (see Fig. 7) . Based on such enzymatic characteristics, it was confirmed that the protease derived from the Bacillus clausii 1-52 fits a variety of industrial uses.
  • the present invention also provides a gene encoding the protease.
  • the sequence of a gene encoding the protease of the invention having the above properties was analyzed. Precisely, chromosomal DNA was isolated from the Bacillus clausii, and PCR was performed using the DNA as a template to obtain DNA fragments of the protease (see Fig. 12 and 13), leading to the determination of nucleotide sequence thereof (SEQ. ID. NO: 3) .
  • the present invention further provides an expression vector containing the gene.
  • a gene SEQ. ID. NO: 3 of the protease having the amino acid sequence represented by SEQ. ID. NO: 5 was introduced into an expression vector.
  • the expression vector constructed thereby was named "pT7-BCAP" (BCAP: Bacillus clausii Alkaline Protease) (see Fig. 15).
  • the present invention also provides a transformant transfected with the expression vector.
  • the constructed expression vector above was introduced into a host cell, resulting in a transformant expressing the protease of the invention as an active form.
  • the vector pT7-BCAP was introduced into E. coli to produce a transformant producing the protease of the invention as a stable active form (see Fig. 16) .
  • the present invention also provides a recombinant expression vector harboring the above gene for the insertion into Bacillus genome.
  • a recombinant expression vector was constructed to integrate an expression cassette represented by SEQ. ID. NO: 2 containing the gene encoding the protease of the invention linked operably to the promoter thereof into Bacillus clauii genome, and the constructed vector was named "pHPS-BCAP" (see Fig. 18) .
  • the recombinant expression vector for genomic insertion was introduced into the chromosome of Bacillus clausii to increase the copy number of BCAP in the chromosome.
  • the present invention also provides a transformant transfected with the recombinant expression vector for genomic insertion.
  • the expression vector pHPS-BCAP was introduced into E. coli JM109, which was deposited at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology on January 26, 2005 (Accession No: KCTC 10772BP) .
  • the present invention also provides the culture supernatant of the Bacillus clausii 1-52 or the transformant.
  • the culture supernatant herein means the liquid composition obtained by eliminating solid components containing cells from centrifugation after liquid-culture of the Bacillus clausii 1-52 or the transformant. If necessary, a partial purification is allowed, and endotoxin such as lipopolysaccharide can be eliminated by methods known in the art. For example, the methods described in Japanese Patent Publication No. 2000- 1897921 A 1, Japanese Patent No. 8193031 and U.S. Patent No. 5,760,177 can be used.
  • the present invention provides a method for purifying the protease of the invention isolated from the transformant or the culture supernatant thereof.
  • the purification method of the invention is basically same as the purification method for the protease of the invention from the Bacillus clauii 1-52, and modifications are allowed as long as they are accepted to those in the art.
  • the invention provides detergent compositions comprising one or more proteases of the invention isolated from the Bacillus clausii 1-52, the transformant or the culture supernatant thereof (referred conveniently as ' microorganism culture supernatant of the invention' hereinafter) , and methods of making and using these compositions.
  • the invention incorporates all methods of making and using detergent compositions, see, e.g., U.S. Patent Nos . 6,413,928; 6,399,561; 6,365,561 and 6,380,147.
  • the detergent compositions can be a one and two part aqueous composition, a non-aqueous liquid composition, a cast solid, a granular form, a particulate form, a compressed tablet, a gel and/or a paste and a slurry form.
  • the proteases of the invention can also be used as a detergent additive product in a solid or a liquid form. Such additive products are intended to supplement or boost the performance of conventional detergent compositions and can be added at any stage of the cleaning process.
  • the invention also provides methods capable of removing gross food wastes, films of food waste sludge and other minor food compositions using these detergent compositions.
  • Proteases of the invention can facilitate the removal of stains by means of catalytic hydrolysis of proteins.
  • the protease of the invention can be used in dishwashing detergents and/or in textile laundering detergents.
  • the actual active enzyme content depends upon the method for manufacturing of a detergent composition and is not critical, assuming the detergent solution has the desired enzymatic activity.
  • the amount of a protease present in the final solution ranges from about 0.001 mg to 0.5 mg per gram of the detergent composition.
  • the particular enzyme chosen for use in the process and products of the present invention depends upon the conditions of final utility, including the physical product form, pH and temperature to be used, and type of wastes to be degraded or altered.
  • the enzyme can be chosen to provide optimum activity and stability for any given set of utility conditions.
  • the proteases of the present invention are active in the pH ranges of from about 7.0 to about 12.0 and in the temperature range of from about 10 °C to about 75 ° C
  • the detergents of the invention can comprise cationic, semi-polar nonionic or zwitterionic surfactants; or, mixtures thereof.
  • Proteases of the invention can be formulated into powdered and liquid detergents having pH between 7.0 and 12.0 at levels of about 0.01% to about 5% (preferably 0.1% to 0.5%) by weight.
  • the microorganism culture supernatant of the invention is dried and powdered by freeze-drying or spray-drying, and then formulated into a powdered detergent having pH between 7.0 and 12.0 at levels of about 0.1% to about 10% by weight.
  • the supernatant can be formulated into a liquid detergent having pH between 7.0 and 12.0 at levels of about 0.1% to about 20% by volume as a liquid form without being dried or concentrated.
  • detergent compositions can also include other enzymes such as proteases, cellulases, lipases or endoglycosidases, endo- beta-1, 4-glucanases, beta-glucanases, endo-beta-1, 3 (4 ) - glucanases, cutinases, peroxidases, laccases, amylases, glucoamylases, pectinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases, mannanases, xyloglucanases, xylanases, pectin acetyl esterases, rhamnogalacturonan acetyl esterases, polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases, pectin methylesterases, cellobiohydrolase
  • proteases of the invention to conventional cleaning compositions does not create any special use limitation.
  • any temperature and pH suitable for the detergent is also suitable for the compositions of the invention as long as the enzyme is active at or tolerant of the pH and/or temperature of the intended use.
  • the proteases of the invention can be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers .
  • the present invention provides cleaning compositions including detergent compositions for cleaning hard surfaces, detergent compositions for cleaning fabrics, dishwashing compositions, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning solutions.
  • the invention provides a method for washing an object comprising contacting the object with the culture supernatant and/or the protease of the invention under conditions sufficient for washing.
  • the culture supernatant and/or the protease of the invention may be included as a detergent additive.
  • the detergent composition of the invention may, for example, be formulated as a hand or machine laundry detergent composition comprising the culture supernatant and/or the protease of the invention.
  • a laundry additive suitable for pre-treatment of stained fabrics can comprise the culture supernatant and/or the protease of the invention.
  • a fabric softener composition can comprise the culture supernatant and/or the protease of the invention.
  • the culture supernatant and/or the protease of the invention can be formulated as a detergent composition for use in general household hard surface cleaning operations .
  • detergent additives and detergent compositions of the invention may comprise one or more other enzymes such as a protease, a lipase, a cutinase, another protease, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a lactase, and/or a peroxidase (see also, above) .
  • the properties of the culture supernatant and/or the protease of the invention are chosen to be compatible with the selected detergent (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.) and the culture supernatant and/ ⁇ r the protease is present in effective amounts.
  • the culture supernatant and/or the protease of the invention is used to remove malodorous materials from fabrics.
  • Various detergent compositions and methods for making them that can be used in practicing the invention are described in, e.g., U.S. Patent Nos . 6,333,301; 6,329,333; 6,326,341; 6,297,038; 6,309,871; 6,204,232; 6,197,070 and 5,856,164.
  • the culture supernatant and/or the protease of the invention can comprise both a surfactant and a builder compound. They can additionally comprise one or more detergent components, e.g., organic polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime- soap dispersants, soil suspension and anti-redeposition agents and corrosion inhibitors. Laundry compositions of the invention can also contain softening agents, as additional detergent components. Such compositions containing carbohydrase can provide fabric cleaning, stain removal, whiteness maintenance, softening, color appearance, dye transfer inhibition and sanitization when formulated as laundry detergent compositions.
  • the density of the laundry detergent compositions of the invention can range from about 200 to 1500 g/t, or, about 400 to 1200 g/t, or, about 500 to 950 q/l, or, 600 to
  • the "compact" form of laundry detergent compositions of the invention is best reflected by density and, in terms of composition, by the amount of inorganic filler salt.
  • Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, the filler salts are present in substantial amounts, typically 17% to 35% by weight of the total composition. "
  • the inorganic filler salts can be selected from the alkali and alkaline-earth-metal salts of sulphates and chlorides, e.g., sodium sulphate.
  • Liquid detergent compositions of the invention can also be in a "concentrated" form.
  • the liquid detergent compositions can contain a lower amount of water, compared to conventional liquid detergents.
  • the water content of the concentrated liquid detergent is less than 40%, or, less than 30%, or, less than 20% by weight of the detergent composition.
  • Detergent compounds of the invention can comprise formulations as described in WO 97/01629.
  • the culture supernatant and/or the protease of the invention can be useful in formulating various cleaning compositions.
  • suitable surfactants including nonionic, anionic, cationic, or zwitterionic detergents, can be used, e.g., as disclosed in U.S. Patent Nos . 4,404,128; 4,261,868; 5,204,015.
  • proteases can be used, for example, in bar or liquid soap applications, dish care formulations, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, textile applications, as fusion-cleavage enzymes in protein production, and the like.
  • protease of the invention can be formulated into known powdered and liquid detergents having pH between 7.0 and 12.0 at levels of about 0.01 to about 5% (for example, about 0.1% to 0.5%) by weight.
  • cleaning compositions can also include other enzymes such as known proteases, amylases, cellulases, lipases or endoglycosidases, as well as builders and stabilizers .
  • the culture supernatant and/or the protease of the invention have numerous applications in food processing industry.
  • the culture supernatant and/or the protease of the invention are used to improve the extraction of oil from oil-rich plant material, e.g., oil-rich seeds, for example, soybean oil from soybeans, olive oil from olives, rapeseed oil from rapeseed and/or sunflower oil from sunflower seeds.
  • the culture supernatant and/or the protease of the invention can be used for separation of components of plant cell materials.
  • the culture supernatant and/or the protease of the invention can be used in the separation of protein-rich material (e.g., plant cells) into components, e.g., sucrose from sugar beet or starch or sugars from potato, pulp or hull fractions.
  • the culture supernatant and/or the protease of the invention can be used to separate protein-rich or oil-rich crops into valuable protein and oil and hull fractions.
  • the separation process may be performed by use of methods known in the art.
  • the culture supernatant and/or the protease of the invention can be used in the preparation of fruit or vegetable juices, syrups, extracts and the like to increase yield.
  • the culture supernatant and/or the protease of the invention can be used in the enzymatic treatment (e.g., hydrolysis of proteins) of various plant cell wall-derived materials or waste materials, e.g., from wine or juice production, or agricultural residues such as vegetable hulls, bean hulls, sugar beet pulp, olive pulp, potato pulp, and the like.
  • the culture supernatant and/or the protease of the invention can be used to modify the consistency and appearance of processed fruit or vegetables.
  • the culture supernatant and/or the protease of the invention can be used to treat plant materials to facilitate processing of plant materials, including foods, facilitate purification or extraction of plant components.
  • the culture supernatant and/or the protease of the invention can be used to improve feed value, decrease the water binding capacity, improve the degradability in waste water plants and/or improve the conversion of a plant material to ensilage, and the like.
  • the invention provides methods for treating animal feeds and foods and food or feed additives using the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention.
  • the animals include mammals (e.g., humans), birds, fish and the like.
  • the invention provides animal feeds, foods, and additives comprising the Bacillus clausii 1-52 strain, the transformant , the culture supernatant thereof and/or the protease isolated therefrom of the invention.
  • treating animal feeds, foods and additives using the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention can help in the availability of nutrients, e.g., starch, in the animal feed or additive.
  • nutrients e.g., starch
  • the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention make nutrients more accessible to other endogenous or exogenous enzymes.
  • the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention can also simply cause the release of readily digestible and easily absorbed nutrients and sugars .
  • the Bacillus clausii 1-52 strain, the transformant , the culture supernatant thereof and/or the protease isolated therefrom of the invention, in the modification of animal feed or a food, can process the food or feed either in vitro (by modifying components of the feed or food) or in vivo.
  • the Bacillus clausii 1-52 strain, the transformant , the culture supernatant thereof and/or the protease isolated therefrom of the invention can be added to animal feed or food compositions containing high amounts of proteins, e.g., feed or food containing plant proteinous materials from soy bean, rape seed, lupin and the like.
  • the protease isolated therefrom of the invention to the feed or food, it significantly improves the in vivo break-down of plant proteinous materials, whereby a better utilization of the plant nutrients by the animal (e.g., human) is achieved.
  • the growth rate and/or feed conversion ratio i.e. the weight of ingested feed relative to weight gain
  • a partially or indigestible galactan-comprising protein is fully or partially degraded by the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention, e.g. in combination with another enzyme, e.g., beta-galactosidase, to peptides and galactose and/or galactooligomers .
  • beta-galactosidase e.g., beta-galactosidase
  • the Bacillus clausii 1-52 strain, the transformant , the culture supernatant thereof and/or the protease isolated therefrom of the invention can contribute to the available energy of the feed or food.
  • the protease of the invention can improve the digestibility and uptake of proteins from the feed or food constituents containing carbohydrate and non- carbohydrate .
  • Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention can be supplied by expressing the enzymes directly in transgenic feed crops (as, e.g., transgenic plants, seeds and the like) , such as corn, soy bean, rape seed, lupin and the like.
  • transgenic feed crops as, e.g., transgenic plants, seeds and the like
  • transgenic feed crops as, soy bean, rape seed, lupin and the like.
  • the nucleic acid is expressed such that the protease of the invention is produced in recoverable quantities.
  • the protease can be recovered from any plant or plant part.
  • the plant or plant part containing the recombinant polypeptide can be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.
  • the invention provides methods of treating fibers and fabrics using the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention.
  • the culture supernatant and/or the protease of the invention can be used in any fiber- or fabric-treating method, which are well known in the art, see, e.g., U.S. Patent Nos . 6,261,828; 6,077,316; 6,024,766; 6,021,536; 6,017,751; 5,980,581; U.S. Patent Publication No. 2002/0142438A1.
  • proteases of the invention can be used in fiber and/or fabric desizing.
  • the feel and appearance of a fabric is improved by a method comprising contacting the fabric with the culture supernatant and/or the protease of the invention in a solution.
  • the fabric is treated with the solution under pressure.
  • the culture supernatant and/or the protease of the invention can be used in the removal of stains.
  • the culture supernatant and/or the protease of the invention is applied during or after the weaving of textiles, or during the desizing stage, or one or more additional fabric processing steps.
  • the threads are exposed to considerable mechanical strain.
  • warp yams Prior to weaving on mechanical looms, warp yams are often coated with sizing starch or starch derivatives in order to increase their tensile strength and to prevent breaking.
  • the culture supernatant and/or the protease of the invention can be applied to remove these sizing starch or starch derivatives.
  • a fabric can proceed to a desizing stage. This can be followed by one or more additional fabric processing steps. Desizing is the act of removing "size" from textiles.
  • the invention provides a method of desizing comprising enzymatic treatment of the "size" by the action of the culture supernatant and/or the protease of the invention.
  • the culture supernatant and/or the protease of the invention can be used to desize fabrics, including cotton- containing fabrics, as detergent additives, e.g., in aqueous compositions.
  • the invention provides methods for producing a stonewashed look on indigo-dyed denim fabric and garments.
  • the fabric can be cut and sewn into clothes or garments. These can be finished before or after the treatment.
  • different enzymatic finishing methods have been developed.
  • the finishing of denim garment normally is initiated with an enzymatic desizing step, during which garments are subjected to the action of amylolytic enzymes in order to provide softness to the fabric and make the cotton more accessible to the subsequent enzymatic finishing steps.
  • the invention provides methods of finishing denim garments (e.g., a "bio- stoning process"), enzymatic desizing and providing softness to fabrics using the culture supernatant and/or the protease of the invention.
  • the invention provides methods for quickly softening denim garments in a desizing and/or finishing process.
  • an alkaline and thermostable amylase and a protease can be combined in a single bath for desizing and bioscouring.
  • exemplary application conditions for desizing and bioscouring are about pH 8.5 to 10.0 and temperatures of about 40 ° Cand up.
  • low enzyme dosages e.g., about 100 grams (g) per ton of cotton, and short reaction times, e.g., about 15 minutes, can be used to obtain efficient desizing and scouring without added calcium.
  • an alkaline and thermostable amylase and a protease are combined in a single bath desizing and bioscouring.
  • desizing and bioscouring can be between about pH 8.5 to pH 10.0 and temperatures at about 40 ° Cand up.
  • Low enzyme dosages e.g., about 100 g per a ton of cotton
  • short reaction times e.g., about 15 minutes
  • the culture supernatant and/or the protease of the invention can be used in combination with other carbohydrate degrading enzymes, e.g., cellulase, arabinanase, xyloglucanase, pectinase, and the like, for the preparation of fibers or for cleaning of fibers. These can be used in combination with detergents.
  • the culture supernatant and/or the protease of the invention can be used in treatments to prevent the graying of a textile.
  • the culture supernatant and/or the protease of the invention can be used to treat any cellulosic material, including fibers (e.g., fibers from cotton, hemp, flax or linen), sewn and unsewn fabrics, e.g., knits, wovens, denims, yarns, and toweling, made from cotton, cotton blends or natural or manmade cellulosics (e.g. originating from xylan-containing cellulose fibers such as from wood pulp) or blends thereof.
  • suitable cellulosic material including fibers (e.g., fibers from cotton, hemp, flax or linen), sewn and unsewn fabrics, e.g., knits, wovens, denims, yarns, and toweling, made from cotton, cotton blends or natural or manmade cellulosics (e.g. originating from xylan-containing cellulose fibers such as from wood pulp) or blends thereof.
  • blends are blends of cotton or rayon
  • polyamide fibers acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers) , and cellulose-containing fibers (e.g. rayon/viscose, ramie, hemp, flax/linen, jute, cellulose acetate fibers, lyocell).
  • cellulose-containing fibers e.g. rayon/viscose, ramie, hemp, flax/linen, jute, cellulose acetate fibers, lyocell.
  • the textile treating processes of the invention can be used in conjunction with other textile treatments, e.g., scouring and bleaching.
  • Scouring is the removal of non-cellulosic material from the cotton fiber, e.g., the cuticle (mainly consisting of waxes) and primary cell wall (mainly consisting of pectin, protein and xyloglucan) .
  • a proper wax removal is necessary for obtaining a high wettability. This is needed for dyeing.
  • Removal of the primary cell walls by the processes of the invention improves wax removal and ensures a more even dyeing. Treating textiles with the processes of the invention can improve whiteness in the bleaching process.
  • Bleaching comprises oxidizing the textile. Bleaching typically involves use of hydrogen peroxide as the oxidizing agent in order to obtain either a fully bleached (white) fabric or to ensure a clean shade of the dye.
  • the invention also provides methods of treating leather using the culture supernatant and/or the protease of the invention.
  • the culture supernatant and/or the protease of the invention can be used in any leather-treating method, which are well known in the art, see, e.g., International Patent Publication No. WO03/008650; Australian Patent No. 696441; U.S. Patent Publication No. 2003/061666.
  • the conventional leather processing is accomplished by the procedure of soaking, liming & deliming and bating.
  • the microorganism culture supernatant and/or the protease of the invention can be treated independently or added to an additive to increase the stability of an enzyme or to be mixed with other materials added to each process. It is preferred to add the protease of the invention for each process, soaking, liming & deliming and bating, at levels of about 0.1% - 15% by weight. And the microorganism culture supernatant of the invention is preferably added to each process, soaking, liming & deliming and bating, at levels of about 0.1% - 50% by volumn .
  • microorganism culture supernatant and/or the protease of the invention enable pro-environmental leather processing because organic and inorganic leather reagents can be significantly reduced thereby.
  • microorganism culture supernatant and/or the protease of the invention can be used not only for the leather processing but also for the elimination of the outer skin, hair and soluble protein left over during the procedure .
  • Soaking is the process that a rawhide salt-treated right after slaughter is transported to a leather processing company where the rawhide is soaked in water in a paddle or a drum for a long while to absorb water enough to eliminate impurities and to recover the protein tissues as soft as before slaughter, and at this time the treatment of the microorganism culture supernatant and/or the protease of the invention helps the elimination of impurities, soluble protein, hair, etc, from the rawhide. Besides, the treatment of the culture supernatant and/or the protease of the invention makes the protein tissues of the leather more soft and the liming & deliming process easy.
  • the waste water produced by liming & deliming affects COD most.
  • the microorganism culture supernatant and/or the protease of the invention decompose cells of stratum germinativum or base cells of hair root, which is based on the principal that the protease or the supernatant decomposes medulla but not cortex of a hair.
  • the supernatant or the protease can eliminate hair roots and follicles, leading to swelling hair poaches to pluck the hairs, so that lime or other reagents can be easily invaded and as a result the doses of those reagents are reduced.
  • the microorganism culture supernatant and/or the protease of the invention eliminates unnecessary proteins included in the rawhide and opens the tissues of the leather, increasing the penetration and coherence of provided reagents after bating. So, the required amount of reagents can be reduced and as a result, the increase of COD or BOD owing to the excessive reagents can be moderated.
  • the treatment of the microorganism culture supernatant and/or the protease of the invention also contribute to the production of very soft leather and thus improve the quality of the final product as well as decrease the waste disposal problem by reducing the dose of dye by eliminating impurities and pollutant and thereby improves the leveling of color.
  • the present invention provides methods of treating waste produced during the leather processing using the microorganism culture supernatant and/or the protease of the invention.
  • the microorganism culture supernatant and/or the protease of the invention treat waste produced during the procedure of soaking, liming & deliming, bating and finishing.
  • the waste can be waste water or a solid waste, and the waste can be re-cycled.
  • microorganism culture supernatant and/or the protease of the invention used for the leather processing can be treated independently or added to an additive to increase the stability of an enzyme or to another material added to each process.
  • microorganism culture supernatant and/or the protease of the invention together with one of lipases and amylases to treat the waste produced during leather processing.
  • the microorganism culture supernatant and/or the protease of the invention can be treated independently to the waste water and a solid waste produced during soaking and liming
  • microorganism culture supernatant and/or the protease of the invention can be applied to such solid waste as fleshing scrap, trimming scrap and fur produced during soaking and liming and pelt scrap produced during bating as well as leather pieces produced during the finishing after drying.
  • one or more enzymes containing a lipase or a glucosidase can be added in addition to the microorganism culture supernatant and/or the protease of the invention.
  • the solid waste of pelt scrap resulted from the bating takes approximately 40% of total waste materials from leather processing.
  • the pelt scrap is composed of 4.0% lipid, 1.5% calcium, 5.5% ash, 50 - 55% water and 35 - 40% protein.
  • the protease of the invention retains its activity even in the presence of a metal ion. In the pelt scrap, numbers of metal ions such as calcium ion are included, so the protease of the invention can be very effective to decompose the protein of the pelt scrap.
  • the microorganism of the invention is highly capable of being growing in the presence of even small amount of carbon source and nitrogen source, and can produce a protease during growth, by which solid leather waste can be decomposed. If the solid waste is a protein, the microorganism culture supernatant and/or the protease of the invention hydrolyze the protein waste into peptides, which will be recycled to produce food, cosmetics and industrial products.
  • the microorganism culture supernatant and/or the protease of the invention is also effective to decompose proteins included in the waste water containing salt produced during soaking and liming & deliming, by which the proteins can also be recycled.
  • the microorganism culture supernatant and/or the protease of the invention can prevent environmental pollution and enables the re-use of solid waste produced from the processing as a secondary product.
  • the invention provides methods of treating paper and paper pulp using the Bacillus clausii 1-52 strain, the transformant , the culture supernatant thereof and/or the protease isolated therefrom of the invention.
  • Bacillus clausii 1-52 strain, the transformant , the culture supernatant thereof and/or the protease isolated therefrom of the invention can be used in any paper-or pulp-treating method, which are well known in the art, see, e.g., U.S. Patent Nos . 6,241,849; 6,066,233; 5,582,681.
  • the invention provides a method for deinking and decolorizing a printed paper containing a dye, comprising pulping a printed paper to obtain a pulp slurry, and dislodging an ink from the pulp slurry in the presence of the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention (other enzymes can also be added) .
  • the invention provides a method for enhancing the freeness of pulp, e.g., pulp made from secondary fiber, by adding an enzymatic mixture comprising the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention (can also include other enzymes, e.g., pectate lyase, cellulase, amylase or glucoamylase enzymes) to the pulp and treating under conditions to cause a reaction to produce an enzymatically treated pulp.
  • the freeness of the enzymatically treated pulp is increased from the initial freeness of the secondary fiber pulp without a loss in brightness .
  • the invention provides chemical and enzymatic deinking processes using the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention.
  • the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention can be used in combination with cellulases, pectate lyases or other enzymes.
  • the paper can be treated by the following three processes: 1) disintegration in the presence of the Bacillus clausii 1-52 strain, the transformant , the culture supernatant thereof and/or the protease isolated therefrom of the invention, 2) disintegration with a deinking chemical and the Bacillus clausii 1-52 strain, the transformant , the culture supernatant thereof and/or the protease isolated therefrom of the invention, and/or 3) disintegration after soaking with the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention.
  • the recycled paper treated with the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention can have a higher brightness due to removal of toner particles as compared to the paper treated with just cellulase. While the invention is not limited by any particular mechanism, the effect of the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention may be due to its behavior as surface-active agents in pulp suspension.
  • the invention provides methods of decomposing organic wastes using the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention.
  • the protease To use a protease industrially, it is required for the protease to be very stable to maintain its activity under severe environmental conditions which are very vulnerable, compared with those in vivo. That is, the severe environmental conditions herein include severe exposure on pH and heavy metals and frequent changes of oxidation-reduction, which are generally believed to affect the enzyme activity significantly. Thus, when an enzyme is out of proper ranges of those conditions, it loses its enzyme activity. Therefore, in order to use a protease industrially, the protease needs to maintain its activity under extremely unstable physical and chemical conditions.
  • the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention can be used in a variety of other industrial applications, e.g., in waste treatment.
  • the invention provides a solid waste digestion process using the Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention.
  • the methods can comprise reducing the mass and volume of substantially untreated solid waste.
  • the Bacillus clausii 1-52 strain and the transformant can be sterilized by autoclaving.
  • Solid waste can be treated with an enzymatic digestive process in the presence of an enzymatic solution (including proteases of the invention) at a controlled temperature. This results in a reaction without appreciable bacterial fermentation from added microorganisms.
  • the solid waste is converted into a liquefied waste and any residual solid waste.
  • the resulting liquefied waste can be separated from said any- residual solidified waste. See e.g., U.S. Patent No. 5,709,796.
  • Bacillus clausii 1-52 strain, the transformant, the culture supernatant thereof and/or the protease isolated therefrom of the invention can be used in the animal rendering industry, to e.g., get rid of feathers, e.g., as described by Yamamura (2002) Biochem. Biophys. Res. Com. 294: 1138-1143.
  • the invention provides oral care products comprising the culture supernatant and/or the protease of the invention.
  • oral care products include toothpastes, dental creams, gels or tooth powders, odontics, mouth washes, pre- or post brushing rinse formulations, chewing gums, lozenges, or candy. See, e.g., U.S. Patent No. 6,264,925.
  • the invention provides methods of brewing (e.g., fermenting) beer comprising the culture supernatant and/or the protease of the invention.
  • starch-containing raw materials are disintegrated and processed to form malt.
  • the culture supernatant and/or the protease of the invention are used at any point in the fermentation process.
  • the culture supernatant and/or the protease of the invention can be used in the processing of barley malt.
  • the major raw material of beer brewing is barley malt. This can be a three-stage process. First, the barley grain can be steeped to increase water content, e.g., to around about 40%.
  • the grain can be germinated by incubation at 15 to 25 "C for 3 to 6 days when enzyme synthesis is stimulated under the control of gibberellins .
  • a culture supernatant and/or a protease of the invention are added at this (or any other) stage of the process.
  • the action of proteases results in an increase in fermentable reducing sugars. This can be expressed as the diastatic power, DP, which can rise from around 80 to 190 degree in 5 days at 12 ° C
  • the culture supernatant and/or the protease of the invention can be used in any beer or alcoholic beverage producing process, as described, e.g., in U.S. Patent Nos .
  • proteases of the invention can be used for cell isolation from tissue for cellular therapies in the same manner that collagenases . Additionally, proteases of the invention can be used as antimicrobial agents, due to their bacteriolytic properties, as described, e.g., in Li, S. et . al . Bacteriolytic Activity and Specificity of Achromobacter b-Lytic Protease, J. Biochem. 124, 332-339 (1998) .
  • Proteases of the invention can also be used therapeutically to cleave and destroy specific proteins.
  • Potential targets include toxin proteins, such as Anthrax,
  • Clostridium botulinum, Ricin, and essential viral or cancer cell proteins Clostridium botulinum, Ricin, and essential viral or cancer cell proteins.
  • Proteases of the invention can also be used in disinfectants, as described, e.g., in J. Gen Microbiol (1991) 137 (5): 1145-1153; Science (2001) 249: 2170-2172.
  • proteases of the invention include lipoma removal, wound debraidment and scar prevention (collagenases) , debriding chronic dermal ulcers and severely burned areas.
  • Proteases of the invention can be used to in sterile enzymatic debriding compositions, e.g., ointments, in one aspect, containing about 250 collagenase units per gram.
  • White petrolatum USP (United States Pharmacopoeia) grade can be a carrier.
  • proteases of the invention can be used in indications similar to Santyl Ointment (BTC, Lynbrook, NY) .
  • Proteases of the invention can also be used in alginate dressings, antimicrobial barrier dressings, burn dressings, compression bandages, diagnostic tools, gel dressings, hydro-selective dressings, hydrocellular (foam) dressings, hydrocolloid dressings, I. V dressings, incise drapes, low adherent dressings, odor absorbing dressings, paste bandages, post operative dressings, scar management, skin care, transparent film dressings and/or wound closure.
  • Proteases of the invention can be used in wound cleansing, wound bed preparation, to treat pressure ulcers, leg ulcers, burns, diabetic foot ulcers, scars, I. V. fixation, surgical wounds and minor wounds .
  • proteases of the invention can be used in proteomics and lab work in general.
  • proteases can be used in the same manner as protein restriction enzymes.
  • the culture supernatant and/or the protease of the invention can be used for peptide synthesis, in the leather industry, e.g., for hide processing, e.g., in hair removal and/or bating, for waste management, e.g., removal of hair from drains, in the photography industry, e.g., for silver recovery from the used X-ray film, in the medical industry, e.g., as discussed above, e.g., for treatment of burns, wounds, carbuncles, furuncles and deep abscesses or to dissolve blood clots by dissolving fibrin, for silk degumming.
  • the culture supernatant and/or the protease of the invention can be used as flavor enhancers in, for example, cheese and pet food, as described, e.g., in Poramer, K., Investigating the impact of enzymes on pet food palatability, Petfood Industry, May 2002, 10-11.
  • the culture supernatant and/or the protease of the invention can be used to increase starch yield from corn wet milling, as described, e.g., in Johnston, D. B., and Singh, V. Use of proteases to Reduce Steep Time and S02 requirements in a corn wet-milling process, Cereal Chem. 78 (4): 405-411.
  • the culture supernatant and/or the protease of the invention can be used in biodefense (e.g., destruction of spores or bacteria) .
  • biodefense e.g., destruction of spores or bacteria
  • Use of proteases in biodefense applications offers a significant benefit, in that they can be very rapidly developed against any currently unknown biological warfare agents of the future.
  • the culture supernatant or the protease of the invention can be used for decontamination of affected environments .
  • the culture supernatant and/or the protease of the invention can be used in biofilm degradation, in biomass conversion to ethanol, and/or in the personal care and cosmetics industry.
  • the culture supernatant and/or the protease of the invention can also be used to enhance enantioselectivity, as described, e.g., in Arisawa, A. et . al. Streptomyces Serine Protease (DHP-A) as a New Biocatalyst Capable of Forming Chiral Intermediates of 1, 4-Diohydropyridine Calcium Antagonists. Appl. Environ. Mircrobiol . 2002 Jun; 68 (6): 2716-2725; Haring, D. et . al.
  • the protease of the invention has a stability not to lose its enzyme activity under severe conditions such as alkaline environment, a surfactant and high temperature, and has a wide substrate specificity enabling the elimination of food and blood or body fluid. Therefore, the protease, the Bacillus clausii 1-52 strain and the transformant producing the protease, and the microorganism culture supernatant containing the same can be used as a detergent component with generally known synthetic surfactants including SDS, octylglucoside, Brij-35, Tritons, Tweens, LAS, AOS ( ⁇ -orephin sulfonate), etc, or with a natural surfactant.
  • synthetic surfactants including SDS, octylglucoside, Brij-35, Tritons, Tweens, LAS, AOS ( ⁇ -orephin sulfonate), etc, or with a natural surfactant.
  • the protease of the invention retains its activity under wide range of pH and oxidation-reduction condition, so that it can be widely used in various fields of processing food, feed, fabrics, natural leather processing, brewing, paper and pulp, and in the production of oral care products and medical applications, in research fields, and in waste treatment because it retains a high activity in the presence of a heavy metal.
  • the detergent composition containing the protease of the invention may be formulated by the conventional method for a laundry detergent or a contact lens detergent. And a processing method or a decomposing method using the protease is not limited. In fact, every method including the step of treating the protease of the invention, according to general manners well known to those in the art, can be included in the criteria of the present invention.
  • Example 1 Isolation of a strain producing a protease from the west coast mudflat
  • the cell culture supernatant was recovered to examine the protease activity. Particularly, 0.5 ni of a substrate solution prepared by dissolving Hammerstan casein (final concentration: 0.5%) in 0.1 M glycine-NaOH buffer (pH 11.0) was mixed well with 0.1 ml of a 100 - 1000 fold diluted enzyme solution, followed by reaction at 60 ° Cfor 10 minutes. Then, 0.5 mi of 10% trichloro acetic acid (TCA) was added thereto in order to terminate the reaction. The reaction solution was put in ice for 10 minutes to precipitate proteins remaining unreacted. Supernatant was obtained by centrifugation and tyrosine was quantified at 275 nm.
  • TCA trichloro acetic acid
  • 1 unit of a protease activity was defined as the amount of enzyme necessary for the production of 1 ⁇ g of tyrosine for one minute.
  • a strain showing the best protease activity was selected, which was confirmed to be a Gram- positive by the Gram-test.
  • the morphological and biochemical characteristics of the selected strain were also investigated.
  • the strain formed a spore and showed catalase and oxidase activities but did not show urease activity.
  • the strain also produced acid and gas by using a sugar such as glucose, fructose, galactose, mannose, maltose, mannitol, sorbitol and starch, and proteins such as casein and gelatin.
  • the morphological and biochemical characteristics of the strain are presented in Table 1. From a nucleotide sequencing, it was confirmed that the strain has 16S ribosomal RNA represented by SEQ. ID. NO: 1, and thereby the strain was further confirmed to be a Bacillus sp. Bacillus clausii strain.
  • the present inventors named the strain selected above as "Bacillus clausii 1-52" and deposited at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB, #52, Oun- dong, Yusong-ku, Taejon, Korea), on Jun 14, 2002 (Accession No: KCTC 10277BP) .
  • KCTC Korean Collection for Type Cultures
  • Example 2 Investigation on factors affecting the protease activity ⁇ 2-l> The effect of pH on the protease activity The present inventors investigated whether pH of a medium could affect the protease activity during the culture of Bacillus clausii 1-52. Particularly, a basal medium was composed of the components listed in Table 2, to which sodium carbonate was added (final concentration: 0- 1%). Seed culture was performed for 20 hours. After inoculating the inoculum by 2%, shaking culture was performed for 40 hours at 37 "C The culture solution was centrifuged to obtain supernatant. The protease activity of the supernatant was measured by the same manner as described in Example 1.
  • the present inventors investigated the effect of an organic nitrogen source on the production of the protease in Bacillus clausii 1-52.
  • a basal medium having the composition of Table 2 was supplemented with each nitrogen source (1%) and sodium carbonate (0.5%), to which the inoculum pre-cultured for 20 hours was inoculated, followed by shaking-culture for 40 hours at 37 ° C
  • the culture solution was centrifuged to obtain a supernatant, and the protease activity of the supernatant was measured by the same manner as described in Example 1.
  • the present inventors investigated the effect of an inorganic nitrogen source on the production of the protease in the Bacillus cla ⁇ sii 1-52.
  • a medium was prepared by adding each inorganic nitrogen source by 0.5% to the basal medium of Table 3 containing casein (1%) and sodium carbonate (0.5%).
  • the inoculum cultured for 20 hours was inoculated in the medium by 2%, followed by shaking culture for 40 hours at 37 ° C
  • the culture solution was centrifuged to obtain a supernatant, and the protease activity of the supernatant was measured by the same manner as described in Example 1.
  • the protease production was increased by the addition of an inorganic nitrogen source except a nitrate.
  • an inorganic nitrogen source except a nitrate.
  • ammonium sulfate was added, the production of the protease was increased most (approximately up to 20%) .
  • the concentration of an inorganic ammonium salt is preferably in the range between 0.1 and 5% (Table 5) .
  • Table 5 Enzyme activities according to inoranic nitrogen
  • the present inventors investigated the effect of a carbon source on the protease activity in Bacillus clausii 1-52.
  • a medium was prepared by adding each carbohydrate by 0.5% to the basal medium containing casein (1%) and sodium carbonate (0.5%), to which the inoculum cultured for 20 hours was inoculated by 2%, followed by shaking culture for 40 hours at 37 ° C
  • the culture solution was centrifuged to obtain supernatant, and the protease activity of the supernatant was measured by the same manner as described in Example 1.
  • the present inventors produced a protease from the Bacillus clausii 1-52 strain selected in Example 1.
  • a medium for the culture of the Bacillus clausii 1-52 was prepared as a liquid medium composed of 1.5% soybean meal, 1% casein, 0.4% potassium phosphate, 0.4% sodium citrate, 0.5 mM magnesium sulfate, 0.5% wheat flour, and autoclaved for 20 minutes at 121 "G
  • 40% sodium carbonate solution and 50% liquid maltose solution were autoclaved separately for 20 minutes at 121 ° C, and the sodium carbonate solution and the liquid maltose solution were added to the medium at a concentration of 0.5% and 1.5% respectively, before a culture.
  • an inoculum from a seed culture using a medium having the same composition as the above medium, which weas cultured in a flask for 24 hours at 37 ° C was inoculated by 2%, followed by culture for 48 hours at 37 ° Cin a 5 L fermentor (NBS, USA) .
  • the airflow speed was 1 vvm and the stirring speed was 400 rpm.
  • Centrifugation was performed at 12,000 g for 20 minutes and supernatant was recovered to measure the protease activity by the same manner as described in Example 1.
  • the culture solution was centrifuged at 12,000 g for 20 minutes to obtain supernatant, and the protease activity of the supernatant was measured by the same manner as described in Example 1.
  • the protease activity was 36,560 U per 1 ml of the Bacillus clausii 1-52 culture supernatant.
  • the protease activity was 52,760 U per 1 m# of the Bacillus clausii 1-52 culture supernatant.
  • the concentration of wheat flour was adjusted to 1% and the concentration of liquid maltose was adjusted to 2%, followed by culture for 48 hours. A supernatant was obtained to examine the protease activity.
  • the protease activity was 80,295 U per 1 mi of the Bacillus clausii 1-52 culture supernatant.
  • Example 3-3 At that time, the airflow speed was 1.5 vvm and the stirring speed was 600 rpm. A supernatant was obtained to examine the protease activity.
  • the protease activity was 123,520 U per liil of the Bacillus clausii 1-52 culture supernatant.
  • Example 4 Separation and purification of the protease from Bacillus clausii 1-52
  • the microorganism was cultured for 48 hours in a medium having the same composition as that of Example 3-4 and the obtained supernatant was used as a crude enzyme solution.
  • Diaion HP20 synthetic absorbent was added to the crude enzyme solution, followed by absorption for 16 hours with stirring. The absorbent was recovered by filtering. The absorbent was then washed with distilled water and active fractions were eluted by sodium phosphate buffer (pH 7,5) containing 30% acetone.
  • Step 2 Phenyl-Sepharose hydrophobic chromatography Phenyl-Sepharose column (5x8 cm, Amersham Pharmarcia) was equilibrated with 0.1 M sodium phosphate buffer (pH 7.5) containing 1 M ammonium sulfate, and then the eluent was loaded to the column. The column was washed until OD 2S o reached almost 0, and active fractions were eluted by 10 mM sodium phosphate buffer (pH 7.5) .
  • DEAE-Sepharose column (2.5x10 cm, Amersham Pharmarcia) was equilibrated with 10 mM sodium phosphate buffer (pH 7.5), then the fraction obtained from the Phenyl-Sepharose column chromatography was loaded onto the DEAE-Sepharose column. The column was washed with the same buffer until OD 2S o reached almost 0.
  • Step 4 CM-Sepharose ion exchange chromatography CM-Sepharose column (2.5x5 cm, Amersham Pharmarcia) was equilibrated with 50 mM sodium phosphate buffer (pH 7.0), then the fraction obtained from the DEAE-Ssepharose column chromatography was loaded onto the CM-Ssepharose column. The column was washed with the same buffer until OD 2 8o reached almost 0. Active fractions were eluted by linear gradient using 100 mi of 50 mM sodium phosphate buffer (pH 7.0) and 100 m ⁇ of 50 mM sodium phosphate buffer (pH 7.0) containing 50 mM NaCl. And, the activity of the fraction was measured. (Step 5) Protease purification by SDS-PAGE and molecular weight determination
  • the enzyme was purified and 5 ⁇ g (20 ⁇ i) of the purified enzyme was electrophoresed in SDS-PAG composed of 15% separating gel and 4% stacking gel. After electrophoresis, the separating gel was loaded on a cassette which was covered with a fiber pad and a filter paper soaked in protein transfer buffer (10 mM CAPS/10% methanol, pH 11.0), and then, PVDF (polyvinylidene difluoride) membrane soaked for one hour in 100% methanol was loaded onto the cassette. Finally, a filter paper and a fiber pad were loaded over again for the close adhesion of the gel and the membrane.
  • protein transfer buffer (10 mM CAPS/10% methanol, pH 11.0
  • Example 5 Optimum conditions for the protease activity ⁇ 5-l> Optimum pH for the protease activity
  • the present inventors investigated optimum pH for the protease activity. Particularly, casein, a substrate, was prepared with buffers having different pH. Each protease activity under different pH conditions was measured as follows: the activity was measured using 0.1 M sodium phosphate buffer under pH 7.0 - 7.5, using 0.1 M Tris-HCl buffer under pH 8.0 - 9.5 and using 0.1 M glycine-NaOH buffer under pH 10.0 - pH 12.0.
  • the protease showed high enzyme activity in pH range between 8.0 and 12.0 and particularly, pH 11.0 was the optimum pH of the protease activity (Fig. 2) .
  • the present inventors investigated the optimum temperature for the protease activity.
  • casein a substrate
  • 0.1 M glycine-NaOH buffer pH 11.0
  • the optimum temperature for the protease activity was confirmed to be 60 - 65 ° C (Fig. 3) .
  • the inventors measured the enzyme activity after treating the enzyme with an anionic surfactant.
  • the protease activity was measured after treating SDS (sodium dodecyl sulfate) with different concentrations such as 1%, 2.5%, 5% and 10%.
  • SDS sodium dodecyl sulfate
  • concentrations such as 1%, 2.5%, 5% and 10%.
  • the protease activity was maintained when 5% SDS was treated thereto for three days, indicating that the enzyme is very stable against SDS (Fig. 4) .
  • the present inventors measured the time-dependent enzyme activity after treating the enzyme with oxidants such as hydrogen peroxide and sodium perborate.
  • oxidants such as hydrogen peroxide and sodium perborate.
  • the protease activity was measured after treating with hydrogen peroxide and sodium perorate, known as strong oxidants, with different concentrations such as 0.1%, 0.5%, 1% and 2.5%.
  • the protease of the invention did not lose the activity after being treated with 2.5% of hydrogen peroxide and sodium perborate for three days, proving the protease to be a very stable enzyme (Fig. 5 and 6) .
  • the present inventors measured the enzyme activity after treating the protein with various organic solvents with 10% or 20%. Particularly, the protease was treated with 10% or 20% of acetone, acetonitrile, ethanol, isopropanol, methanol, dimethylformamide or dimethylsulfoxide, followed by measuring the time-dependent enzyme activity.
  • the protease retained the activity after treating with 20% of various organic solvents for three days, proving the protease to be a very stable enzyme against an organic solvent (Table 7) .
  • the result also suggests that the protease of the invention can be effectively used for the biosynthesis of an oligopeptide.
  • the concentration of NaCl in the reaction solution containing the protease was adjusted differently and then the protease activity was measured.
  • Inoculum precultured for 24 hours was inoculated on the culture medium (soybean meal 2%, wheat flour 1%, liquid maltose 2%, sodium citrate 0.5%, potassium phosphate 0.5%, magnesium sulfate 0.5 mM, sodium carbonate 0.6%), followed by shaking culture for 48 hours at 37 ° C
  • the culture solution was centrifuged to obtain a supernatant. NaCl was added to the supernatant by 0 20%, and the enzyme activity was measured by the same manner as described in Example 5.
  • the Bacillus clausii derived protease did not lose the enzyme activity under the high concentration of salt (more than 10%), in particular, the protease retained more than 75% of the activity even fewer than 20% concentration of salt.
  • the above results indicate that the Bacillus clausii derived protease is a halophilic protease, which can be effectively used as a detergent additive and for the food processing.
  • the present inventors treated various metal ions to the reaction solution and measured the enzyme activity.
  • the protease was treated with 1 mM of calcium, cadmium, cobalt, chrome, copper, magnesium or manganese and measured the enzyme activity thereafter.
  • the protease of the invention was not affected by the metal ions, even by the heavy metals such as cadmium, cobalt, chrome, etc (Fig. 8) .
  • the present inventors examined the stability of the protease against a commercial detergent.
  • the protease was treated with 4 different commercial detergents (BrightTM, Mukunahwa, Korea; One ScoopTM, LG Household & Health Care Ltd., Korea; SparkTM, Aekyung Ind., Korea; Super TiTM, LG Household & Health Care Ltd., Korea) on market by the concentration of 2.5% each and stood for 4 weeks at room temperature. As a result, the protease retained the enzyme activity, suggesting that the protease is a very stable enzyme and thus can be used as a detergent additive (Fig. 8) .
  • the present inventors performed detergency test of the protease of the invention, which was separated and purified in Example 4. As a soiled cloth, cotton test cloth (EMPA 116) contaminated with blood and milk, and mixed fiber test cloth (EMPA 117) contaminated with blood and milk were used to examine the detergency of the protease.
  • the protease showed excellent detergency in the presence of the commercial surfactant LAS (linear alkylbenzene sulfonates) 15%, indicating that the protease can be used as a detergent additive (Fig. 9) .
  • the present invention investigated soybean meal decomposing capacity of the protease separated and purified in Example 4. Soybean meal was added to distilled water at a concentration of 10%, to which the protease was added by 0.5% for the total soybean meal volume, followed by stirring. Soybean meal decomposition was measured by SDS-PAGE. As a result, the protease of the invention was proved to decompose soybean meal efficiently within 2 hours (Fig. 10) .
  • the protease of the invention was proved to inactivate the soybean trypsin inhibitor approximately 80% within 2 hours and when the protease was treated for 16 hours, the soybean trypsin inhibitor was completely inactivated (Fig. 11) .
  • the microorganism of the invention Bacillus clausii 1-52, was inoculated into 3 ml of tryptic soy broth (TSB), followed by shaking culture at 37 ° C with 250 rpm.
  • the microorganism was inoculated again into a 500 ml Erlenmeyer flask containing 100 ml of TSB, followed by further culture for 24 hours.
  • the culture solution was centrifuged at 5,000 x g, and lysozyme and proteinase K were added to the resultant Bacillus, followed by reaction for 18 hours at room temperature. Same volume of phenol was added thereto and well mixed for 20 minutes at room temperature.
  • Centrifugation was performed at 13,000 x g for 20 minutes to obtain supernatant.
  • the experiments were repeated and same volume of chloroform/isoamyl alcohol mixture (24:1) was added thereto, which was also mixed for 20 minutes at room temperature.
  • Centrifugation was performed again at 13,000 x g for 20 minutes to obtain a supernatant. Same amount of chloroform was added thereto, followed by stirring for 20 minutes at room temperature.
  • Centrifugation was performed at 13,000 x g for 20 minutes to obtain a supernatant, to which 2X volume of ethanol was added, followed by slow stirring at room temperature until DNA precipitate was obtained.
  • the genomic DNA in the reaction solution was recovered by using a sterilized Pasteur pipette and the DNA was washed with 70% ethanol to remove the remaining salts.
  • the DNA was dried in a clean bench and then resuspended in 1 ml TE buffer (10 mM Tris- HCl, pH 8.2/1 mM EDTA). The resultant DNA was examined by 1% agarose gel electrophoresis.
  • the present inventors prepared primers with well-preserved regions of the sequence of alkaline serine protease by Blast searching. And the primers were designed to include the entire amino acid sequence of the alkaline serine protease.
  • forward primers F2 and F3, represented by SEQ. ID. NO: 6 and 7, respectively, and reverse primers Rl, R2 and R3, represented by SEQ. ID. NO: 8, 9 and 10, respectively were synthesized (Fig. 12) .
  • PCR was performed by using the Bacillus clausii genomic DNA extracted in Example ⁇ 9-l> as a template and various combinations of the primers as follows: pre- denaturation at 94 ° Cfor 2 minutes with Taq DNA polymerase, denaturation at 94 ° Cfor 1 minute, annealing at 60°Cfor 1 minute, polymerization at 72 ° C for 1 minute, 30 cycles from denaturation to polymerization.
  • PCR products according to different primer combinations were obtained (Fig. 13) .
  • the PCR product obtained by the primer combination F3/R2 was cloned into a PCR cloning vector, pGEM T easy (Promega, USA) (Fig. 14), followed by nucleotide sequencing.
  • the whole DNA size was 2280 bp (SEQ. ID. NO: 3), TCTACT (872-877) was -35 sequence and TACAAT (897-902) was -10 sequence, which contained a promoter region.
  • the active protease region was composed of 275 amino acid residues represented by SEQ. ID. NO: 5 and three active catalytic triads Asp32, His64 and Ser221, which are common in serine proteases, were also included therein. Thus, the protease was confirmed to be one of typical serine proteases.
  • the active protease region represented by SEQ. ID. NO: 5 was named Bacillus clausii alkaline protease (BCAP) .
  • Example 10 Construction of an expression vector for an active protease and transformation
  • PCR was performed by using pGEM T easy-BCAP as a template with primers represented by SEQ. ID. NO: 11 and 12 as follows: pre-denaturation at 94 ° Cfor 2 minutes with Taq DNA polymerase, denaturation at 94 ° C for 1 minute, annealing at 60 ° Cfor 1 minute, polymerization at 72°Cfor 1 minute, 30 cycles from denaturation to polymerization.
  • the amplified approximately 870 bp DNA fragment, which includes 825 bp DNA sequence encoding 275 amino acid residues represented by SEQ. ID.
  • Example 11 Inducement of the expression of a recombinant protein
  • E. coli BL21 (DE3) transformed with the expression vector pT7-BCAP constructed in Example 8 was inoculated into a LB medium containing ampicillin (50 ⁇ g/ ⁇ Jt), followed by culture for overnight at 37 "Q which was used as an inoculum. 1 ml of the inoculum was inoculated into 100 ml LB medium (500 ml Erlenmeyer flask) containing ampicillin, followed by further culture until OD 60 O reached 0.6 - 0.8. To induce protein synthesis, IPTG was added (final concentration, 1 inM) , and then the culture solution was taken every hour by 1 ml per hour.
  • the cell pellet was suspended in 20 ⁇ i of IX SDS-PAGE sample buffer 0.05 M Tris-HCl, pH 6.8, 0.1 M DTT, 2% SDS, 10% glycerol and 0.1% bromophenol blue), followed by heating at 100 ° Cfor 5 minutes to lysate the cells. Electrophoresis was performed according to the method of Laemmli, et al (Laemmli, U.K., 1970, Nature, 227, 680-685). The gel was stained with Coomassie brilliant blue R-250 to confirm the protein produced in the E. coli transformed with pT7-BCAP.
  • the mature protease of the invention was produced by ITPG induction as time went by (Fig. 17) .
  • Example 12 Construction of a plasmid for the insertion of a protease gene containing a promoter pGEM-T easy plasmid with the DNA sequence represented by SEQ. ID. NO: 3 which contains the protease promoter derived from the Bacillus clausii of the invention and pHPS9 (ACTC) plasmid for the insertion of Bacillus genome were treated with EcoR I, followed by electrophoresis. Then, DNAs were extracted by using DNA elution kit. After ligation, transformation of E. coli JM109 was performed according to the same manner as described in Example 9. Chloramphenicol was used as a selection marker instead of ampicillin.
  • SEQ. ID. NO: 3 which contains the protease promoter derived from the Bacillus clausii of the invention and pHPS9 (ACTC) plasmid for the insertion of Bacillus genome were treated with EcoR I, followed by electrophoresis. Then, DNAs were extracted by using DNA
  • a plasmid was extracted from the transformant, which was digested with a restriction enzyme corresponding to the cloning site to confirm the insertion of DNA fragment represented by SEQ. ID. NO: 3.
  • the transformation vector with the insertion of DNA fragment represented by SEQ. ID. NO: 3 was named "pHPS-BCAP" (Fig. 18) .
  • the expression vector was introduced into E.
  • Example 13 Measurement of the enzyme activity of the recombinant protease
  • Centrifugation was performed to obtain supernatant, followed by measuring the amount of tyrosine produced at 275 nm.
  • 1 unit of the protease means the amount of enzyme necessary for the production of 1 ⁇ g of tyrosine for one minute.
  • the titer was 160,000 U per 1 mi of the transformant culture solution.
  • the protease of the invention is so much stable that it retains the enzyme activity under alkaline condition, in the presence of a surfactant and at the high temperature.
  • the protease of the invention has a broad substrate specificity, so that it can eliminate food, blood or body fluid.
  • the protease, the Bacillus clausii 1-52 strain and the transformant producing the protease, and the microorganism culture supernatant containing the same can be used as a detergent component with generally known synthetic surfactants including SDS, octylglucoside, Brij-35, Tritons, Tweens, LAS, AOS or with a natural surfactant.
  • the protease of the invention retains the enzyme activity even under wide range of pH and oxidation-reduction condition, so that it can be widely used in various fields of processing food, feed, fabrics, natural leather, brewing, paper and pulp, and in the production of oral care products and medical applications, in research fields, and in waste treatment because it retains a high activity in the presence of a heavy metal.
  • SEQ. ID. NO: 1 is a nucleotide sequence of 16S rDNA of the Bacillus clausii 1-52 strain of the invention.
  • the SEQ. ID. NO: 2 is an amino acid sequence of N- terminal of the protease of the invention.
  • the SEQ. ID. NO: 3 is a nucleotide sequence of a polynucleotide fragment containing a gene encoding the protease of the invention.
  • the SEQ. ID. NO: 4 is an amino acid sequence corresponding to the full length of the protease of the invention .
  • the SEQ. ID. NO: 5 is an amino acid sequence of the active form of the protease of the invention generated by the digestion of the leader sequence and the pre-pro sequence of the protease represented by SEQ. ID. NO: 3.
  • the SEQ. ID. NO: 6 - 10 are promoter sequences used for the cloning of a gene encoding the protease of the invention .
  • the SEQ. ID. NO: 11 and 12 are primer sequences used for the construction of an expression vector for the active form of the protease of the invention.

Landscapes

  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Mechanical Engineering (AREA)
  • Combustion & Propulsion (AREA)
  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Bacillus clausii I-52, protéase isolée à partir de cette espèce, gène isolé assurant son codage et utilisation. En particulier, Bacillus clausii I-52 isolé donnant une protéase qui en conserve l'activité dans des conditions extrêmes, protéase isolée de cette espèce et ayant l'activité en question, gène isolant assurant son codage, et utilisations industrielles par exemple dans les industries suivantes : blanchissage, cuir, alimentation, produits alimentaires pour animaux et traitement des déchets. La protéase conserve l'activité enzymatique dans des conditions extrêmes comme dans le cas d'une solution alcaline, de la présence de détergent et dans le cas d'une température élevée. De plus, elle offre une large spécificité de substrat permettant d'éliminer les aliments, le sang ou les fluides organiques, et on peut l'utiliser comme principe actif dans les détergents sur les tissus, pour la cuisine ou pour les lentilles de contact et pour le traitement des aliments, des aliments pour animaux ou du cuir et le traitement des déchets organiques.
PCT/KR2006/000545 2005-02-18 2006-02-17 Bacillus sp. i-52, permettant d'isoler une protease haloalcalophile stable vis-a-vis des detergents et des oxydants, gene assurant son codage et utilisation WO2006088325A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020050013731A KR100750659B1 (ko) 2005-02-18 2005-02-18 계면활성제 및 산화제에 매우 안정한 호염, 호알카리성 단백질 분해효소 및 이를 암호화하는 유전자
KR10-2005-0013731 2005-02-18

Publications (1)

Publication Number Publication Date
WO2006088325A1 true WO2006088325A1 (fr) 2006-08-24

Family

ID=36916694

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2006/000545 WO2006088325A1 (fr) 2005-02-18 2006-02-17 Bacillus sp. i-52, permettant d'isoler une protease haloalcalophile stable vis-a-vis des detergents et des oxydants, gene assurant son codage et utilisation

Country Status (2)

Country Link
KR (1) KR100750659B1 (fr)
WO (1) WO2006088325A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103849583A (zh) * 2013-12-31 2014-06-11 成都大学 短小芽孢杆菌（Bacillus pumilus）LDS33及其应用
CN110484598A (zh) * 2019-08-30 2019-11-22 贵州大学 酪蛋白平板法在蛋白酶酶活的定量分析中的应用
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109517522A (zh) * 2018-11-20 2019-03-26 萍乡亨厚新材科技有限公司 一种建筑用超疏水自洁涂料
KR102260512B1 (ko) 2019-10-15 2021-06-04 장정순 염증반응 억제 또는 조절 활성을 나타내는 효소조성물
KR20230046626A (ko) 2021-09-30 2023-04-06 장정순 항염증 활성을 나타내는 알칼리성 단백질 분해효소를 포함하는 조성물

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987004461A1 (fr) * 1986-01-15 1987-07-30 Amgen ANALOGUES DE SUBTILISINE THERMIQUEMENT STABLE ET STABLE AU pH, ET LEUR PROCEDE DE PRODUCTION
US4914031A (en) * 1987-04-10 1990-04-03 Amgen, Inc. Subtilisin analogs
KR100465852B1 (ko) * 2002-06-29 2005-01-13 인하대학교 산학협력단 고활성의 알카리성 단백질 분해효소를 대량 생산하는 바실러스 속 ｉ-５２

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR0127099B1 (ko) * 1994-05-24 1997-12-29 김은영 신균주 바실러스 속(Bacillus sp.) NS-70(KCTC 8555P)와 이를 이용한 신규한 단백질 가수분해효소의 제조방법
US7081359B2 (en) 2002-05-17 2006-07-25 Enzymes Technology Limited Recombinant bacillus proteases and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987004461A1 (fr) * 1986-01-15 1987-07-30 Amgen ANALOGUES DE SUBTILISINE THERMIQUEMENT STABLE ET STABLE AU pH, ET LEUR PROCEDE DE PRODUCTION
US4914031A (en) * 1987-04-10 1990-04-03 Amgen, Inc. Subtilisin analogs
KR100465852B1 (ko) * 2002-06-29 2005-01-13 인하대학교 산학협력단 고활성의 알카리성 단백질 분해효소를 대량 생산하는 바실러스 속 ｉ-５２

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOO H.S. ET AL.: "Oxidant and SDS-stable alkaline protease from a halo-tolerant Bacillus clausii I-52: enhanced production and simple purification", J. APPL. MICROBIOL., vol. 98, no. 2, December 2004 (2004-12-01), pages 491 - 497 *
JOO H.S. ET AL.: "Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties", J. APPL. MICROBIOL., vol. 95, no. 2, 2003, pages 267 - 272 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103849583A (zh) * 2013-12-31 2014-06-11 成都大学 短小芽孢杆菌（Bacillus pumilus）LDS33及其应用
CN110484598A (zh) * 2019-08-30 2019-11-22 贵州大学 酪蛋白平板法在蛋白酶酶活的定量分析中的应用
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

Also Published As

Publication number Publication date
KR100750659B1 (ko) 2007-08-20
KR20060092663A (ko) 2006-08-23

Similar Documents

Publication Publication Date Title
JP5357514B2 (ja) クリソスポリウムセルラーゼ及び使用方法
EP1456368B1 (fr) Nouvelle protease alcaline obtenue a partir de bacillus sp. (dsm 14392) et agents de lavage et de nettoyage contenant cette protease alcaline
JP5193997B2 (ja) エンドグルカナーゼｐｐｃｅおよびそれを含んでなるセルラーゼ調製物
EP1994148B1 (fr) Polypeptides presentant une activite endoglucanase et polynucleotides codant pour eux
KR101739770B1 (ko) 신규 진균 프로테아제 및 이의 용도
WO1998008940A1 (fr) Nouvelle endoglucanase
WO2006088325A1 (fr) Bacillus sp. i-52, permettant d'isoler une protease haloalcalophile stable vis-a-vis des detergents et des oxydants, gene assurant son codage et utilisation
WO2011111836A1 (fr) Cellulase alcaline mutée
DE60029955T2 (de) Endo-beta-1,4-glukanasen
WO2001049859A1 (fr) Xylanase de trichoderma reesei
CN111851077A (zh) 具有内切葡聚糖酶活性的多肽以及对其进行编码的多核苷酸
WO1994004664A1 (fr) Nouvelles xylanases a stabilite et a activite elevees dans des conditions alcalines et a hautes temperatures
JPWO2002042474A1 (ja) セルロース結合領域を欠失した接合菌由来エンドグルカナーゼ酵素
JP2003507039A (ja) マルブランケア属Ｍａｌｂｒａｎｃｈｅａ由来アルカリ性キシログルカナーゼ
JP2002345472A (ja) 新規ヘキセンウロニダーゼ、それをコードする遺伝子、およびそれらの使用
JP4681283B2 (ja) 新規な高アルカリプロテアーゼ及びその利用
Irfan et al. Cloning, purification and characterization of halotolerant xylanase from Geobacillus thermodenitrificans C5
JP2004313022A (ja) エンドグルカナーゼｍｔｅ１およびそれを含んでなるセルラーゼ調製物
CN104583394A (zh) 用内切葡聚糖酶处理纺织品的方法
WO1998006857A1 (fr) Composition de traitement de tissus vegetaux
Nedwin Green chemistry: Using enzymes as benign substitutes for synthetic chemicals and harsh conditions in industrial processes
RU2790443C2 (ru) Варианты протеаз и пути их применения
JP2004121257A (ja) 耐熱性キシラナーゼ
JP5714830B2 (ja) 変異アルカリセルラーゼ
Demattè Microbial enzymes for hair removal: an integrated biotechnological approach for application in the leather tanning industry

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06715996

Country of ref document: EP

Kind code of ref document: A1