CN115247165A - Cellulase mutant with improved specific activity and thermal stability - Google Patents
Cellulase mutant with improved specific activity and thermal stability Download PDFInfo
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- CN115247165A CN115247165A CN202210443408.0A CN202210443408A CN115247165A CN 115247165 A CN115247165 A CN 115247165A CN 202210443408 A CN202210443408 A CN 202210443408A CN 115247165 A CN115247165 A CN 115247165A
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- Prior art keywords
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- 108010059892 Cellulase Proteins 0.000 title claims abstract description 86
- 229940106157 cellulase Drugs 0.000 title claims abstract description 78
- 230000000694 effects Effects 0.000 title claims abstract description 65
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- 102000004190 Enzymes Human genes 0.000 claims abstract description 41
- 229940088598 enzyme Drugs 0.000 claims abstract description 41
- 239000000203 mixture Substances 0.000 claims abstract description 36
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 22
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 17
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 15
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 15
- 239000002157 polynucleotide Substances 0.000 claims abstract description 15
- 241000499912 Trichoderma reesei Species 0.000 claims abstract description 14
- 239000013612 plasmid Substances 0.000 claims abstract description 9
- 239000012634 fragment Substances 0.000 claims description 36
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 33
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 27
- 229920001184 polypeptide Polymers 0.000 claims description 26
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 26
- 150000007523 nucleic acids Chemical group 0.000 claims description 15
- 230000035772 mutation Effects 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 239000003599 detergent Substances 0.000 claims description 6
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- 238000009988 textile finishing Methods 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 abstract description 11
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- 230000004048 modification Effects 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
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- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38645—Preparations containing enzymes, e.g. protease or amylase containing cellulase
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
- D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
- D21H17/005—Microorganisms or enzymes
Abstract
The invention belongs to the technical field of protein engineering modification, and discloses a cellulase mutant with improved specific activity and thermostability, a polynucleotide for coding the cellulase variant, a composition containing the cellulase variant, a recombinant plasmid and/or an engineering strain; and methods for making and/or using the cellulase variants and/or compositions containing the cellulase variants. When the cellulase mutant is expressed in trichoderma reesei, the enzyme activity can reach 374U/mL, the enzyme activity can reach 2412U/mL after amplification on a tank, the expression amount is 13.53g/L, and the specific activity is 178U/mg. Compared with wild neutral cellulase, the expression amount and specific activity of the mutant cellulase are improved by 33.8 percent and 29.9 percent, under the same application effect, the addition amount of the mutant cellulase is 53 percent of that of the wild cellulase, and the enzyme dosage is obviously reduced. The improvement of the expression quantity and specific activity of the neutral cellulase mutant is beneficial to reducing the production and cost of the neutral cellulase, and can be widely applied to the textile industry.
Description
Technical Field
The invention belongs to the technical field of protein engineering modification, and particularly discloses a cellulase variant with improved specific activity and thermostability, a polynucleotide for coding the cellulase variant, a composition containing the cellulase variant, a recombinant plasmid and/or an engineering strain; and methods for making and/or using the cellulase variants and/or compositions containing the cellulase variants.
Background
Cellulase enzymes are a general term for a group of enzyme systems that hydrolyze cellulose to glucose. Cellulase enzymes can be classified into 3 categories according to their catalytic properties: exo-beta-glucanases (EC 3.2.1.91), endo-beta-glucanases (EC 3.2.1.4) and beta-glucosidases (EC 3.2.1.21). The endo-beta-glucanase is one of the important components of the cellulase, has the function of hydrolyzing natural cellulose into amorphous cellulose, can loosen the cellulose structure of cotton fabrics when being applied to a detergent, is beneficial to the detergent components to enter fiber gaps to fully contact dirt, thereby greatly improving the washing effect, and the clothes washed by the detergent added with the cellulase have bright and soft color and small damage to the clothes.
Endo- β -glucanase NCE4 was originally disclosed in Japanese Kongku Kogyo, patent No. 1154721C endoglucanase and an enzyme-containing cellulase preparation, and NCE4 was homologously expressed in Humicola insolens, yielding 4.5g/L. However, humicola does not serve as a host for filamentous fungi commonly used in industrial production, and greatly limits its application and production. In patent CN110093332A, a cellulase mutant and a high-yield strain are disclosed, trichoderma reesei is used as a host, a wild cellulase NCE4 and a mutant KDNE-D with amino acid deletion at positions 219-235 are heterologously expressed, although the enzyme activity, stability and expression quantity are improved to a certain extent compared with the wild type, under the same application effect, the addition amount of the mutant cellulase is 67% of the wild type, and the obtained enzyme activity and expression quantity are still to be improved.
Disclosure of Invention
In order to solve the problems of the prior art, the invention carries out protein engineering on cellulase NCE4 derived from specific Humicola insolens to obtain a series of mutant proteins. When the xylanase is expressed in trichoderma reesei, the enzyme activity can reach 374U/mL, the enzyme activity can reach 2412U/mL after the xylanase is amplified on a tank, the expression quantity is 13.53g/L, and the specific activity is 178U/mg. Compared with wild neutral cellulase NCE4-WT, the expression amount and specific activity are improved by 33.8% and 29.9%, and under the same application effect, the addition amount of the mutant cellulase is 53% of that of the wild cellulase. The expression quantity and specific activity of the neutral cellulase mutant are improved, so that the production and cost of the neutral cellulase are reduced, and the neutral cellulase mutant is widely applied to the textile industry.
In particular, the method comprises the following steps of,
in one aspect, the present invention provides a cellulase variant or an active fragment thereof with improved specific activity and thermostability, characterised in that the amino acid sequence of the variant or the active fragment thereof comprises a mutation selected from the group consisting of at one or more of the following positions corresponding to SEQ ID NO: 1:
(1)38、41、56、64、75、98、104、113、120、134、138、153、158、185、202、263；
(2)38L、41G/T、56C、64M、75C、98P/C、104Y、113I、120I、134C、138V、153S、158R、185C、202C、263D；
wherein the variant or active fragment thereof has endoglucanase activity and wherein the amino acid position of the variant or active fragment thereof is numbered by corresponding to the amino acid sequence of SEQ ID NO. 1.
In some embodiments of the invention, the cellulase variant or active fragment thereof comprises at least one of the following mutations: 38L, 41G, 41T, 64M, 98P, 104Y, 113I, 120I, 138V, 153S, 158R, 263D, 41G +64M, 41T +104Y, 38L +158R, 120I +263D, 98C +134C, 56C +202C, 75C +185C.
In other embodiments of the invention, the variant or active fragment thereof comprises at least one of the following mutations: 153S, 158R, 263D, 41G +64M, 41T +104Y, 38L +158R, preferably, said variant or active fragment thereof comprises the following mutation 41G +64M.
In still other embodiments of the present invention, the cellulase variant or active fragment thereof has an amino acid sequence as shown in SEQ ID NO. 3 to SEQ ID NO. 21; preferably, the amino acid sequence is shown as SEQ ID NO 12-17; more preferably, the amino acid sequence is as shown in SEQ ID NO. 15.
The cellulase variant or active fragment thereof of the present invention has at least one improved property selected from the group consisting of increased enzyme activity, increased protein expression, increased thermostability when compared to the parent polypeptide.
The invention also provides a composition comprising the cellulase variant or an active fragment thereof.
The invention also relates to a polynucleotide comprising a nucleic acid sequence encoding said cellulase variant or an active fragment thereof.
The invention also provides a recombinant plasmid carrying the polynucleotide.
The invention further provides a Trichoderma reesei engineering strain carrying the recombinant plasmid.
In another aspect, the present invention provides a method of producing the cellulase variants or active fragments thereof provided by the invention.
In another aspect, the invention provides the use of a cellulase variant or an active fragment thereof according to the invention in an enzyme composition, a detergent composition, a fabric care composition, a textile finishing process or a paper and pulp process.
The invention has the beneficial technical effects that:
the cellulase mutant provided by the invention has the advantages that the enzymatic activity, the specific activity, the thermal stability and the expression quantity are all remarkably improved, under the same application effect, the addition amount of the cellulase mutant is 53% of that of a wild type, the production and application cost of the cellulase can be greatly reduced in actual production, the wide application of the cellulase in the field of textile washing can be accelerated, the market prospect is wide, and the cellulase has important economic value.
The following terms are defined for clarity. Undefined terms shall be accorded the conventional meaning as used in the art. For example, technical and scientific terms not defined herein have The same meaning as commonly understood by one of ordinary skill (see, e.g., singleton and Sainsbury, dictionary of Microbiology and Molecular Biology, 2 nd edition, john Willi parent publishing company, new York, 1994; and Hale and Marham, harbourine Biology Dictionary (The Harper Collins Dictionary of Biology), huber permanent Press, new York, 1991).
The singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.
The term "about" when used in conjunction with a numerical value refers to a range of-10% to +10% of the numerical value.
The compositions of the invention comprise an effective amount of a cellulase variant or an active fragment thereof as described herein. In some embodiments, an effective amount of a cellulase variant or an active fragment thereof is from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% cellulase by weight of the composition. In other embodiments, an effective amount of a cellulase variant or active fragment thereof is from about 0.001% to about 5.0 weight percent of the composition. In still other embodiments, an effective amount of a cellulase variant or an active fragment thereof is from about 0.001% to about 4.5% by weight of the composition. In still other embodiments, an effective amount of a cellulase variant or active fragment thereof is from about 0.001% to about 4.0% by weight of the composition. In yet even other embodiments, an effective amount of a cellulase variant or active fragment thereof is from about 0.001% to about 3.5, 3.6, 3.7, 3.8, or 3.9 weight percent of the composition.
The composition of the present invention may further comprise necessary auxiliary components. The term "adjunct ingredient" means any liquid, solid or gaseous material selected for the particular type of detergent or fabric care composition desired, as well as the product form (e.g., liquid, granular, powder, bar, paste, tablet, gel, unit dose, flake or foam composition), which is also preferably compatible with the cellulase variant or active fragment thereof used in the composition. In some embodiments, the granular composition is in "compact" form, while in other embodiments, the liquid composition is in "concentrated" form.
In some embodiments, the compositions of the present invention are in a form selected from: powders, liquids, granules, rods, solids, semisolids, gels, pastes, emulsions, tablets, capsules, unit doses, flakes, and foams. In even further embodiments, the composition is in a form selected from the group consisting of: liquids, powders, granular solids, tablets, wafers, and unit doses. In some embodiments, the compositions described herein are provided in unit dosage forms, including tablets, capsules, sachets, pouches, sheets, and multi-compartment bags. In some embodiments, the unit dosage form is designed to provide controlled release of the ingredients within a multi-compartment pouch (or other unit dosage form). In some embodiments, the unit dosage form is provided by a tablet wrapped with a water-soluble film or a water-soluble pouch.
In some embodiments, the compositions of the present invention further comprise one or more surfactants. In some other embodiments, the surfactant is selected from the group consisting of nonionic, amphoteric, semi-polar, anionic, cationic, zwitterionic, and combinations and mixtures thereof. In other embodiments, the surfactant is selected from anionic, cationic, nonionic, and zwitterionic compounds. In some embodiments, the composition comprises from about 0.1% to about 60%, about 1% to about 50%, or about 5% to about 40%, by weight of the composition, of a surfactant.
The term "cellulase variant" refers to a recombinant polypeptide derived from a parent polypeptide or a reference polypeptide by substitution, addition or deletion of one or more amino acids. Cellulase variants may differ from a parent polypeptide by a small number of amino acid residues and may be defined by their level of primary amino acid sequence homology/identity to the parent polypeptide. For example, a cellulase variant has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less than 100% amino acid sequence identity to a parent (or reference) polypeptide.
The term "mutation" refers to any change or alteration in an amino acid sequence, including substitution of an amino acid at an identified position of the amino acid sequence with an amino acid different from the starting amino acid, deletion of an amino acid at an identified position of the amino acid sequence, insertion of an amino acid at an identified position of the amino acid sequence, substitution of an amino acid side chain in the amino acid sequence, and/or chemical modification of the amino acid sequence.
The term "expression vector" refers to a DNA construct comprising a DNA sequence encoding a particular polypeptide, and is operably linked to suitable control sequences capable of effecting the expression of the polypeptide in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some cases, integrate into the genome itself. In some embodiments, the expression vector may be provided in a heterologous host cell suitable for expressing the cellulase variants or active fragments thereof described herein or for propagating the expression vector prior to introducing the expression vector into a suitable host cell. In some embodiments, the polynucleotide is contained in an expression cassette and/or cloned into a suitable expression vector by standard molecular cloning techniques. Such expression cassettes or vectors contain sequences that aid in transcription initiation and termination (e.g., promoters and terminators), and typically contain a selectable marker.
The term "host cell" generally refers to a prokaryotic or eukaryotic host transformed or transfected with a vector constructed using recombinant DNA techniques known in the art. The transformed host cell is capable of replicating vectors encoding protein variants or expressing desired protein variants. In the case of vectors encoding protein variant pre-forms (pre-form) or forms (pro-form), these variants are typically secreted from the host cell into the host cell culture medium when expressed. In some embodiments, the host cell, wherein the cellulase variant or active fragment thereof described herein is expressed in an organism other than a heterologous organism species. Exemplary heterologous organisms include, for example: bacillus subtilis (b.subtilis), bacillus licheniformis (b.licheniformis), bacillus lentus (b.lentus), bacillus brevis (b.brevis), geobacillus stearothermophilus (Geobacillus (formerly bacillus), stearothermophilus), bacillus alkalophilus (b.alkalophilus), bacillus amyloliquefaciens (b.amyloliquefaciens), bacillus coagulans (b.coemulans), bacillus circulans (b.circulans), bacillus lautus (b.lautus), bacillus megaterium (b.megaterium), bacillus thuringiensis (b.thunbergensis), streptomyces lividans (s.lividans), streptomyces murinus(s), pseudomonas fluorescens (p.fluorescuescens), pseudomonas stus (p.zeazerei), saccharomyces mirabilis (p.mirabilis), saccharomyces cerevisiae(s), lactobacillus acidophilus (s.lactis), lactobacillus acidophilus (e.sp.sp., lactobacillus), lactobacillus acidophilus (s.sp.sp., lactobacillus), lactobacillus acidophilus(s), lactobacillus acidophilus (r. Lactis), lactobacillus sp.sp.sp. such as Saccharomyces cerevisiae (s.cerevisiae), cryptosporidium ruknowense (c.lucknowense) and filamentous fungi such as Aspergillus species (Aspergillus spp.), e.g. Aspergillus oryzae (a.oryzae) or Aspergillus niger (a.niger), humicola grisea (h.grisea), humicola insolens and trichoderma reesei (t.reesei.) methods for transforming nucleic acids into these organisms are well known in the art. 2011, appl.environ.microbiol. [ applied and environmental microbiology ] 77.
The term "hybridization" refers to the process of joining a nucleic acid strand to a complementary strand by base pairing, as is known in the art.
The term "hybridization conditions" refers to conditions under which a hybridization reaction is performed. These conditions are generally classified according to the "stringency" of the conditions under which hybridization is measured. Stringency can be based on, for example, the melting temperature (Tm) of the nucleic acid to which the complex or probe is bound. For example, "maximum stringency" typically occurs at about Tm-5 ℃ (5 ℃ below the Tm of the probe); "high stringency" occurs at about 5 ℃ to 10 ℃ below Tm; "moderately stringent" occurs at about 10 ℃ to 20 ℃ below the Tm of the probe; and "low stringency" occurs at about 20 ℃ to 25 ℃ below Tm. Alternatively, or in addition, hybridization conditions may be based on salt or ionic strength conditions for hybridization, and/or one or more stringent washes, such as: 6X SSC = very low stringency; 3X SSC = low to medium stringency; 1X SSC = medium stringency; and 0.5X SSC = high stringency. Functionally, maximum stringency conditions can be used to identify nucleic acid sequences that are strictly or nearly strictly identical to the hybridization probes; while high stringency conditions are used to identify nucleic acid sequences that have about 80% or greater sequence identity with the probe. For applications requiring high selectivity, it is often desirable to use relatively stringent conditions to form the hybrid (e.g., using relatively low salt and/or high temperature conditions).
The term "polynucleotide" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. The nucleic acid may be single-stranded or double-stranded, and may have chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Since the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the compositions and methods of the invention encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5 'to 3' orientation.
The term "polypeptide" refers to a molecule comprising a plurality of amino acids linked by peptide bonds. The terms "polypeptide", "peptide" and "protein" are used interchangeably. Proteins may optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, and sulfonated) to add functionality. When such amino acid sequences exhibit activity, they may be referred to as "enzymes". Amino acid sequences represented in the standard amino-to-carboxyl terminal orientation (i.e., N → C) are used using the conventional one-letter or three-letter code for amino acid residues.
The term "recombinant" refers to genetic material (i.e., nucleic acids, polypeptides they encode, and vectors and cells comprising such polynucleotides) that is modified to alter its sequence or expression characteristics, such as, for example, by: mutating a coding sequence to produce an altered polypeptide, fusing the coding sequence to the coding sequence of another gene, placing the genes under the control of different promoters, expressing the genes in a heterologous organism, expressing the genes at reduced or elevated levels, and conditionally or constitutively expressing the genes in a manner different from their native expression profile. Typically, recombinant nucleic acids, polypeptides and cells based thereon have been manipulated by man such that they are not identical to the relevant nucleic acids, polypeptides and/or cells found in nature.
The term "signal sequence" refers to an amino acid sequence that binds to the N-terminal portion of a polypeptide and facilitates secretion of the mature form of the protein from the cell. The mature form of the extracellular protein lacks a signal sequence that is cleaved off during secretion.
The term "derived from" encompasses the terms "originating from," "obtained from," "obtainable from," "isolated from," and "generated from," and generally means that one specified material finds its origin in, or has a characteristic that can be described with reference to, another specified material.
The term "endoglucanase" refers to an endo-1, 4- (1, 3, 1, 4) - β -D-glucan 4-glucanohydrolase (e.c. 3.2.1.4) that catalyzes the endo-hydrolysis of β -1,4- β -D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, mixed β -1,3 glucans, such as cereal β -D-glucans or xyloglucans, and other plant materials containing a cellulose component. Endoglucanase activity may be determined according to the procedures described in the examples.
The term "introduced" in the context of inserting a nucleic acid sequence into a cell means transformation, transduction, or transfection. Transformation means include protoplast transformation, calcium chloride precipitation, electroporation, naked DNA, and the like as known in the art.
The term "signal sequence" or "signal peptide" refers to an amino acid sequence that binds to the N-terminal portion of a polypeptide and facilitates secretion of the mature form of the protein from the cell. The mature form of the extracellular protein lacks a signal sequence that is cleaved off during secretion.
The term "wild-type" or "parent" refers to a naturally occurring polypeptide that does not include artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the term "wild-type" or "parent" refers to a naturally occurring polynucleotide that does not include artificial substitutions, insertions, or deletions at one or more nucleosides. However, a polynucleotide encoding a wild-type or parent polypeptide is not limited to a naturally occurring polynucleotide, and encompasses any polynucleotide encoding a wild-type or parent polypeptide.
The term "naturally-occurring" refers to any substance (e.g., a polypeptide or nucleic acid sequence) that is found in nature. In contrast, the term "non-naturally occurring" refers to anything not found in nature (e.g., modifications of recombinant nucleic acid and polypeptide sequences or wild-type sequences produced in the laboratory).
Amino acid substitutions described herein use one or more of the following nomenclature: position or starting amino acid position one or more substituted amino acids. Reference to only one position encompasses any starting amino acid at that position that may be present in a reference polypeptide, parent or wild-type molecule, as well as any amino acid with which such starting amino acid may be substituted (i.e., amino acid substitution excludes the starting amino acid of such reference polypeptide, parent or wild-type molecule). Reference to a substituted amino acid or a starting amino acid may further be indicated as several substituted amino acids or several starting amino acids separated by a slash ("/"). For example, X130A/N-209-213 represents a combination of three amino acid substitutions, wherein X is any starting amino acid at position 130 that can be substituted with alanine (A) or asparagine (N); 209 represents any position at which the starting amino acid can be substituted with an amino acid other than the starting amino acid; and 213 represents a position at which any starting amino acid can be substituted by an amino acid other than the starting amino acid. By way of further example, E/Q/S101F/G/H/T/V represents five possible substitutions at position 101, wherein the starting amino acid glutamic acid (E), glutamine (Q), or serine (S) can be substituted with phenylalanine (F), glycine (G), histidine (H), threonine (T), or valine (V).
Drawings
FIG. 1 is a structural diagram of vector pHEC-NCE 4;
FIG. 2 is an SDS-PAGE analysis of yeast expression parent NCE4-WT and its mutant NCE4-S13 fermentation broth.
Detailed Description
The process of the present invention is further illustrated below with reference to examples. The examples are illustrative only and not limiting. The experimental procedures, for which specific conditions are not noted in the following examples, can generally be run under conventional conditions, such as those described in molecular cloning guidelines written by J.Sambruke (Sambrook), et al, or as recommended by the manufacturer. The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples. However, the protection of the invention and the scope of the claims are not limited to the examples provided. The process of the present invention is further illustrated by the following examples.
Example 1: expression of NCE4 and mutants thereof
1.1 construction of recombinant vectors
The amino acid sequence of cellulase NCE4 (CN 110093332A) was selected as parent cellulase NCE4-WT (amino acid sequence shown in SEQ ID NO: 1), delivered to general biosystems (Anhui) Ltd for codon optimization and synthesis, and the synthesized NCE4-WT gene (nucleotide sequence shown in SEQ ID NO: 2) was located on the vector pHEC-NCE4 between the CBHI promoter and CBHI terminator. The structure of the vector pHEC-NCE4 is shown in FIG. 1.
Using molecular biology techniques known in the art, multiple amino acid mutations (substitutions, insertions) were introduced into the parent NCE4-WT using the vector pHEC-NCE4 as a template using the site-directed mutagenesis Kit TaKaRa MutanBEST Kit (Code NO. R401) of TAKARA, to obtain expression vectors containing various NCE4 mutants.
The parent NCE4-WT and NCE4 mutant expression vectors were introduced into a Trichoderma reesei host, respectively, by reference to the protoplast transformation method reported in the literature (Penttila M, nevalaine H, ratto M, et al. A.versatilis transformation system for the cellular viral genes Trichoderma reesei [ J ]. Gene,1987,61 (2): 155-164.). The numbering, mutation sites and SEQ ID NO of the constructed parent NCE4-WT and NCE4 mutant strains are shown in Table 1.
TABLE 1 NCE4-WT and NCE4 mutant numbering, mutation sites and SEQ ID NO
| Mutant numbering | Mutation site | SEQ ID NO |
| NCE4-WT | Not mutated | 1 |
| NCE4-S1 | 38L | 3 |
| NCE4-S2 | 41G | 4 |
| NCE4-S3 | 41T | 5 |
| NCE4-S4 | 64M | 6 |
| NCE4-S5 | 98P | 7 |
| NCE4-S6 | 104Y | 8 |
| NCE4-S7 | 113I | 9 |
| NCE4-S8 | 120I | 10 |
| NCE4-S9 | 138V | 11 |
| NCE4-S10 | 153S | 12 |
| NCE4-S11 | 158R | 13 |
| NCE4-S12 | 263D | 14 |
| NCE4-S13 | 41G+ |
15 |
| NCE4-S14 | 41T+104Y | 16 |
| NCE4-S15 | 38L+158R | 17 |
| NCE4-S16 | 120I+263D | 18 |
| NCE4-S17 | 98C+134C | 19 |
| NCE4-S18 | 56C+ |
20 |
| NCE4-S19 | 75C+185C | 21 |
1.2 construction and screening of recombinant strains
(1) Preparation of protoplasts
Trichoderma reesei mycelia were inoculated onto PDA plates (triumphanthus cantonensis, 24g PDB powder is dissolved in 1000mL purified water, 2% agar powder, natural pH), and cultured at 30 ℃ for 6-7 days. After a large amount of spores are produced by the flat plate thallus, a proper amount of spores are taken to inoculate 50mL PDB, and the culture is carried out for 24h at 30 ℃ and 220 rpm.
The mycelium is pumped out by using a sand core funnel, and the mycelium is washed once by using sterile water; the cells were placed in a flask containing 2mL of 2% yatalase enzymatic hydrolysate (Takara) and reacted at 30 ℃ and 100rpm for 1 hour; the number of protoplast productions was observed microscopically every half hour. The enzymolysis liquid is prepared by using a solution I, and the formula is as follows: 1.2M sorbitol, 50mM CaCl2, 50mM Tris-HCl, pH 7.5.
Pre-cooling the centrifuge; and filtering the enzymolysis reaction solution by using sterile absorbent cotton to obtain a protoplast solution. The protoplast solution was centrifuged at 500g for 5min at 4 ℃ and the supernatant was removed and the protoplast pellet retained. The pellet was washed twice with pre-cooled solution I, resuspended protoplasts adjusted to a concentration of 108/mL and 200. Mu.L was aliquoted.
(2)PEG/CaCl 2 Mediated protoplast transformation
200. Mu.L of the protoplast solution was taken, 10. Mu.L of plasmid (plasmid concentration > 1. Mu.g/. Mu.L) was added, and incubation was carried out at 4 ℃ for 10min.
Add 50. Mu.L of solution II and incubate at 4 ℃ for 30min. The formula of the solution II is as follows: 60% PEG4000, 50mM CaCl 2 ，10mM Tris-HCl，pH 7.5。
Add 1mL of solution II and mix well and incubate at room temperature for 20min.
Adding 1mL of solution I, mixing, coating with a hypertonic screening culture medium, and performing inverted culture at 30 ℃ for 4-7 days. The formula of the hypertonic screening culture medium is as follows: 1.2M sorbitol, 2% glucose, 0.3% NaNO 3 ，0.2％KCl，0.05％MgSO 4 ，0.1％KH 2 PO 4 ，0.001％FeSO 4 2% agar, pH adjusted to 5.5, and hygromycin added to a final concentration of 120. Mu.g/. Mu.L just prior to use.
(3) Transformant validation
After 4 days of culture, transformants were grown successively on the transformation plates, and marginal cells were picked and spotted on PDA resistant plates (containing 120. Mu.g/. Mu.L hygromycin) and cultured at 30 ℃ for 2 days. And selecting transformants which grow vigorously on the resistant plates, continuously inoculating the fresh resistant plates, and further purifying the transformants.
Transformants purified by two-generation resistance plate were cultured for 2 days at 30 ℃ and 220rpm with hyphae picked up to 5mL of PDB.
A small amount of thallus is taken to be put into a 2mL EP tube, 800 mu L of filamentous fungus lysate and 1/3EP tube volume quartz sand are added, and the mixture is violently shaken for 5min under the condition of 30 Hz on a ball mill. mu.L of the supernatant was removed, 50. Mu.L of 3M sodium acetate and 1mL of absolute ethanol were added, and the mixture was frozen at-20 ℃ for 20min. Centrifugation was carried out at 12000rpm at 4 ℃ for 5min to remove the supernatant. Washing the precipitate with 700 μ L of 75% ethanol, centrifuging at 4 deg.C and 12000rpm for 5min, and removing supernatant; repeating the above steps once. And finally blowing dry the residual ethanol, resuspending the precipitate in 50 mu L of enzyme-free water, and freezing and storing the obtained genome at-20 ℃ after the detected concentration.
The genome obtained above can be used for PCR verification of a target gene, and the primer Pcbhi-F: ctcacaactcagatcctcctcccagga and NCE4-R: GCAACCGGTACGGGCAACCAGCT. PCR procedure: 5min at 95 ℃; 30s at 95 ℃; 30s at 58 ℃; 30s at 72 ℃ for 30 cycles; 5min at 72 ℃. The PCR enzyme used was DreamTaq Green PCR (2X) premix (Thermo Scientific) and the negative control was Trichoderma reesei blank host cell genome. The size of the amplified band is verified by agarose gel electrophoresis, and the amplified 752bp band is a positive transformant.
Example 2 Trichoderma reesei transformant Shake flask fermentation and enzyme Activity detection
2.1 transformant shake flask fermentation
The positive transformants were inoculated on PDA plates and cultured for 7 days at 30 ℃. The spores were washed with sterile physiological saline to prepare a spore suspension, which was inoculated into 50mL of a seed medium (lactose 20g/L, calcium chloride 1g/L, magnesium sulfate 0.6g/L, potassium dihydrogen phosphate 5g/L, ammonium sulfate 2.5g/L, corn steep liquor 20g/L, ammonia adjusted pH 4.5), and 50. Mu.L of a Tr2 solution (boric acid 2g/L, zinc sulfate monohydrate 15g/L, copper sulfate heptahydrate 8g/L, cobalt chloride hexahydrate 20g/L, ferrous sulfate 50g/L, manganese sulfate monohydrate 16 g/L) was added and cultured at 28 ℃ for 26-28h at 220 rpm. 5mL of the cultured seed bacterial solution was transferred to 50mL of a fermentation medium (5 g/L glucose, 13g/L lactose, 0.5g/L calcium chloride, 1g/L magnesium sulfate, 5.9g/L potassium dihydrogen phosphate, 5.6g/L ammonium sulfate, 2.7g/L corn steep liquor, pH4.5 adjusted with ammonia) and 20. Mu.L of Tr2 solution and 20. Mu.L of Tr1 solution (5.76 g/L ferric citrate, 0.768g/L zinc acetate, 0.81g/L EDTA) were added and cultured at 220rpm at 28 ℃. The pH was adjusted to 4.5 every 24h with ammonia. After 168 hours of fermentation, the mixture was centrifuged at 4000rpm at 4 ℃ for 15min to collect the fermentation supernatant.
2.2 detection of enzyme Activity in fermentation broth
The enzyme activity of the neutral cellulase is defined as that the enzyme amount required for degrading and releasing 1 mu mol of reducing sugar in glucose equivalent from CMC-Na solution with the concentration of 20mg/mL per minute is 1U under the conditions of 50 ℃ and pH 6.0. The results of shake flask enzyme activity assays for the parent NCE4-WT and NCE4 mutant strains are shown in Table 2. The fermentation broth of NCE4-WT and NCE4 mutant were each collected in an equal amount and subjected to SDS-PAGE to detect a protein band of about 42KD. The results are shown in FIG. 2. The ratio of total protein in the fermentation broth supernatants of the parent stce1-WT and stce1 mutants was calculated by Gel analysis pro software according to SDS-PAGE bands to obtain the protein concentrations of the parent stce1-WT and stce1 mutants, and further the specific activities of the stce1-WT and stce1 mutants (specific activity = enzyme activity/protein concentration) as shown in Table 2.
TABLE 2 results of shake flask enzyme activities of the NCE4-WT and NCE4 mutant strains
| Mutant numbering | Mutation site | Enzyme activity (U/mL) |
| NCE4-WT | Not mutated | 214 |
| NCE4-S1 | 38L | 302 |
| NCE4-S2 | 41G | 298 |
| NCE4-S3 | 41T | 317 |
| NCE4-S4 | 64M | 335 |
| NCE4-S5 | 98P | 346 |
| NCE4-S6 | 104Y | 367 |
| NCE4-S7 | 113I | 352 |
| NCE4-S8 | 120I | 272 |
| NCE4-S9 | 138V | 302 |
| NCE4-S10 | 153S | 325 |
| NCE4-S11 | 158R | 365 |
| NCE4-S12 | 263D | 309 |
| NCE4-S13 | 41G+64M | 374 |
| NCE4-S14 | 41T+104Y | 361 |
| NCE4-S15 | 38L+158R | 337 |
| NCE4-S16 | 120I+263D | 278 |
| NCE4-S17 | 98C+134C | 289 |
| NCE4-S18 | 56C+202C | 334 |
| NCE4-S19 | 75C+185C | 320 |
2.3 optimal pH detection
Diluting the NCE4 fermentation supernatant with buffer solutions with different pH values (3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.5, 7.0, 7.5 and 8.0), measuring the enzyme activity of the recombinant NCE4 under the condition of different pH values (3.0-8.0) according to a standard neutral cellulase activity measuring method at the measuring temperature of 50 ℃, and determining the optimum pH value of the reaction. The highest enzyme activity is 100%, and the results show that the optimum pH values of the NCE4-WT and the NCE4 mutant are both 6.0, and more than 80% of the enzyme activity can be maintained within the range of pH 5.0-7.0.
2.4 Heat resistance test
After the enzyme solution is respectively treated at 60 ℃ and 70 ℃ for 1h, the residual enzyme activity of the recombinant NCE4 is determined according to a standard neutral cellulase activity determination method, the enzyme activity of an untreated control group is 100%, and partial enzyme activity results are shown in Table 3, wherein the heat resistance of the mutant NCE4-S13 is optimal.
TABLE 3 thermotolerance of NCE4-WT and NCE4 mutant strains
Example 3 amplification culture of Trichoderma reesei
Spore plates with vigorous growth states of NCE4-WT and the mutant NCE4-S13 were inoculated with appropriate amounts of spores to 50mL of PDB and cultured at 28 ℃ and 220rpm for 24 hours, respectively. Transferring 1L of secondary culture medium (glucose 3-5g/L, lactose 30-45g/L, calcium chloride 0.5-0.8g/L, magnesium sulfate 1-1.3g/L, potassium dihydrogen phosphate 5.5-6.5g/L, ammonium sulfate 5.0-6.5g/L, corn steep liquor 2.5-4g/L, defoaming agent 0.2g/L, ammonia water to adjust pH 4.7-4.9) according to 3%, and respectively adding 1mL of Tr2 and Tr3 (sodium molybdate 10 g/L) solution. The secondary culture conditions were 220rpm at 28 ℃ for 24h.
The secondary culture solution was transferred to a 50L fermentor (glucose 3-,5g/L, lactose 30-45g/L, calcium chloride 0.5-0.8g/L, magnesium sulfate 1-1.3g/L, potassium dihydrogen phosphate 5.5-6.5g/L, ammonium sulfate 5.0-6.5g/L, corn steep liquor 2.5-4g/L, antifoaming agent 0.2g/L, ammonia adjusted pH 4.7-4.9, liquid contents of 20L,20mL Tr2 and Tr3 solutions) by flame inoculation. Culturing at 28 deg.C under 0.03 + -0.01 Mpa with air flow rate upper limit 3500m3/h and rotation speed upper limit 130rpm; adding 25% ammonia water to adjust the pH value to 3.5-4.5, adding lactose at the upper limit of 1.3g/L x h, and fermenting for 140-220 h to obtain the fermentation liquor.
And (3) centrifuging the fermentation liquor to obtain fermentation supernatant, and performing neutral cellulase enzyme activity detection (the method refers to example 2.2) and protein content detection (SDS-PAGE), wherein the molecular weight of the recombinant NCE4 is about 42KDa. The result shows that the enzyme activity of the NCE4-WT expressed by the trichoderma reesei is 1387U/mL, the protein content is 10.11g/L, and the specific activity is 137.2U/mg; the enzyme activity of the variant NCE4-S13 was 2412U/mL, the protein content was 13.53g/L, and the specific activity was 178U/mg.
Example 4 detection of thermostability of recombinant NCE4
Adding glycerol with final concentration of 20% as protectant into the fermentation supernatant of NCE4-WT and mutant NCE4-S13, and storing at-80 deg.C for 3 months, which is a control group; the fermentation supernatant was added to 20% final concentration glycerol and kept at 37 ℃ for 3 months, which was the experimental group. Three months later, the cellulase activity is respectively detected, and the result shows that: the enzyme activity of the control group stored at low temperature is not obviously reduced, and the enzyme activities of the experiment group NCE4-WT and the mutant NCE4-S13 stored at 37 ℃ are respectively reduced by 52 percent and 26 percent. According to the enzyme activity result, the enzyme activity of the mutant NCE4-S13 is reduced to a lower extent under the same condition, so that the long-term storage of the enzyme is facilitated, and the product effective period is prolonged.
| Sample numbering | Initial enzyme activity (U/mL) | Final enzyme activity (U/mL) | Percentage of remaining enzyme activity |
| NCE 4-WT-control group | 1135 | 1087 | - |
| NCE 4-S13-control group | 1917 | 1864 | - |
| NCE 4-WT-Experimental group | 1135 | 553 | 48％ |
| NCE 4-S13-Experimental group | 1917 | 1418 | 74％ |
Example 5 application of recombinant NCE4 in textile industry
The recombinant cellulase provided by the invention is mainly applied to the raising and hair removal of denim fabric, and the process comprises the following steps: adding 10L of water into each cylinder of a washing machine, heating to 40-60 deg.C, keeping the temperature at pH6.0-8.0 for 50min, sampling, washing with water, dewatering, and oven drying.
The denim fabric treated by the recombinant cellulase mutant provided by the invention has good smoothness and clean hair removal; the front side has clear spots and strong stereoscopic impression, and the re-dyeing phenomenon is not easy to occur through enzyme washing, and the blue-white contrast is good. The same effects as above were obtained with NCE4-WT in an amount of 7.5g/L and the mutant NCE4-S13 in an amount of 4g/L.
The cellulase provided by the invention can be widely applied to the textile processing industry, and has good and stable application effect under the conditions of pH8.0 and 50 ℃. Under the same application effect, the addition amount of the mutant cellulase is 53 percent of that of the wild cellulase, the enzyme dosage is obviously reduced, and the cost is obviously reduced.
In the description of the specification, reference to the description of "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Yichangdong sunshine Biochemical pharmaceutical Co., ltd
<120> a cellulase mutant having improved specific activity and heat resistance
<130> 2021
<160> 21
<170> PatentIn version 3.5
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<212> PRT
<213> Humicola insolens
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Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
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Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
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Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
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Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
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Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
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Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
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Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
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Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
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Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
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Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
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Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
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atgcgctcct cccctctcct gcgcagcgcc gttgtggccg ctctccctgt gctggccctc 60
gctgctgatg gcaagagcac ccgctactgg gattgctgca agcctagctg cggctgggcc 120
aagaaggccc ctgtcaacca gcctgtgttc tcctgcaacg ccaacttcca gcgcctcacc 180
gacttcgacg ccaagtccgg ctgcgagcct ggcggtgtgg cctactcctg cgccgatcag 240
accccttggg ccgtgaacga tgacttcgcc ttcggcttcg ccgccaccag catcgccggc 300
tctaacgagg ccggctggtg ctgcgcctgc tacgagctga ccttcacctc cggccctgtc 360
gccggcaaga agatggtggt gcagagcacc tccaccggcg gcgacctcgg ttccaaccac 420
ttcgacctca acatccctgg cggcggcgtc ggcatcttcg acggttgcac ccctcagttc 480
ggcggcctcc ccggtcagcg ttacggcggt atcagcagcc gcaacgagtg cgaccgcttc 540
cccgatgccc tgaagcccgg ctgctactgg cgcttcgatt ggttcaagaa cgccgacaac 600
cctagcttca gcttccgcca ggtgcagtgc cccgccgagc tggttgcccg taccggttgc 660
cgccgcaacg atgatggcaa cttccctgcc gtccagatcc cttccagcag caccagctcc 720
cccgtcggcc agcctaccag cacctccact acctccacct ccaccacctc cagccccccc 780
gtgcagccta ccaccccttc cggttgcacc gccgagcgct gggctcagtg cggtggtaac 840
ggctggagcg gctgcaccac ctgcgtggct ggctccacct gcaccaagat caacgactgg 900
taccatcagt gcctctga 918
<210> 3
<211> 305
<212> PRT
<213> Artificial sequence
<400> 3
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Leu Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 4
<211> 305
<212> PRT
<213> Artificial sequence
<400> 4
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Gly Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 5
<211> 305
<212> PRT
<213> Artificial sequence
<400> 5
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Thr Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 6
<211> 305
<212> PRT
<213> Artificial sequence
<400> 6
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Met
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 7
<211> 305
<212> PRT
<213> Artificial sequence
<400> 7
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Pro Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 8
<211> 305
<212> PRT
<213> Artificial sequence
<400> 8
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Tyr Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 9
<211> 305
<212> PRT
<213> Artificial sequence
<400> 9
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Ile Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 10
<211> 305
<212> PRT
<213> Artificial sequence
<400> 10
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Ile Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 11
<211> 305
<212> PRT
<213> Artificial sequence
<400> 11
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Val Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 12
<211> 305
<212> PRT
<213> Artificial sequence
<400> 12
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Ser Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 13
<211> 305
<212> PRT
<213> Artificial sequence
<400> 13
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Arg Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 14
<211> 305
<212> PRT
<213> Artificial sequence
<400> 14
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Asp Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 15
<211> 305
<212> PRT
<213> Artificial sequence
<400> 15
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Gly Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Met
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Asp Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 16
<211> 305
<212> PRT
<213> Artificial sequence
<400> 16
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Thr Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Tyr Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Asp Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 17
<211> 305
<212> PRT
<213> Artificial sequence
<400> 17
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Leu Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Arg Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Asp Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 18
<211> 305
<212> PRT
<213> Artificial sequence
<400> 18
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Ile Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Asp Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 19
<211> 305
<212> PRT
<213> Artificial sequence
<400> 19
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Cys Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Cys Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 20
<211> 305
<212> PRT
<213> Artificial sequence
<400> 20
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Cys Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Cys Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
<210> 21
<211> 305
<212> PRT
<213> Artificial sequence
<400> 21
Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro
1 5 10 15
Val Leu Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys
20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala
50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Cys Ser Cys Ala Asp Gln
65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr
85 90 95
Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
115 120 125
Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175
Cys Asp Arg Phe Pro Asp Ala Leu Cys Pro Gly Cys Tyr Trp Arg Phe
180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val
195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser
225 230 235 240
Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
260 265 270
Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys
290 295 300
Leu
305
Claims (10)
1. A cellulase variant or an active fragment thereof with improved specific activity and thermostability, characterised in that the amino acid sequence of the variant or the active fragment thereof comprises a mutation selected from the group consisting of the following at one or more positions corresponding to SEQ ID NO: 1:
(1)38、41、56、64、75、98、104、113、120、134、138、153、158、185、202、263；
(2)38L、41G/T、56C、64M、75C、98P/C、104Y、113I、120I、134C、138V、153S、158R、185C、202C、263D；
wherein the variant or active fragment thereof has endoglucanase activity and wherein the amino acid position of the variant or active fragment thereof is numbered by corresponding to the amino acid sequence of SEQ ID NO. 1.
2. The cellulase variant or active fragment thereof according to claim 1, wherein the variant or active fragment comprises at least one of the following mutations: 38L, 41G, 41T, 64M, 98P, 104Y, 113I, 120I, 138V, 153S, 158R, 263D, 41G +64M, 41T +104Y, 38L +158R, 120I +263D, 98C +134C, 56C +202C, 75C +185C;
preferably, said variant or active fragment thereof comprises at least one of the following mutations: 153S, 158R, 263D, 41G +64M, 41T +104Y, 38L +158R;
more preferably, said variant or active fragment thereof comprises the following mutation 41G +64M.
3. The cellulase variant or active fragment thereof according to claim 1 or 2, having an amino acid sequence as shown in SEQ ID No. 3 to SEQ ID No. 21, preferably having an amino acid sequence as shown in SEQ ID No. 12 to SEQ ID No. 17, more preferably having an amino acid sequence as shown in SEQ ID No. 15.
4. The cellulase variant or active fragment thereof of any one of claims 1-3, wherein said variant has at least one improved property selected from the group consisting of increased enzyme activity, increased protein expression, increased thermostability when compared to a parent polypeptide.
5. A composition comprising the cellulase variant or an active fragment thereof according to any one of claims 1-3.
6. A polynucleotide comprising a nucleic acid sequence encoding the cellulase variant or an active fragment thereof of any one of claims 1-3.
7. A recombinant plasmid carrying the polynucleotide of claim 6.
8. A Trichoderma reesei engineered strain carrying the recombinant plasmid of claim 7.
9. A method of producing the cellulase variant or an active fragment thereof of any one of claims 1-3.
10. Use of the cellulase variant or active fragment thereof according to any one of claims 1-3 in an enzyme composition, a detergent composition, a fabric care composition, a textile finishing process or a paper and pulp process.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110452274 | 2021-04-26 | ||
| CN2021104522744 | 2021-04-26 |
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| Publication Number | Publication Date |
|---|---|
| CN115247165A true CN115247165A (en) | 2022-10-28 |
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|---|---|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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2022
- 2022-04-25 CN CN202210443408.0A patent/CN115247165A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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