CN109337888A - Cellobiohydrolase mutant and its production method and application - Google Patents

Cellobiohydrolase mutant and its production method and application Download PDF

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Publication number
CN109337888A
CN109337888A CN201811340287.7A CN201811340287A CN109337888A CN 109337888 A CN109337888 A CN 109337888A CN 201811340287 A CN201811340287 A CN 201811340287A CN 109337888 A CN109337888 A CN 109337888A
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gly
asp
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pro
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CN109337888B (en
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蔡文生
宗志友
邵学广
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Nankai University
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Nankai University
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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
    • 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
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • 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)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

Abstract

The invention discloses a kind of cellobiohydrolase mutant, its activity provides the function of degradation in cellulose degradation, protein of the enzyme for following (a) or (b): (a) the 393rd threonine of the amino acid sequence as shown in SEQ ID NO:1 is substituted by other amino acid, other amino acid are lysine, arginine or glutamic acid, and (b) amino acid sequence in (a) is by replacing, missing or adding one or several amino acid and with the protein as derived from (a) of cellobiohydrolase activity.The invention also discloses the compositions containing one or more of enzymes.The invention also discloses the application of cellobiohydrolase mutant or composition in cellulose hydrolysis.Cellobiohydrolase mutant of the invention has cellulolytic activity, and, the mutant of active raising has been obtained, the hydrolysis effect to cellulose is improved.

Description

Cellobiohydrolase mutant and its production method and application
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of cellobiohydrolase mutant, the combination containing enzyme The production method and its application in cellulose degradation of object, DNA molecular, recombinant vector, host cell and the mutant.
Background technique
Currently, cleaning, reproducible wood fibre quality alcohol fuel have been to be concerned by more and more people.Utilize cellulose The biobased products that enzyme lignocellulose degradation finally produces including ethyl alcohol are most important to human kind sustainable development.But It is that the cost of cellulose degraded lignocellulosic is still very high, cost is primarily limited to the hydrolysis efficiency of cellulase.Cause This says, during cellulose degraded lignocellulosic, the hydrolysis efficiency of cellulase is a master of cellulose conversion Want bottleneck.So in the research direction for obtaining the high cellulase of hydrolysis efficiency, researcher always it is diligent constantly It explores.
Summary of the invention
It is excellent it is an object of the invention to solve at least the above problems and/or defect, and provide at least to will be described later Point.
It is a still further object of the present invention to provide a kind of cellobiohydrolase mutant, to solve the hydrolysis of cellulase The problem of low efficiency, the hydrolysis efficiency of accelerating fibers disaccharide-hydrolysing enzymes.
A further object of the present invention is to provide the production method of cellobiohydrolase mutant.
The present invention separately has a purpose to be to provide application of the cellobiohydrolase mutant in cellulose degradation.
For this purpose, technical solution provided by the invention are as follows:
A kind of cellobiohydrolase mutant, protein of the enzyme for following (a) or (b):
(a) the 393rd threonine of the amino acid sequence as shown in SEQ ID NO:1 is substituted by other amino acid, Other described amino acid be lysine, arginine or glutamic acid,
(b) amino acid sequence in (a) is by replacing, missing or adding one or several amino acid and having fiber two The active protein as derived from (a) of glycosylhydrolase；The activity of the cellobiohydrolase mutant is in cellulose degradation The function of degradation is provided.
Composition containing one or more of enzymes.The enzyme is the cellobiohydrolase mutant.This is several One of kind of enzyme or it is any several and meanwhile in enzyme system compounding for degraded cellulose.
A kind of DNA molecular, the DNA molecular coding enzyme.
Preferably, in the DNA molecular, the base sequence of the DNA molecular is as shown in SEQ ID NO:4.
A kind of recombinant vector, contains the DNA molecular and what is be operably connected with the DNA molecular is used to express Adjusting sequence.
Host cell, the host cell contain the DNA molecular or the recombinant vector.
The production method of cellobiohydrolase mutant includes the following steps: the host cell in culture medium Middle culture is produced in the host cell based on the cellobiohydrolase mutant gene coding contained by recombinant vector Cellobiohydrolase mutant.
The application of the cellobiohydrolase mutant or the composition in cellulose hydrolysis.
The present invention is include at least the following beneficial effects:
Enzyme provided by the invention has the function of good degraded cellulose, cellobiose yield can be improved 9.52%~ 28.57%, improve the hydrolysis effect to cellulose.It can individually or with other mutant combinations be used, to improve hydrolysis Efficiency.The expression quantity of enzyme of the invention is high and activity is high, can be used in enzyme system compounding, and balance hydrolysis enzyme system improves transformation efficiency. The hydrolysis efficiency of cellobiohydrolase in the unit time can be improved in the raising of percent hydrolysis, reduces enzyme dosage.
To facilitate the understanding of the present invention, the meaning of term and phrase of the present invention is defined as follows for definition:
" gene " refers to that a kind of DNA molecular, gene are the main matters of hereditary variation, be control biological traits it is basic Hereditary unit, the base sequence of coding RNA or protein is known as structural gene in gene, and so-called gene is structure in the present invention Gene.
" carrier ", is the nucleic acid molecules for referring to transport another nucleic acid connected to it, and a type of carrier is " matter Grain ", plasmid is that other DNA fragmentations can circular double stranded DNA ring connected to it.Another type of carrier is viral vectors, Other DNA fragmentations can be connected to viral genome.Certain vector integrations are able to and host into host cell gene group Genome replicates together.Also, certain carriers can instruct the expression for the gene being operatively connected with it, and what is generally used is such Expression vector is plasmid form.
" recombinant vector " refers to the expression vector for having had connected gene.In the present invention, it can be used interchangeably " recombinant plasmid " " recombinant vector ".
" cellulase ", cellulase refer to energy degraded cellulose Isosorbide-5-Nitrae-glucoside bond, cellulose are made to become fiber two The general name of one group of enzyme of sugar and glucose has been the multicomponent enzyme system of synergistic effect.The main component of cellulase is inscribe 1, 4- glucolase, exoglucanase and cellobiohydrolase.First two enzyme mainly dissolves fiber, and latter enzyme is by fiber two Sugar is converted into glucose, and the ratio of these three main ingredient activities, has been realized in appropriate regulation composition (i.e. to component enzyme system) At the degradation of cellulose.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this The research and practice of invention and be understood by the person skilled in the art.
Detailed description of the invention
Fig. 1 is gel electrophoresis figure after cellobiohydrolase of the present invention and its mutant expression；
Fig. 2 is the histogram that cellobiohydrolase mutant T393K of the present invention produces cellobiose；
Fig. 3 is the histogram that cellobiohydrolase and its mutant of the present invention produce cellobiose.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text Word can be implemented accordingly.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more The presence or addition of a other elements or combinations thereof.
The present invention provides a kind of cellobiohydrolase mutant, protein of the enzyme for following (a) or (b):
(a) the 393rd threonine of the amino acid sequence as shown in SEQ ID NO:1 is substituted by other amino acid, Other described amino acid be lysine, arginine or glutamic acid,
(b) amino acid sequence in (a) is by replacing, missing or adding one or several amino acid and having fiber two The active protein as derived from (a) of glycosylhydrolase；The activity of the cellobiohydrolase mutant is in cellulose degradation The function of degradation is provided.
The present invention also provides the composition containing one or more of enzymes, the enzyme is the cellobiohydrolase Mutant.Enzyme of the invention can be individually used for degraded cellulose, can also be same with any one or more of other several mutant When be used for degraded cellulose, improve the hydrolysis efficiency of cellulose.The enzyme provided by the invention is used for degraded cellulose with other Such as the enzyme collective effect of β-Isosorbide-5-Nitrae-glucolase, endoglucanase, it is used for degraded cellulose.Enzyme provided by the invention can also It is individually used for degraded cellulose.
The present invention also provides a kind of DNA molecular, the DNA molecular coding enzyme.
It is of the invention in one embodiment, preferably, the base sequence of the DNA molecular such as SEQ ID NO: Shown in 4, encoding amino acid sequence sequence as shown in SEQ ID NO:3 is the amino acid sequence as shown in SEQ ID NO:1 The 393rd threonine be substituted by the enzyme mutant of lysine.Since there are degeneracies for amino acid codes, correspond to same A kind of different codons of amino acid are known as synonym (synonymous codon).Even if degeneracy makes those passwords Base is changed in son, but still can encode out original acid.The degeneracy of codon also make on DNA molecular base composition have compared with The variation in big leeway.So DNA molecular of the present invention include but is not limited to more than polynucleotide sequence, there is also very Multipotency enough encodes the possible polynucleotide sequence of the enzyme, is not enumerating here.
The present invention also provides a kind of recombinant vector, contains the DNA molecular and operationally connect with the DNA molecular The adjusting sequence for expression connect, the sequence of the DNA molecular are selected from: SEQ ID NO:4,6 and 10.It is used used in the present invention In the pPICZ α A carrier of expression, it is of course also possible to using the expression vector of other nucleotide sequences for being suitable for encoding the enzyme, It can be expressed in a variety of eukaryotic host cells.
The present invention also provides host cell, the host cell contains the DNA molecular or the recombinant vector.It should DNA molecular contained by recombinant vector can encode the enzyme.In a preferred embodiment, which is to finish red ferment Female (Pichia pastoris) cell.
The present invention also provides the production method of cellobiohydrolase mutant, include the following steps: the host Cell is cultivated in the medium, is produced in the host cell based on the cellobiohydrolase mutation contained by recombinant vector The cellobiohydrolase mutant of body gene coding.
The present invention also provides the cellobiohydrolase mutant or the composition in cellulose hydrolysis Using.
Technical solution in order to enable those skilled in the art to better understand the present invention now provides following embodiment and is said It is bright:
Amino acid-deficient culture medium (YPDS) purchase from zeocin (Thermo FisherScientific, Runcorn, Cheshire, UK), formula are as follows: 1% yeast extract (yeast extract), 2% peptone (peptone), 2% grape Sugared (glucose), 1M sorbierite (sorbitol), 2% agar (agar).
Resiliency complexity culture medium (Buffered Glycerol-complex Medium) containing glycerol, formula are as follows:
1% yeast extract (yeast extract), 2% peptone (peptone), 100mM potassium phosphate pH 6.0 (potassium phosphate), 1.34% without amino acid nitrogen source and 1% glycerol (glycerol).
Buffered Minimal Methanol (BMMH) medium, formula are as follows:
100mM potassium phosphate (potassium phosphate) pH 6.0,1.34% without amino acid nitrogen source, 0.5% methanol (methanol)。
Wild type cellobiohydrolase derives from Talaromyces emersonii (Talaromyces emersonii), amino Acid sequence is as shown in SEQ ID NO:1, and DNA molecular sequence is as shown in SEQ ID NO:2.
Cellobiohydrolase mutant provided by the invention is by compiling to the DNA molecular as shown in SEQ ID NO:2 The protein of code is mutated.Obtain cellobiohydrolase mutant T393K (SEQ ID NO:3), T393R (SEQ ID NO:5), T393D (SEQ ID NO:7), T393E (SEQ ID NO:9), T393Q (SEQ ID NO:11) and T393A (SEQ ID NO:12).
The engineered strain of wild type cellobiohydrolase and its mutant contains cellobiohydrolase mutant T393K (SEQ ID NO:3), T393R (SEQ ID NO:5), T393D (SEQ ID NO:7), T393E (SEQ ID NO:9), The carrier of T393Q (SEQ ID NO:11) and T393A is respectively as follows: pPICZ α A-T393K, pPICZ α A-T393R, pPICZ α A- T393D,pPICZαA-T393E,pPICZαA-T393Q,pPICZαA-T393A.Expression vector used in above-mentioned construction method Refer to pPICZ α A.The carrier for being connected with wild type cellobiohydrolase and its mutant is transferred to Pichia pastoris competent cell, is chosen Positive colony is taken to get the engineered strain containing mutated gene of the invention is arrived.
Expression and protein purification containing cellobiohydrolase mutant genetically engineered bacteria of the present invention
The engineered strain coated plate of obtained wild type cellobiohydrolase and its mutant is won to containing 100 μ g/ml In the amino acid-deficient culture medium of Lay mycin, and cultivated 3 days at 30 DEG C.Positive bacterium colony is inoculated into containing glycerol later Resiliency complexity culture medium (Buffered Glycerol-complex Medium) in 240rpm, 30 DEG C be incubated overnight to OD600=2-6 is (with UNICO UV2102 ultraviolet-uisible spectrophotometer, to cultivate the resiliency complexity culture containing glycerol Base is blank control.Culture solution is centrifuged in 4000g, after collecting thallus, with Buffered Minimal Methanol (BMMH) Thallus is resuspended in medium.Later every 12 hours 0.5% methanol of supplement, cultivated 3 days under the conditions of 240rpm, 30 DEG C.
Crude enzyme liquid is purified by Ni-NTA column chromatography, and imidazole concentration is 300mM in eluent, elutes 10 cylinders Product.The albumen obtained later reaches the requirement of SDS-PAGE purity after testing.Cellobiohydrolase is obtained by above method Albumen (amino acid sequence SEQ ID NO:1) and cellobiohydrolase mutant (amino acid sequence SEQ ID NO:3).
Using the enzyme activity of ultra-performance liquid chromatography measurement albumen, the survey really carried out using microcrystalline cellulose as substrate It is fixed, cellobiohydrolase mutant T393K activity and percent hydrolysis shown in amino acid sequence SEQ ID NO:3 are measured, specifically Method and steps are as follows:
Sample: 1% (w/v) microcrystalline cellulose preheats 5-10min in 60 DEG C of constant-temperature metal baths, and 1.33 μM of enzyme dilution is added Liquid.
Water-bath: the control group of uniformly mixed sample, substrate and enzyme is placed in 60 DEG C of constant-temperature metal baths simultaneously, is reacted 60h。
Inactivation: at the end of reaction, the control group of sample, substrate and enzyme is boiled to 5min together inactivates enzyme.
Measurement: cellobiose absorption peak is measured using Composition distribution, and calculates fiber using cellobiose standard curve The content of disaccharides.As shown in Fig. 2, wild type releases 0.21g cellobiose, and mutant T393K is released after hydrolysis 60 hours 0.27g cellobiose, improves 28.6%.
According to above-mentioned same method, respectively by albumen T393R, T393D, T393E, T393Q and T393A carrier of building It has been transformed into Pichia pastoris competent cell respectively, has obtained engineered strain, and carried out protein expression, measured cellobiose water Solve enzyme mutant T393K (SEQ ID NO:3), T393R (SEQ ID NO:5), T393D (SEQ ID NO:7), T393E (SEQ ID NO:9), the cellobiose yield of T393Q (SEQ ID NO:11) and T393A (SEQ ID NO:13), according to it is above-mentioned efficiently The method of the enzyme activity of liquid chromatography for measuring albumen measures protein active and its percent hydrolysis, cellobiose yield is calculated, such as Shown in Fig. 3, when hydrolyzing 60h, the fiber two of wild type and mutant T393K, T393R, T393D, T393E, T393Q and T393A Candy output is respectively 0.21g, 0.27g, 0.25g, 0.22g, 0.23g, 0.22g and 0.21g.
In the present invention, sequence table and corresponding sequence are as follows:
Fiber in the unit time can be improved in the raising of the enzymatic activity of cellobiohydrolase mutant provided by the invention The hydrolysis efficiency of disaccharide-hydrolysing enzymes reduces enzyme dosage, improves the effect that balanced enzyme in enzyme system compounding improves overall hydrolysis rate, It is with a wide range of applications.
Module number and treatment scale described herein are for simplifying explanation of the invention.To fiber two of the invention The application of glycosylhydrolase mutant and its application, modifications and variations will be readily apparent to persons skilled in the art.
As described above, according to the present invention, the enzyme and combinations thereof improved due to the provision of enzymatic activity, when unit can be improved The hydrolysis efficiency of interior cellobiohydrolase reduces enzyme dosage, improves balanced enzyme in enzyme system compounding and improves integral hydrolysis The effect of rate, is with a wide range of applications.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited In specific details and legend shown and described herein.
<110>Nankai University
<120>cellobiohydrolase mutant and its production method and application
<130> 2017
<160> 14
<170> PatentIn version 3.5
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<213>Talaromyces emersonii (Talaromyces emersonii)
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Thr Phe Thr Ala Ser Asn Pro Pro Gly Gly Gly Thr Thr Thr Thr Thr
435 440 445
Thr Thr Thr Thr Ser Lys Pro Ser Gly Pro Thr Thr Thr Thr Asn Pro
450 455 460
Ser Gly Pro Gln Gln Thr Met Trp Gly Gln Cys Gly Gly Gln Gly Trp
465 470 475 480
Thr Gly Pro Thr Ala Cys Gln Ser Pro Ser Thr Cys His Val Ile Asn
485 490 495
Asp Phe Tyr Ser Gln Cys Phe
500
<210> 4
<211> 1512
<212> DNA
<213>artificial sequence
<400> 4
caacaggcag gaacagctac agccgaaaat catcccccgt taacatggca agaatgtacg 60
gcacccggtt cctgtacaac acagaatggt gcagtagtat tagatgccaa ttggcgttgg 120
gtgcatgatg ttaacgggta tactaactgt tataccggaa atacctggga ccccacatac 180
tgcccagacg atgaaacgtg cgctcaaaac tgtgcattgg atggtgcaga ttacgaagga 240
acttacggag tgacatcctc cggtagcagt ttaaaattga attttgtgac tgggtccaac 300
gttggttcaa gactgtatct attgcaggat gatagcacct atcagatttt caaacttttg 360
aatcgcgagt tcagtttcga cgttgatgtt tctaacttgc cttgcggttt aaatggtgct 420
ttatactttg ttgctatgga cgccgacggt ggtgtatcca agtaccccaa taacaaagcg 480
ggtgcgaagt atgggaccgg atactgtgac agccaatgtc caagagattt gaagtttatt 540
gatggcgaag ccaacgtaga aggctggcag cctagctcca acaacgcaaa taccggcata 600
ggagatcatg gctcatgttg tgcagaaatg gatgtttggg aggctaactc aatcagtaat 660
gctgttaccc cccatccatg cgatactcca ggacaaacga tgtgttccgg cgacgattgc 720
ggaggtacat attcgaatga tagatatgcc ggaacctgtg atcctgatgg ttgtgatttc 780
aacccatata gaatgggtaa cacgtctttt tatggtccgg gtaaaattat agatacaaca 840
aagccattca ctgttgttac ccagtttctt accgatgacg gtaccgacac agggacactt 900
agcgagatca aaagatttta tattcagaac tcaaacgtta ttcctcaacc aaatagtgac 960
ataagcggtg ttactggcaa ctctattacg actgaatttt gtacggctca aaaacaagcc 1020
tttggagata ccgatgattt tagtcagcat gggggactgg ctaaaatggg ggcagctatg 1080
caacagggta tggttttagt tatgtcatta tgggatgatt acgctgcaca aatgctttgg 1140
ttagattccg attacccgac tgatgccgat ccaacaaaac ctggtatcgc gcgtggaaca 1200
tgtccgactg actctggcgt tcctagcgac gttgaatctc agagtcctaa tagctatgtc 1260
acatactcca atataaaatt tggtcctatc aattcaacat tcaccgccag caatccccca 1320
ggtggtggta ctacaacaac aaccaccaca actaccagta agccgtcagg tccaacgaca 1380
actacgaacc catccggacc acagcagacg atgtggggac aatgcggggg tcaaggttgg 1440
accggtccta cagcctgtca gagtccttcg acctgtcacg taatcaacga cttttactct 1500
caatgtttct aa 1512
<210> 5
<211> 503
<212> PRT
<213>artificial sequence
<400> 5
Gln Gln Ala Gly Thr Ala Thr Ala Glu Asn His Pro Pro Leu Thr Trp
1 5 10 15
Gln Glu Cys Thr Ala Pro Gly Ser Cys Thr Thr Gln Asn Gly Ala Val
20 25 30
Val Leu Asp Ala Asn Trp Arg Trp Val His Asp Val Asn Gly Tyr Thr
35 40 45
Asn Cys Tyr Thr Gly Asn Thr Trp Asp Pro Thr Tyr Cys Pro Asp Asp
50 55 60
Glu Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr Glu Gly
65 70 75 80
Thr Tyr Gly Val Thr Ser Ser Gly Ser Ser Leu Lys Leu Asn Phe Val
85 90 95
Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Gln Asp Asp Ser
100 105 110
Thr Tyr Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe Ser Phe Asp Val
115 120 125
Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val
130 135 140
Ala Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Asn Asn Lys Ala
145 150 155 160
Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp
165 170 175
Leu Lys Phe Ile Asp Gly Glu Ala Asn Val Glu Gly Trp Gln Pro Ser
180 185 190
Ser Asn Asn Ala Asn Thr Gly Ile Gly Asp His Gly Ser Cys Cys Ala
195 200 205
Glu Met Asp Val Trp Glu Ala Asn Ser Ile Ser Asn Ala Val Thr Pro
210 215 220
His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Ser Gly Asp Asp Cys
225 230 235 240
Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr Cys Asp Pro Asp
245 250 255
Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Thr Ser Phe Tyr Gly
260 265 270
Pro Gly Lys Ile Ile Asp Thr Thr Lys Pro Phe Thr Val Val Thr Gln
275 280 285
Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr Leu Ser Glu Ile Lys
290 295 300
Arg Phe Tyr Ile Gln Asn Ser Asn Val Ile Pro Gln Pro Asn Ser Asp
305 310 315 320
Ile Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Glu Phe Cys Thr Ala
325 330 335
Gln Lys Gln Ala Phe Gly Asp Thr Asp Asp Phe Ser Gln His Gly Gly
340 345 350
Leu Ala Lys Met Gly Ala Ala Met Gln Gln Gly Met Val Leu Val Met
355 360 365
Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp Leu Asp Ser Asp
370 375 380
Tyr Pro Thr Asp Ala Asp Pro Thr Arg Pro Gly Ile Ala Arg Gly Thr
385 390 395 400
Cys Pro Thr Asp Ser Gly Val Pro Ser Asp Val Glu Ser Gln Ser Pro
405 410 415
Asn Ser Tyr Val Thr Tyr Ser Asn Ile Lys Phe Gly Pro Ile Asn Ser
420 425 430
Thr Phe Thr Ala Ser Asn Pro Pro Gly Gly Gly Thr Thr Thr Thr Thr
435 440 445
Thr Thr Thr Thr Ser Lys Pro Ser Gly Pro Thr Thr Thr Thr Asn Pro
450 455 460
Ser Gly Pro Gln Gln Thr Met Trp Gly Gln Cys Gly Gly Gln Gly Trp
465 470 475 480
Thr Gly Pro Thr Ala Cys Gln Ser Pro Ser Thr Cys His Val Ile Asn
485 490 495
Asp Phe Tyr Ser Gln Cys Phe
500
<210> 6
<211> 1512
<212> DNA
<213>artificial sequence
<400> 6
caacaggcag gaacagctac agccgaaaat catcccccgt taacatggca agaatgtacg 60
cacccggtt cctgtacaac acagaatggt gcagtagtat tagatgccaa ttggcgttgg 120
gtgcatgatg ttaacgggta tactaactgt tataccggaa atacctggga ccccacatac 180
tgcccagacg atgaaacgtg cgctcaaaac tgtgcattgg atggtgcaga ttacgaagga 240
acttacggag tgacatcctc cggtagcagt ttaaaattga attttgtgac tgggtccaac 300
gttggttcaa gactgtatct attgcaggat gatagcacct atcagatttt caaacttttg 360
aatcgcgagt tcagtttcga cgttgatgtt tctaacttgc cttgcggttt aaatggtgct 420
ttatactttg ttgctatgga cgccgacggt ggtgtatcca agtaccccaa taacaaagcg 480
ggtgcgaagt atgggaccgg atactgtgac agccaatgtc caagagattt gaagtttatt 540
gatggcgaag ccaacgtaga aggctggcag cctagctcca acaacgcaaa taccggcata 600
ggagatcatg gctcatgttg tgcagaaatg gatgtttggg aggctaactc aatcagtaat 660
gctgttaccc cccatccatg cgatactcca ggacaaacga tgtgttccgg cgacgattgc 720
ggaggtacat attcgaatga tagatatgcc ggaacctgtg atcctgatgg ttgtgatttc 780
aacccatata gaatgggtaa cacgtctttt tatggtccgg gtaaaattat agatacaaca 840
aagccattca ctgttgttac ccagtttctt accgatgacg gtaccgacac agggacactt 900
agcgagatca aaagatttta tattcagaac tcaaacgtta ttcctcaacc aaatagtgac 960
ataagcggtg ttactggcaa ctctattacg actgaatttt gtacggctca aaaacaagcc 1020
tttggagata ccgatgattt tagtcagcat gggggactgg ctaaaatggg ggcagctatg 1080
caacagggta tggttttagt tatgtcatta tgggatgatt acgctgcaca aatgctttgg 1140
ttagattccg attacccgac tgatgccgat ccaacacgcc ctggtatcgc gcgtggaaca 1200
tgtccgactg actctggcgt tcctagcgac gttgaatctc agagtcctaa tagctatgtc 1260
acatactcca atataaaatt tggtcctatc aattcaacat tcaccgccag caatccccca 1320
ggtggtggta ctacaacaac aaccaccaca actaccagta agccgtcagg tccaacgaca 1380
actacgaacc catccggacc acagcagacg atgtggggac aatgcggggg tcaaggttgg 1440
accggtccta cagcctgtca gagtccttcg acctgtcacg taatcaacga cttttactct 1500
caatgtttct aa 1512
<210> 7
<211> 503
<212> PRT
<213>artificial sequence
<400> 7
Gln Gln Ala Gly Thr Ala Thr Ala Glu Asn His Pro Pro Leu Thr Trp
1 5 10 15
Gln Glu Cys Thr Ala Pro Gly Ser Cys Thr Thr Gln Asn Gly Ala Val
20 25 30
Val Leu Asp Ala Asn Trp Arg Trp Val His Asp Val Asn Gly Tyr Thr
35 40 45
Asn Cys Tyr Thr Gly Asn Thr Trp Asp Pro Thr Tyr Cys Pro Asp Asp
50 55 60
Glu Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr Glu Gly
65 70 75 80
Thr Tyr Gly Val Thr Ser Ser Gly Ser Ser Leu Lys Leu Asn Phe Val
85 90 95
Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Gln Asp Asp Ser
100 105 110
Thr Tyr Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe Ser Phe Asp Val
115 120 125
Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val
130 135 140
Ala Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Asn Asn Lys Ala
145 150 155 160
Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp
165 170 175
Leu Lys Phe Ile Asp Gly Glu Ala Asn Val Glu Gly Trp Gln Pro Ser
180 185 190
Ser Asn Asn Ala Asn Thr Gly Ile Gly Asp His Gly Ser Cys Cys Ala
195 200 205
Glu Met Asp Val Trp Glu Ala Asn Ser Ile Ser Asn Ala Val Thr Pro
210 215 220
His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Ser Gly Asp Asp Cys
225 230 235 240
Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr Cys Asp Pro Asp
245 250 255
Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Thr Ser Phe Tyr Gly
260 265 270
Pro Gly Lys Ile Ile Asp Thr Thr Lys Pro Phe Thr Val Val Thr Gln
275 280 285
Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr Leu Ser Glu Ile Lys
290 295 300
Arg Phe Tyr Ile Gln Asn Ser Asn Val Ile Pro Gln Pro Asn Ser Asp
305 310 315 320
Ile Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Glu Phe Cys Thr Ala
325 330 335
Gln Lys Gln Ala Phe Gly Asp Thr Asp Asp Phe Ser Gln His Gly Gly
340 345 350
Leu Ala Lys Met Gly Ala Ala Met Gln Gln Gly Met Val Leu Val Met
355 360 365
Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp Leu Asp Ser Asp
370 375 380
Tyr Pro Thr Asp Ala Asp Pro Thr Asp Pro Gly Ile Ala Arg Gly Thr
385 390 395 400
Cys Pro Thr Asp Ser Gly Val Pro Ser Asp Val Glu Ser Gln Ser Pro
405 410 415
Asn Ser Tyr Val Thr Tyr Ser Asn Ile Lys Phe Gly Pro Ile Asn Ser
420 425 430
Thr Phe Thr Ala Ser Asn Pro Pro Gly Gly Gly Thr Thr Thr Thr Thr
435 440 445
Thr Thr Thr Thr Ser Lys Pro Ser Gly Pro Thr Thr Thr Thr Asn Pro
450 455 460
Ser Gly Pro Gln Gln Thr Met Trp Gly Gln Cys Gly Gly Gln Gly Trp
465 470 475 480
Thr Gly Pro Thr Ala Cys Gln Ser Pro Ser Thr Cys His Val Ile Asn
485 490 495
Asp Phe Tyr Ser Gln Cys Phe
500
<210> 8
<211> 1512
<212> DNA
<213>artificial sequence
<400> 8
caacaggcag gaacagctac agccgaaaat catcccccgt taacatggca agaatgtacg 60
gcacccggtt cctgtacaac acagaatggt gcagtagtat tagatgccaa ttggcgttgg 120
gtgcatgatg ttaacgggta tactaactgt tataccggaa atacctggga ccccacatac 180
tgcccagacg atgaaacgtg cgctcaaaac tgtgcattgg atggtgcaga ttacgaagga 240
acttacggag tgacatcctc cggtagcagt ttaaaattga attttgtgac tgggtccaac 300
gttggttcaa gactgtatct attgcaggat gatagcacct atcagatttt caaacttttg 360
aatcgcgagt tcagtttcga cgttgatgtt tctaacttgc cttgcggttt aaatggtgct 420
ttatactttg ttgctatgga cgccgacggt ggtgtatcca agtaccccaa taacaaagcg 480
ggtgcgaagt atgggaccgg atactgtgac agccaatgtc caagagattt gaagtttatt 540
gatggcgaag ccaacgtaga aggctggcag cctagctcca acaacgcaaa taccggcata 600
ggagatcatg gctcatgttg tgcagaaatg gatgtttggg aggctaactc aatcagtaat 660
gctgttaccc cccatccatg cgatactcca ggacaaacga tgtgttccgg cgacgattgc 720
ggaggtacat attcgaatga tagatatgcc ggaacctgtg atcctgatgg ttgtgatttc 780
aacccatata gaatgggtaa cacgtctttt tatggtccgg gtaaaattat agatacaaca 840
aagccattca ctgttgttac ccagtttctt accgatgacg gtaccgacac agggacactt 900
agcgagatca aaagatttta tattcagaac tcaaacgtta ttcctcaacc aaatagtgac 960
ataagcggtg ttactggcaa ctctattacg actgaatttt gtacggctca aaaacaagcc 1020
tttggagata ccgatgattt tagtcagcat gggggactgg ctaaaatggg ggcagctatg 1080
caacagggta tggttttagt tatgtcatta tgggatgatt acgctgcaca aatgctttgg 1140
ttagattccg attacccgac tgatgccgat ccaacagatc ctggtatcgc gcgtggaaca 1200
tgtccgactg actctggcgt tcctagcgac gttgaatctc agagtcctaa tagctatgtc 1260
acatactcca atataaaatt tggtcctatc aattcaacat tcaccgccag caatccccca 1320
ggtggtggta ctacaacaac aaccaccaca actaccagta agccgtcagg tccaacgaca 1380
actacgaacc catccggacc acagcagacg atgtggggac aatgcggggg tcaaggttgg 1440
accggtccta cagcctgtca gagtccttcg acctgtcacg taatcaacga cttttactct 1500
caatgtttct aa 1512
<210> 9
<211> 503
<212> PRT
<213>artificial sequence
<400> 9
Gln Gln Ala Gly Thr Ala Thr Ala Glu Asn His Pro Pro Leu Thr Trp
1 5 10 15
Gln Glu Cys Thr Ala Pro Gly Ser Cys Thr Thr Gln Asn Gly Ala Val
20 25 30
Val Leu Asp Ala Asn Trp Arg Trp Val His Asp Val Asn Gly Tyr Thr
35 40 45
Asn Cys Tyr Thr Gly Asn Thr Trp Asp Pro Thr Tyr Cys Pro Asp Asp
50 55 60
Glu Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr Glu Gly
65 70 75 80
Thr Tyr Gly Val Thr Ser Ser Gly Ser Ser Leu Lys Leu Asn Phe Val
85 90 95
Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Gln Asp Asp Ser
100 105 110
Thr Tyr Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe Ser Phe Asp Val
115 120 125
Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val
130 135 140
Ala Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Asn Asn Lys Ala
145 150 155 160
Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp
165 170 175
Leu Lys Phe Ile Asp Gly Glu Ala Asn Val Glu Gly Trp Gln Pro Ser
180 185 190
Ser Asn Asn Ala Asn Thr Gly Ile Gly Asp His Gly Ser Cys Cys Ala
195 200 205
Glu Met Asp Val Trp Glu Ala Asn Ser Ile Ser Asn Ala Val Thr Pro
210 215 220
His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Ser Gly Asp Asp Cys
225 230 235 240
Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr Cys Asp Pro Asp
245 250 255
Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Thr Ser Phe Tyr Gly
260 265 270
Pro Gly Lys Ile Ile Asp Thr Thr Lys Pro Phe Thr Val Val Thr Gln
275 280 285
Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr Leu Ser Glu Ile Lys
290 295 300
Arg Phe Tyr Ile Gln Asn Ser Asn Val Ile Pro Gln Pro Asn Ser Asp
305 310 315 320
Ile Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Glu Phe Cys Thr Ala
325 330 335
Gln Lys Gln Ala Phe Gly Asp Thr Asp Asp Phe Ser Gln His Gly Gly
340 345 350
Leu Ala Lys Met Gly Ala Ala Met Gln Gln Gly Met Val Leu Val Met
355 360 365
Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp Leu Asp Ser Asp
370 375 380
Tyr Pro Thr Asp Ala Asp Pro Thr Glu Pro Gly Ile Ala Arg Gly Thr
385 390 395 400
Cys Pro Thr Asp Ser Gly Val Pro Ser Asp Val Glu Ser Gln Ser Pro
405 410 415
Asn Ser Tyr Val Thr Tyr Ser Asn Ile Lys Phe Gly Pro Ile Asn Ser
420 425 430
Thr Phe Thr Ala Ser Asn Pro Pro Gly Gly Gly Thr Thr Thr Thr Thr
435 440 445
Thr Thr Thr Thr Ser Lys Pro Ser Gly Pro Thr Thr Thr Thr Asn Pro
450 455 460
Ser Gly Pro Gln Gln Thr Met Trp Gly Gln Cys Gly Gly Gln Gly Trp
465 470 475 480
Thr Gly Pro Thr Ala Cys Gln Ser Pro Ser Thr Cys His Val Ile Asn
485 490 495
Asp Phe Tyr Ser Gln Cys Phe
500
<210> 10
<211> 1512
<212> DNA
<213>artificial sequence
<400> 10
caacaggcag gaacagctac agccgaaaat catcccccgt taacatggca agaatgtacg 60
gcacccggtt cctgtacaac acagaatggt gcagtagtat tagatgccaa ttggcgttgg 120
gtgcatgatg ttaacgggta tactaactgt tataccggaa atacctggga ccccacatac 180
tgcccagacg atgaaacgtg cgctcaaaac tgtgcattgg atggtgcaga ttacgaagga 240
acttacggag tgacatcctc cggtagcagt ttaaaattga attttgtgac tgggtccaac 300
gttggttcaa gactgtatct attgcaggat gatagcacct atcagatttt caaacttttg 360
aatcgcgagt tcagtttcga cgttgatgtt tctaacttgc cttgcggttt aaatggtgct 420
ttatactttg ttgctatgga cgccgacggt ggtgtatcca agtaccccaa taacaaagcg 480
ggtgcgaagt atgggaccgg atactgtgac agccaatgtc caagagattt gaagtttatt 540
gatggcgaag ccaacgtaga aggctggcag cctagctcca acaacgcaaa taccggcata 600
ggagatcatg gctcatgttg tgcagaaatg gatgtttggg aggctaactc aatcagtaat 660
gctgttaccc cccatccatg cgatactcca ggacaaacga tgtgttccgg cgacgattgc 720
ggaggtacat attcgaatga tagatatgcc ggaacctgtg atcctgatgg ttgtgatttc 780
aacccatata gaatgggtaa cacgtctttt tatggtccgg gtaaaattat agatacaaca 840
aagccattca ctgttgttac ccagtttctt accgatgacg gtaccgacac agggacactt 900
agcgagatca aaagatttta tattcagaac tcaaacgtta ttcctcaacc aaatagtgac 960
ataagcggtg ttactggcaa ctctattacg actgaatttt gtacggctca aaaacaagcc 1020
tttggagata ccgatgattt tagtcagcat gggggactgg ctaaaatggg ggcagctatg 1080
caacagggta tggttttagt tatgtcatta tgggatgatt acgctgcaca aatgctttgg 1140
ttagattccg attacccgac tgatgccgat ccaacagaac ctggtatcgc gcgtggaaca 1200
tgtccgactg actctggcgt tcctagcgac gttgaatctc agagtcctaa tagctatgtc 1260
acatactcca atataaaatt tggtcctatc aattcaacat tcaccgccag caatccccca 1320
ggtggtggta ctacaacaac aaccaccaca actaccagta agccgtcagg tccaacgaca 1380
actacgaacc catccggacc acagcagacg atgtggggac aatgcggggg tcaaggttgg 1440
accggtccta cagcctgtca gagtccttcg acctgtcacg taatcaacga cttttactct 1500
caatgtttct aa 1512
<210> 11
<211> 503
<212> PRT
<213>artificial sequence
<400> 11
Gln Gln Ala Gly Thr Ala Thr Ala Glu Asn His Pro Pro Leu Thr Trp
1 5 10 15
Gln Glu Cys Thr Ala Pro Gly Ser Cys Thr Thr Gln Asn Gly Ala Val
20 25 30
Val Leu Asp Ala Asn Trp Arg Trp Val His Asp Val Asn Gly Tyr Thr
35 40 45
Asn Cys Tyr Thr Gly Asn Thr Trp Asp Pro Thr Tyr Cys Pro Asp Asp
50 55 60
Glu Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr Glu Gly
65 70 75 80
Thr Tyr Gly Val Thr Ser Ser Gly Ser Ser Leu Lys Leu Asn Phe Val
85 90 95
Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Gln Asp Asp Ser
100 105 110
Thr Tyr Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe Ser Phe Asp Val
115 120 125
Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val
130 135 140
Ala Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Asn Asn Lys Ala
145 150 155 160
Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp
165 170 175
Leu Lys Phe Ile Asp Gly Glu Ala Asn Val Glu Gly Trp Gln Pro Ser
180 185 190
Ser Asn Asn Ala Asn Thr Gly Ile Gly Asp His Gly Ser Cys Cys Ala
195 200 205
Glu Met Asp Val Trp Glu Ala Asn Ser Ile Ser Asn Ala Val Thr Pro
210 215 220
His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Ser Gly Asp Asp Cys
225 230 235 240
Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr Cys Asp Pro Asp
245 250 255
Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Thr Ser Phe Tyr Gly
260 265 270
Pro Gly Lys Ile Ile Asp Thr Thr Lys Pro Phe Thr Val Val Thr Gln
275 280 285
Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr Leu Ser Glu Ile Lys
290 295 300
Arg Phe Tyr Ile Gln Asn Ser Asn Val Ile Pro Gln Pro Asn Ser Asp
305 310 315 320
Ile Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Glu Phe Cys Thr Ala
325 330 335
Gln Lys Gln Ala Phe Gly Asp Thr Asp Asp Phe Ser Gln His Gly Gly
340 345 350
Leu Ala Lys Met Gly Ala Ala Met Gln Gln Gly Met Val Leu Val Met
355 360 365
Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp Leu Asp Ser Asp
370 375 380
Tyr Pro Thr Asp Ala Asp Pro Thr Gln Pro Gly Ile Ala Arg Gly Thr
385 390 395 400
Cys Pro Thr Asp Ser Gly Val Pro Ser Asp Val Glu Ser Gln Ser Pro
405 410 415
Asn Ser Tyr Val Thr Tyr Ser Asn Ile Lys Phe Gly Pro Ile Asn Ser
420 425 430
Thr Phe Thr Ala Ser Asn Pro Pro Gly Gly Gly Thr Thr Thr Thr Thr
435 440 445
Thr Thr Thr Thr Ser Lys Pro Ser Gly Pro Thr Thr Thr Thr Asn Pro
450 455 460
Ser Gly Pro Gln Gln Thr Met Trp Gly Gln Cys Gly Gly Gln Gly Trp
465 470 475 480
Thr Gly Pro Thr Ala Cys Gln Ser Pro Ser Thr Cys His Val Ile Asn
485 490 495
Asp Phe Tyr Ser Gln Cys Phe
500
<210> 12
<211> 1512
<212> DNA
<213>artificial sequence
<400> 12
caacaggcag gaacagctac agccgaaaat catcccccgt taacatggca agaatgtacg 60
gcacccggtt cctgtacaac acagaatggt gcagtagtat tagatgccaa ttggcgttgg 120
gtgcatgatg ttaacgggta tactaactgt tataccggaa atacctggga ccccacatac 180
tgcccagacg atgaaacgtg cgctcaaaac tgtgcattgg atggtgcaga ttacgaagga 240
acttacggag tgacatcctc cggtagcagt ttaaaattga attttgtgac tgggtccaac 300
gttggttcaa gactgtatct attgcaggat gatagcacct atcagatttt caaacttttg 360
aatcgcgagt tcagtttcga cgttgatgtt tctaacttgc cttgcggttt aaatggtgct 420
ttatactttg ttgctatgga cgccgacggt ggtgtatcca agtaccccaa taacaaagcg 480
ggtgcgaagt atgggaccgg atactgtgac agccaatgtc caagagattt gaagtttatt 540
gatggcgaag ccaacgtaga aggctggcag cctagctcca acaacgcaaa taccggcata 600
ggagatcatg gctcatgttg tgcagaaatg gatgtttggg aggctaactc aatcagtaat 660
gctgttaccc cccatccatg cgatactcca ggacaaacga tgtgttccgg cgacgattgc 720
ggaggtacat attcgaatga tagatatgcc ggaacctgtg atcctgatgg ttgtgatttc 780
aacccatata gaatgggtaa cacgtctttt tatggtccgg gtaaaattat agatacaaca 840
aagccattca ctgttgttac ccagtttctt accgatgacg gtaccgacac agggacactt 900
agcgagatca aaagatttta tattcagaac tcaaacgtta ttcctcaacc aaatagtgac 960
ataagcggtg ttactggcaa ctctattacg actgaatttt gtacggctca aaaacaagcc 1020
tttggagata ccgatgattt tagtcagcat gggggactgg ctaaaatggg ggcagctatg 1080
caacagggta tggttttagt tatgtcatta tgggatgatt acgctgcaca aatgctttgg 1140
ttagattccg attacccgac tgatgccgat ccaacacaac ctggtatcgc gcgtggaaca 1200
tgtccgactg actctggcgt tcctagcgac gttgaatctc agagtcctaa tagctatgtc 1260
acatactcca atataaaatt tggtcctatc aattcaacat tcaccgccag caatccccca 1320
ggtggtggta ctacaacaac aaccaccaca actaccagta agccgtcagg tccaacgaca 1380
actacgaacc catccggacc acagcagacg atgtggggac aatgcggggg tcaaggttgg 1440
accggtccta cagcctgtca gagtccttcg acctgtcacg taatcaacga cttttactct 1500
caatgtttct aa 1512
<210> 13
<211> 503
<212> PRT
<213>artificial sequence
<400> 13
Gln Gln Ala Gly Thr Ala Thr Ala Glu Asn His Pro Pro Leu Thr Trp
1 5 10 15
Gln Glu Cys Thr Ala Pro Gly Ser Cys Thr Thr Gln Asn Gly Ala Val
20 25 30
Val Leu Asp Ala Asn Trp Arg Trp Val His Asp Val Asn Gly Tyr Thr
35 40 45
Asn Cys Tyr Thr Gly Asn Thr Trp Asp Pro Thr Tyr Cys Pro Asp Asp
50 55 60
Glu Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr Glu Gly
65 70 75 80
Thr Tyr Gly Val Thr Ser Ser Gly Ser Ser Leu Lys Leu Asn Phe Val
85 90 95
Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Gln Asp Asp Ser
100 105 110
Thr Tyr Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe Ser Phe Asp Val
115 120 125
Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val
130 135 140
Ala Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Asn Asn Lys Ala
145 150 155 160
Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp
165 170 175
Leu Lys Phe Ile Asp Gly Glu Ala Asn Val Glu Gly Trp Gln Pro Ser
180 185 190
er Asn Asn Ala Asn Thr Gly Ile Gly Asp His Gly Ser Cys Cys Ala
195 200 205
Glu Met Asp Val Trp Glu Ala Asn Ser Ile Ser Asn Ala Val Thr Pro
210 215 220
His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Ser Gly Asp Asp Cys
225 230 235 240
Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr Cys Asp Pro Asp
245 250 255
Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Thr Ser Phe Tyr Gly
260 265 270
Pro Gly Lys Ile Ile Asp Thr Thr Lys Pro Phe Thr Val Val Thr Gln
275 280 285
Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr Leu Ser Glu Ile Lys
290 295 300
Arg Phe Tyr Ile Gln Asn Ser Asn Val Ile Pro Gln Pro Asn Ser Asp
305 310 315 320
Ile Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Glu Phe Cys Thr Ala
325 330 335
Gln Lys Gln Ala Phe Gly Asp Thr Asp Asp Phe Ser Gln His Gly Gly
340 345 350
Leu Ala Lys Met Gly Ala Ala Met Gln Gln Gly Met Val Leu Val Met
355 360 365
Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp Leu Asp Ser Asp
370 375 380
Tyr Pro Thr Asp Ala Asp Pro Thr Ala Pro Gly Ile Ala Arg Gly Thr
385 390 395 400
Cys Pro Thr Asp Ser Gly Val Pro Ser Asp Val Glu Ser Gln Ser Pro
405 410 415
Asn Ser Tyr Val Thr Tyr Ser Asn Ile Lys Phe Gly Pro Ile Asn Ser
420 425 430
Thr Phe Thr Ala Ser Asn Pro Pro Gly Gly Gly Thr Thr Thr Thr Thr
435 440 445
Thr Thr Thr Thr Ser Lys Pro Ser Gly Pro Thr Thr Thr Thr Asn Pro
450 455 460
Ser Gly Pro Gln Gln Thr Met Trp Gly Gln Cys Gly Gly Gln Gly Trp
465 470 475 480
Thr Gly Pro Thr Ala Cys Gln Ser Pro Ser Thr Cys His Val Ile Asn
485 490 495
Asp Phe Tyr Ser Gln Cys Phe
500
<210> 14
<211> 1512
<212> DNA
<213>artificial sequence
<400> 14
caacaggcag gaacagctac agccgaaaat catcccccgt taacatggca agaatgtacg 60
gcacccggtt cctgtacaac acagaatggt gcagtagtat tagatgccaa ttggcgttgg 120
gtgcatgatg ttaacgggta tactaactgt tataccggaa atacctggga ccccacatac 180
tgcccagacg atgaaacgtg cgctcaaaac tgtgcattgg atggtgcaga ttacgaagga 240
acttacggag tgacatcctc cggtagcagt ttaaaattga attttgtgac tgggtccaac 300
gttggttcaa gactgtatct attgcaggat gatagcacct atcagatttt caaacttttg 360
aatcgcgagt tcagtttcga cgttgatgtt tctaacttgc cttgcggttt aaatggtgct 420
ttatactttg ttgctatgga cgccgacggt ggtgtatcca agtaccccaa taacaaagcg 480
ggtgcgaagt atgggaccgg atactgtgac agccaatgtc caagagattt gaagtttatt 540
gatggcgaag ccaacgtaga aggctggcag cctagctcca acaacgcaaa taccggcata 600
ggagatcatg gctcatgttg tgcagaaatg gatgtttggg aggctaactc aatcagtaat 660
gctgttaccc cccatccatg cgatactcca ggacaaacga tgtgttccgg cgacgattgc 720
ggaggtacat attcgaatga tagatatgcc ggaacctgtg atcctgatgg ttgtgatttc 780
aacccatata gaatgggtaa cacgtctttt tatggtccgg gtaaaattat agatacaaca 840
aagccattca ctgttgttac ccagtttctt accgatgacg gtaccgacac agggacactt 900
agcgagatca aaagatttta tattcagaac tcaaacgtta ttcctcaacc aaatagtgac 960
ataagcggtg ttactggcaa ctctattacg actgaatttt gtacggctca aaaacaagcc 1020
tttggagata ccgatgattt tagtcagcat gggggactgg ctaaaatggg ggcagctatg 1080
caacagggta tggttttagt tatgtcatta tgggatgatt acgctgcaca aatgctttgg 1140
ttagattccg attacccgac tgatgccgat ccaacagccc ctggtatcgc gcgtggaaca 1200
tgtccgactg actctggcgt tcctagcgac gttgaatctc agagtcctaa tagctatgtc 1260
acatactcca atataaaatt tggtcctatc aattcaacat tcaccgccag caatccccca 1320
ggtggtggta ctacaacaac aaccaccaca actaccagta agccgtcagg tccaacgaca 1380
actacgaacc catccggacc acagcagacg atgtggggac aatgcggggg tcaaggttgg 1440
accggtccta cagcctgtca gagtccttcg acctgtcacg taatcaacga cttttactct 1500
caatgtttct aa 1512

Claims (8)

1. a kind of cellobiohydrolase mutant, protein of the enzyme for following (a) or (b):
(a) the 393rd threonine of the amino acid sequence as shown in SEQ ID NO:1 is substituted by other amino acid, described Other amino acid be lysine, arginine or glutamic acid,
(b) amino acid sequence in (a) is by replacing, missing or adding one or several amino acid and having cellobiose water Solve the protein as derived from (a) of enzymatic activity；
The activity of the cellobiohydrolase mutant provides the function of degradation in cellulose degradation.
2. the composition containing one or more of enzymes as described in claim 1.
3. a kind of DNA molecular, the DNA molecular encodes enzyme as claimed in claim 1 or 2.
4. DNA molecular as claimed in claim 3, the base sequence of the DNA molecular is as shown in SEQ ID NO:4.
5. a kind of recombinant vector is operably connected containing DNA molecular as claimed in claim 3 and with the DNA molecular Adjusting sequence for expression.
6. host cell, the host cell contains the load of recombination described in DNA molecular as claimed in claim 3 or claim 5 Body.
7. the production method of cellobiohydrolase mutant includes the following steps: to exist host cell as claimed in claim 6 It is cultivated in culture medium, production is based on the cellobiohydrolase mutant gene contained by recombinant vector in the host cell The cellobiohydrolase mutant of coding.
8. cellobiohydrolase mutant described in claim 1 or composition as claimed in claim 2 are hydrolyzed in cellulose In application.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102844430A (en) * 2010-02-11 2012-12-26 南方化学知识产权有限公司 Optimized cellulase enzymes
CN103068976A (en) * 2009-11-06 2013-04-24 诺维信股份有限公司 Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
CN103348005A (en) * 2011-01-31 2013-10-09 帝斯曼知识产权资产管理有限公司 Mutant cellobiohydrolase
WO2016138167A2 (en) * 2015-02-24 2016-09-01 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
CN107002056A (en) * 2014-09-05 2017-08-01 诺维信公司 Carbohydrate binding module variant and the polynucleotides for encoding them

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103068976A (en) * 2009-11-06 2013-04-24 诺维信股份有限公司 Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
CN102844430A (en) * 2010-02-11 2012-12-26 南方化学知识产权有限公司 Optimized cellulase enzymes
CN103348005A (en) * 2011-01-31 2013-10-09 帝斯曼知识产权资产管理有限公司 Mutant cellobiohydrolase
CN107002056A (en) * 2014-09-05 2017-08-01 诺维信公司 Carbohydrate binding module variant and the polynucleotides for encoding them
WO2016138167A2 (en) * 2015-02-24 2016-09-01 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same

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