CN107916257B

CN107916257B - T1 lipase mutant and application

Info

Publication number: CN107916257B
Application number: CN201810017359.8A
Authority: CN
Inventors: 王方华; 王永华; 张慧; 黄伟谦; 蓝东明; 杨博
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2018-01-09
Filing date: 2018-01-09
Publication date: 2020-07-28
Anticipated expiration: 2038-01-09
Also published as: CN107916257A

Abstract

The invention discloses a T1 lipase mutant and application thereof, wherein the mutant at least has any one of the following mutations at an amino acid substitution position in a sequence SEQ ID NO.1, wherein (1) the amino acid substitution position of the original amino acid-position-substituted amino acid is adopted to represent the amino acid L359S, I362E or V364N of single point mutation in the lipase mutant, and (2) the mutation is carried out by adopting an amino acid sequence fragment replacement mode, and the activity of the amino acid residue Asn-Ala-Phe-Ser at position 288 and 291 of a wild type T1 is replaced by Glu-Phe-Thr-L ys. mutant hydrolysis glyceride is improved to different degrees and is respectively 6, 4, 6, 3 and 5 times of the wild type T1, while the optimal reaction temperature and the pH of the mutant are not obviously changed.

Description

T1 lipase mutant and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a T1 lipase mutant with improved catalytic activity, which is obtained by utilizing a molecular biology technology.

Background

The lipase (EC3.1.1.3), namely triacylglycerol hydrolase, can catalyze and hydrolyze natural oil to generate fatty acid, glycerol and monoglyceride or diglyceride, the types of catalytic reactions involving the lipase are wide, including catalytic fat degradation, ester exchange, ester synthesis and the like, and the lipase is widely applied to the fields of feed additives, oil processing, Food industry, biomedicine, daily chemical industry and the like, the high-temperature resistant lipase has higher optimal reaction temperature and is very suitable for modification of refractory oil in industry, for example, palm stearin, the oil is quite cheap but contains higher calorie and saturated fatty acid and has a melting point as high as 44-56 ℃, so that the development of the high-temperature resistant lipase has important industrial application value in the field, the T1 lipase is a heat-resistant lipase derived from Geobacillus zanihou (Geobacillus zaile strain T1), can better hydrolyze medium-long-chain glyceride, the optimal reaction temperature of the heat-resistant lipase T29 is as high as possible, the optimal reaction temperature of 60 ℃, the heat-resistant lipase has better application in oil modification industrial application, the European strain T20129, the European strain, the lipase T20132, the lipase has been proved to be a high-resistant lipase, the industrial production cost of the wild strain lipase is proved, the wild strain of the enzyme, the wild strain lipase (EP 120, the strain of the strain, the strain of the strain, the strain of the strain, the strain of the strain, the strain of the strain, the strain of the.

Protein engineering is a feasible method capable of improving the characteristics of enzyme proteins, and the protein is subjected to rational modification and site-directed mutagenesis by means of molecular biology and bioinformatics, so that an enzyme mutant with improved performance is obtained. The method has the advantages of small workload and high probability of obtaining positive mutants, so the method is widely applied to the field of enzyme molecule modification.

Disclosure of Invention

The technical problem solved by the invention is to provide a T1 lipase mutant with improved activity.

The technical scheme of the invention is as follows:

the lipase mutant with improved activity is constructed by rational design, Geobacillus zalihae T1 lipase with an amino acid sequence of SEQ ID No.1(GenBank: AAO92067.2) is used as a starting parent, mutation sites are selected and subjected to calculation simulation of mutation amino acid mutation by analyzing a protein structure (PDB: 2DSN), and finally, the lipase mutant is constructed by a site-directed mutation method, the amino acid substitution position in the wild type T1 amino acid sequence SEQ ID NO: 1 is mutated, the amino acid of single-point mutation in the lipase mutant is represented by 'original amino acid-position-substituted amino acid', the first 3 heat-resistant T1 lipase mutants are T1L 359S, T I362E and T1V364N, the fourth mutant is mutated by adopting an amino acid sequence fragment substitution mode, the 288-amino acid residue Asn-Ala-Phe-Ser-291-Ser-to Glu-L ys, the fifth mutant is introduced into the wild type T865 mutant with Ser 291-288S 2, Ser-288S-based mutant, and the fifth mutant is expressed as Ser-9S-1, 11S-9, 11, 9, 1, 11, 1, 9, 11.

The amino acid sequences of the six mutants are respectively SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO.5, SEQ NO.6 and SEQ NO. 7.

The DNA sequences of the six mutants are respectively SEQ NO.9, SEQ NO.10, SEQ NO.11, SEQ NO.12, SEQ NO.13 and SEQ NO. 14.

The construction method of the six heat-resistant T1 lipase mutant gene recombinant expression plasmids and strains comprises the following steps:

(1) construction of six heat-resistant T1 lipase mutant expression plasmids

The first four mutants are mutated by a site-directed mutagenesis method for amplifying full-length plasmids by one-step PCR, firstly, wild type T1 recombinant expression plasmids pET23a-CBD-T1 are used as templates, and PCR amplification is respectively carried out on mutation primer pairs (T1-L359S-F/T1-L359S-R and the like) corresponding to the first four mutants, and amplification products are pET23a-CBD-T1 mutant recombinant expression plasmids containing corresponding mutation site T1 gene sequences.

Digesting the PCR product of the amplified gene by enzyme digestion of DpnI, converting the product into an escherichia coli E.coli DH5 α competent cell, and carrying out sequencing verification to obtain a mutant recombinant expression plasmid;

the fifth mutant T1S 288-291-L359S recombinant expression plasmid is obtained by performing one-step PCR with the first mutant L359S primer pair as the amplification primer and the fourth mutant T1S288-291 plasmid as the template, according to the above method, after obtaining the fourth mutant T1S288-291 recombinant expression plasmid.

The sixth mutant T1S 288-291-L359S-V364N recombinant expression plasmid is obtained by performing a second PCR using the third mutant V364N primer pair as an amplification primer, the fourth mutant T1S288-291 recombinant expression plasmid as a template, and the first mutant L359S primer pair as an amplification primer after the fourth mutant T1S288-291 recombinant expression plasmid is obtained.

(2) Preparation of recombinant strains using mutant expression plasmids:

using CaCl for the mutant expression plasmid of the step (1)₂Transforming and expressing host bacterium Escherichia coli B L21 (DE3) competent cells by transformation method, spreading the transformation solution on a plate containing 100 μ g/ml ampicillin L B, culturing at 37 deg.C for 12 hr to obtain Escherichia coli single colony as recombinant strain;

the corresponding primer sequences of each mutant in the step (1) are as follows:

T1-L359S-F 5'-GATATGGGAACGTACAACGTTGACCATTCTGAAATCATCG-3'

T1-L359S-R 5'-CGGATTCGGGTCAACGCCGATGATTTCAGAATGGTCAACGT-3'

T1-I362E-F 5'-GTTGACCATTTGGAAATCGAAGGCGTTGACCCGAATCC-3'

T1-I362E-R 5'-GGATTCGGGTCAACGCCTTCGATTTCCAAATGGTCAAC-3'

T1-V364N-F 5'-CCATTTGGAAATCATCGGCAATGACCCGAATCCGTCATTTG-3'

T1-V364N-R 5'-CAAATGACGGATTCGGGTCATTGCCGATGATTTCCAAATGG-3'

T1-S288-291-F

5'-CTCACAGGCAACCATTATCCCGAACTCGGAATGGAATTTACCAAAGCGGT-3'

T1-S288-291-R

5'-TACGAACCGAGAAACGGAGCGCATACGACCGCTTTGGTAAATTCCATTCCG-3'

the invention relates to a method for constructing a recombinant expression strain of a lipase mutant, which can adopt any appropriate vector, wherein the appropriate vector comprises but is not limited to prokaryotic expression vectors pET23a, pF L-B62 cl, pF L-B13 cl, pET28, pET22 and the like, and comprises but is not limited to eukaryotic expression vectors pPIC9K, pPICZ α, pPIC3.5K, pPICZ, pYDI or p L IZG7 and the like.

In the method for preparing the lipase mutant, the obtained lipase mutant gene can be expressed in prokaryotic cells or eukaryotic cells, wherein the prokaryotic cells comprise but are not limited to escherichia coli, bacillus subtilis and streptomyces. Such eukaryotic cells include, but are not limited to, Pichia pastoris, Saccharomyces cerevisiae, and Aspergillus niger, and extracellular expression in prokaryotic or eukaryotic cells can also be achieved using any other suitable method known in the art.

The enzyme activity and the optimum reaction temperature of the mutant are determined by an olive oil emulsification method, the enzyme activity of the T1L 359S, T1I362E, T1V364N, T1S288-291, T1S 288-291-L359S and T1S 288-291-L359S-V364N mutant are respectively improved by 6, 4, 6, 3 and 5 times compared with the wild type, and the optimum reaction temperature and the pH value are kept unchanged with the wild type.

The lipase mutant can be applied to industrial production of detergents, additives, foods, pharmacy, paper making, biological energy and the like.

Compared with the prior art, the invention has the following beneficial effects:

the mutants T1L 359S, T1I362E, T1V364N, T1S288-291 and T1S 288-291-L359 ST1S 288-291-L359S-V364N obtained by the invention are mutants with improved hydrolytic activity of heat-resistant T1 lipase, and meanwhile, the optimal reaction temperature and pH are not changed, the hydrolytic activity of the T1L 359S, T1I362E, T1V364N, T1S288-291 and T1S 288-291-L359S, T1S 288-291-L359S-V364N mutants is improved to different degrees after being modified, and the improved hydrolytic activity is respectively 6 times, 4 times, 6 times, 3 times and 5 times of wild-type T1 lipase, so that the mutant is more suitable for application in biochemical industry, in particular for modification of grease.

Drawings

FIG. 1 shows protein purification of lipase T1 and mutants, wherein the Marker lane is protein molecular weight Marker, and lanes 1-7 are purified wild-type T1, T1L 359S, T1I362E, T1V364N, T1S288-291, T1S 288-291-L359S and T1S 288-291-L359S-364N.

FIG. 2 is a graph showing the results of measuring the optimum reaction temperature of lipase T1 and the mutant.

FIG. 3 is a graph showing the results of pH measurement of the optimum reaction of lipase T1 and the mutant.

FIG. 4 is a graph showing the relative lipase activities of lipase T1 and the mutant, in which the activity of the Wild Type (WT) was 100%.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.

Example 1: construction of T1 Lipase wild type E.coli expression vector

According to the amino acid sequence (GenBank: AAO92067.2) of heat-resistant T1 lipase of Geobacillus thermophilus (Geobacillus zalihae) of Genbank, a signal peptide prediction analysis software is used for accurately predicting a signal peptide and deleting the signal peptide from the complete sequence to obtain a mature peptide coding sequence of the lipase GZE L, the amino acid sequence is shown as SEQ ID NO.1, Shanghai bio-engineering is entrusted to optimize the sequence according to the preference of escherichia coli codes, KpnI and XhoI enzyme cutting sites are respectively introduced at the upstream and downstream of the mature peptide coding sequence, a gene coding the amino acid sequence is synthesized by an artificial synthesis method, after gene products are purified by a DNA purification kit, restriction enzymes KpnI and XhoI are respectively used for double enzyme cutting digestion of a purified gene fragment and pET 23-CBD, the gene fragments are connected and transformed into escherichia coli DH pE 48364 competent cells, the escherichia coli E39L B (containing 100ug/m positive penicillin) is coated on a plate L ampicillin, and the wild plasmid DNA sequence is cloned by a double enzyme cutting plasmid 6778, and the wild plasmid DNA sequence is detected, and the wild plasmid is obtained.

Example 2: construction of T1 lipase amino acid site mutant

Constructing mutants T1L 359S, T1I362E, T1V364N and T1S288-291 by adopting a site-directed mutagenesis technology, introducing a target mutagenesis site by a one-step PCR method, respectively carrying out PCR reaction by using mutation primer pairs (T1-L359S-F/T1-L359S-R and the like) corresponding to the first 4 mutants by using a T1 wild-type gene pET23a-CBD-T1 as a template, and carrying out site-directed mutagenesis reaction conditions:

primers used, see table below:

PCR amplification conditions: 3min at 94 ℃; 30 cycles of 10s at 98 ℃, 15s at 58 ℃ and 5min at 72 ℃; 10min at 72 ℃.

The PCR amplification product was then digested with DpnI enzyme at 37 ℃ for 2h, conditions for the DpnI digestion reaction:

coli DH5 α competent cells, spread on L B (containing 100ug/m L ampicillin) plates, after 12h of growth, 10 single colonies on the plates were picked for colony PCR, and positive clones were picked for gene sequencing, which indicated that the correct mutant expression plasmids pET23 a-CBD-T1-L359S, pET23a-CBD-T1-I362E, pET23a-CBD-T1-V364N and pET23a-CBD-T1-S288-291 were obtained.

The recombinant expression plasmid of the fifth mutant T1S 288-291-L359S is obtained by performing one-step PCR using the first mutant L359S primer pair (T1-L359S-F/T1-L359S-R) and the fourth mutant pET23a-CBD-T1S288-291 plasmid as a template after obtaining the fourth mutant T1S288-291 recombinant expression plasmid of the fourth mutant T1S 288-291.

Example 3: recombinant expression and purification of lipase T1 and mutant thereof

The mutant expression plasmid is transformed into E.coli B L21 (DE3) competent cells, the colony PCR is carried out on L B (containing 100 mug/m L ampicillin) plates, 10 single colonies on the plates are picked after 15h of growth, and positive clones are picked and subjected to gene sequencing, so that the correct T1-L359S, T1-I362E, T1-V364N, T1-S288-291, T1S 288-291-L359S and T1S 288-291-L359S-V364N mutant positive Escherichia coli B L21 (DE3) strains are obtained.

Wild type T1 expressing strain and each T1 lipase mutant strain are inoculated into 5ml L B (containing 100 mug/M L ampicillin) culture medium, after shaking culture at 37 ℃ and 200rpm for 12h, 2ml of bacterial liquid is inoculated into 200M L L B (containing 100 mug/M L ampicillin) culture medium, after shaking culture at 20 ℃ and 200rpm until OD600 is equal to 0.6, 40 mug L M IPTG is added for induction expression, after shaking culture at 20 ℃ and 200rpm for 30h, T1 wild type and mutant strains fermented bacterial cells are collected, PBS buffer (137mM NaCl,2.7mM KCl,10mM Na2HPO4,2mM KH2PO4, pH7.4) with the volume of original fermentation liquor of 40% is used for resuspension, the crushed bacterial cells are ultrasonically crushed for 10.11000g, 10min, after centrifugation at 4 ℃, the crushed supernatant is collected, then the supernatant is removed by 0.22μm concentration on nickel column, after filtration, the supernatant is eluted by using filter column, the eluent of the eluent containing target protein is further eluted by using 100mM filter (see the flow rate of HCl-8. mu. M) and the eluent is eluted by using Tris-pHG, the eluent of the target protein is eluted by using 100mM filter (see the eluent) and the eluent, the eluent of the eluent, the eluent of the eluent, the eluent is further, the eluent, the.

Example 4: activity measurement of lipase T1 and mutant thereof

The method comprises the steps of measuring the activity of wild lipase GZE L and mutant lipase by adopting an olive oil emulsification method, adding a certain amount of olive oil into a certain amount of 4% polyvinyl alcohol solution according to the volume ratio of the olive oil to the 4% polyvinyl alcohol solution of 1:3, homogenizing by using a high-speed homogenizer, and using after complete homogenization, wherein the lipase activity is defined as the enzyme amount required by protease catalyzed hydrolysis of the olive oil substrate emulsion substrate to generate 1 micromole (mu mo L) of free fatty acid at a specific temperature and pH condition every minute, namely a lipase activity unit, and is represented by U.

The lipase activity is measured by the following conditions that 1ml of enzyme solution +4ml of substrate (olive oil emulsion) +5ml of 50mM Tris-HC L Buffer pH 9.0 is subjected to shaking table reaction in water bath at 60 ℃ for 15min, then 10m L95% ethanol is added to stop the reaction, 2 drops of phenolphthalein are added, 50mM NaOH is used for titration, and the volume delta V of consumed 50mM NaOH solution is recorded (NaOH), and the enzyme activity is calculated according to the following formula that the enzyme activity (U/m L) is 50 × delta V (NaOH)/15.

(1) Determination of optimum reaction temperature of enzyme

The optimum reaction temperature conditions of the wild type lipase and the mutant lipase are determined as follows: according to the method for measuring the lipase activity, under the condition that the pH value of a fixed reaction system is 9.0, olive oil substrate emulsion is used as a substrate, buffer solutions with corresponding pH values are added, the reaction system is placed under different temperature conditions for reaction (30.0-80.0 ℃), and the lipase activity of mutant enzyme liquid and wild enzyme liquid under different reaction temperature conditions is measured. The control and sample groups were run in triplicate. And calculating the relative enzyme activity of the lipase under different temperature conditions by taking the highest point of the respective activity as 100%. The temperature set point is used as the abscissa and the relative activity is used as the ordinate to plot. As shown in FIG. 2, the optimum reaction temperatures of the wild-type lipase and the mutant T1 lipase were 60 ℃ and were not significantly changed.

(2) pH determination of enzyme optimum reaction

The method for determining the optimum reaction pH condition of the activity of the wild type lipase and the mutant lipase comprises the following steps: according to the method for measuring the lipase activity, the fixed reaction temperature condition is 60 ℃, olive oil substrate emulsion is used as a substrate, buffer solutions (4.0-8.0) with different pH values (the buffer solutions with different pH values are pH 5.0, 0.05M citric acid-0.05M sodium citrate, pH6.0-7.0, 0.05M sodium dihydrogen phosphate-0.05M disodium hydrogen phosphate, pH8.0-9.0, 0.05M Tris-HCl and pH 10.0, 0.05M Gly-NaOH) are added, the enzyme is reacted under different pH environmental conditions, and the lipase activity of each mutant enzyme solution under different pH environmental conditions is measured. The control and sample groups were run in triplicate. The pH value is plotted on the abscissa and the corresponding enzyme specific activity is plotted on the ordinate. And calculating the relative enzyme activity of the lipase under different pH conditions by taking the highest point of the activity as 100%. As a result, as shown in FIG. 3, the optimum pH of the wild-type T1 lipase was 9.0 for each mutant, and the site mutation did not cause a change in the optimum pH.

As shown in FIG. 4, the activity of each mutant was increased 6, 4, 6, 3, and 5 times higher than that of the wild type under the conditions of the optimum reaction temperature and the optimum reaction pH.

In conclusion, compared with the wild type lipase, the lipase mutant obtained by the invention has the advantages that the enzyme activity of the lipase mutant is obviously improved under the condition of not changing the optimal action temperature and pH, the lipase mutant is more suitable for being applied to the industrial fields of detergents, additives, foods, pharmacy, papermaking, biological energy sources and the like, and has wide market space.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> university of southern China's science

<120> T1 lipase mutant and application

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<170>SIPOSequenceListing 1.0

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Ala Ser Leu Arg Ala Asn Asp Ala Pro Ile Val Leu Leu His Gly Phe

1 5 10 15

Thr Gly Trp Gly Arg Glu Glu Met Phe Gly Phe Lys Tyr Trp Gly Gly

20 25 30

Val Arg Gly AspIle Glu Gln Trp Leu Asn Asp Asn Gly Tyr Arg Thr

35 40 45

Tyr Thr Leu Ala Val Gly Pro Leu Ser Ser Asn Trp Asp Arg Ala Cys

50 55 60

Glu Ala Tyr Ala Gln Leu Val Gly Gly Thr Val Asp Tyr Gly Ala Ala

65 70 75 80

His Ala Ala Lys His Gly His Ala Arg Phe Gly Arg Thr Tyr Pro Gly

85 90 95

Leu Leu Pro Glu Leu Lys Arg Gly Gly Arg Ile His Ile Ile Ala His

100 105 110

Ser Gln Gly Gly Gln Thr Ala Arg Met Leu Val Ser Leu Leu Glu Asn

115 120 125

Gly Ser Gln Glu Glu Arg Glu Tyr Ala Lys Ala His Asn Val Ser Leu

130 135 140

Ser Pro Leu Phe Glu Gly Gly His His Phe Val Leu Ser Val Thr Thr

145 150 155 160

Ile Ala Thr Pro His Asp Gly Thr Thr Leu Val Asn Met Val Asp Phe

165 170 175

Thr Asp Arg Phe Phe Asp Leu Gln Lys Ala Val Leu Glu Ala Ala Ala

180 185 190

Val Ala Ser Asn Val Pro Tyr Thr Ser Gln Val Tyr Asp Phe Lys Leu

195 200 205

Asp Gln Trp Gly Leu Arg Arg Gln Pro Gly Glu Ser Phe Asp His Tyr

210 215 220

Phe Glu Arg Leu Lys Arg Ser Pro Val Trp Thr Ser Thr Asp Thr Ala

225 230 235 240

Arg Tyr Asp Leu Ser Val Ser Gly Ala Glu Lys Leu Asn Gln Trp Val

245 250 255

Gln Ala Ser Pro Asn Thr Tyr Tyr Leu Ser Phe Ser Thr Glu Arg Thr

260 265 270

Tyr Arg Gly Ala Leu Thr Gly Asn His Tyr Pro Glu Leu Gly Met Asn

275 280 285

Ala Phe Ser Ala Val Val Cys Ala Pro Phe Leu Gly Ser Tyr Arg Asn

290 295 300

Pro Thr Leu Gly Ile Asp Asp Arg Trp Leu Glu Asn Asp Gly Ile Val

305 310 315 320

Asn Thr Val Ser Met Asn Gly Pro Lys Arg Gly Ser Ser Asp Arg Ile

325 330 335

Val Pro Tyr Asp Gly Thr Leu Lys Lys Gly Val Trp Asn Asp Met Gly

340 345350

Thr Tyr Asn Val Asp His Leu Glu Ile Ile Gly Val Asp Pro Asn Pro

355 360 365

Ser Phe Asp Ile Arg Ala Phe Tyr Leu Arg Leu Ala Glu Gln Leu Ala

370 375 380

Ser Leu Gln Pro

385

<210>2

<211>388

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Ala Ser Leu Arg Ala Asn Asp Ala Pro Ile Val Leu Leu His Gly Phe

1 5 10 15

Thr Gly Trp Gly Arg Glu Glu Met Phe Gly Phe Lys Tyr Trp Gly Gly

20 25 30

Val Arg Gly Asp Ile Glu Gln Trp Leu Asn Asp Asn Gly Tyr Arg Thr

35 40 45

Tyr Thr Leu Ala Val Gly Pro Leu Ser Ser Asn Trp Asp Arg Ala Cys

50 55 60

Glu Ala Tyr Ala Gln Leu Val Gly Gly Thr Val Asp Tyr Gly Ala Ala

65 70 75 80

His Ala Ala Lys His Gly His Ala Arg Phe Gly Arg Thr Tyr Pro Gly

85 90 95

Leu Leu Pro Glu Leu Lys Arg Gly Gly Arg Ile His Ile Ile Ala His

100 105 110

Ser Gln Gly Gly Gln Thr Ala Arg Met Leu Val Ser Leu Leu Glu Asn

115 120 125

Gly Ser Gln Glu Glu Arg Glu Tyr Ala Lys Ala His Asn Val Ser Leu

130 135 140

Ser Pro Leu Phe Glu Gly Gly His His Phe Val Leu Ser Val Thr Thr

145 150 155 160

Ile Ala Thr Pro His Asp Gly Thr Thr Leu Val Asn Met Val Asp Phe

165 170 175

Thr Asp Arg Phe Phe Asp Leu Gln Lys Ala Val Leu Glu Ala Ala Ala

180 185 190

Val Ala Ser Asn Val Pro Tyr Thr Ser Gln Val Tyr Asp Phe Lys Leu

195 200 205

Asp Gln Trp Gly Leu Arg Arg Gln Pro Gly Glu Ser Phe Asp His Tyr

210 215 220

Phe Glu Arg Leu Lys Arg Ser Pro Val Trp Thr Ser Thr Asp Thr Ala

225 230 235 240

Arg Tyr Asp Leu Ser Val Ser Gly Ala Glu Lys Leu Asn Gln Trp Val

245 250 255

Gln Ala Ser Pro Asn Thr Tyr Tyr Leu Ser Phe Ser Thr Glu Arg Thr

260 265 270

Tyr Arg Gly Ala Leu Thr Gly Asn His Tyr Pro Glu Leu Gly Met Asn

275 280 285

Ala Phe Ser Ala Val Val Cys Ala Pro Phe Leu Gly Ser Tyr Arg Asn

290 295 300

Pro Thr Leu Gly Ile Asp Asp Arg Trp Leu Glu Asn Asp Gly Ile Val

305 310 315 320

Asn Thr Val Ser Met Asn Gly Pro Lys Arg Gly Ser Ser Asp Arg Ile

325 330 335

Val Pro Tyr Asp Gly Thr Leu Lys Lys Gly Val Trp Asn Asp Met Gly

340 345 350

Thr Tyr Asn Val Asp His Ser Glu Ile Ile Gly Val Asp Pro Asn Pro

355 360 365

Ser Phe Asp Ile Arg Ala Phe Tyr Leu Arg Leu Ala Glu Gln Leu Ala

370 375 380

Ser Leu Gln Pro

385

<210>3

<211>388

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Ala Ser Leu Arg Ala Asn Asp Ala Pro Ile Val Leu Leu His Gly Phe

1 5 10 15

Thr Gly Trp Gly Arg Glu Glu Met Phe Gly Phe Lys Tyr Trp Gly Gly

20 25 30

Val Arg Gly Asp Ile Glu Gln Trp Leu Asn Asp Asn Gly Tyr Arg Thr

35 40 45

Tyr Thr Leu Ala Val Gly Pro Leu Ser Ser Asn Trp Asp Arg Ala Cys

50 55 60

Glu Ala Tyr Ala Gln Leu Val Gly Gly Thr Val Asp Tyr Gly Ala Ala

65 70 75 80

His Ala Ala Lys His Gly His Ala Arg Phe Gly Arg Thr Tyr Pro Gly

85 90 95

Leu Leu Pro Glu Leu Lys Arg Gly Gly Arg Ile His Ile Ile Ala His

100 105 110

Ser Gln Gly Gly Gln Thr Ala Arg Met Leu Val Ser Leu Leu Glu Asn

115 120 125

Gly Ser Gln Glu Glu Arg Glu Tyr Ala Lys Ala His Asn Val Ser Leu

130 135 140

Ser Pro Leu Phe Glu Gly Gly His His Phe Val Leu Ser Val Thr Thr

145 150 155 160

Ile Ala Thr Pro His Asp Gly Thr Thr Leu Val Asn Met Val Asp Phe

165 170 175

Thr Asp Arg Phe Phe Asp Leu Gln Lys Ala Val Leu Glu Ala Ala Ala

180 185 190

Val Ala Ser Asn Val Pro Tyr Thr Ser Gln Val Tyr Asp Phe Lys Leu

195 200 205

Asp Gln Trp Gly Leu Arg Arg Gln Pro Gly Glu Ser Phe Asp His Tyr

210 215 220

Phe Glu Arg Leu Lys Arg Ser Pro Val Trp Thr Ser Thr Asp Thr Ala

225 230 235 240

Arg Tyr Asp Leu Ser Val Ser Gly Ala Glu Lys Leu Asn Gln Trp Val

245 250 255

Gln Ala Ser Pro Asn Thr Tyr Tyr Leu Ser Phe Ser Thr Glu Arg Thr

260 265 270

Tyr Arg Gly Ala Leu Thr Gly Asn His Tyr Pro Glu Leu Gly Met Asn

275 280 285

Ala Phe Ser Ala Val Val Cys Ala Pro Phe Leu Gly Ser Tyr Arg Asn

290 295 300

Pro Thr Leu Gly Ile Asp Asp Arg Trp Leu Glu Asn Asp Gly Ile Val

305 310 315 320

Asn Thr Val Ser Met Asn Gly Pro Lys Arg Gly Ser Ser Asp Arg Ile

325 330 335

Val Pro Tyr Asp Gly Thr Leu Lys Lys Gly Val Trp Asn Asp Met Gly

340 345 350

Thr Tyr Asn Val Asp His Leu Glu Ile Glu Gly Val Asp Pro Asn Pro

355 360 365

Ser Phe Asp Ile Arg Ala Phe Tyr Leu Arg Leu Ala Glu Gln Leu Ala

370 375 380

Ser Leu Gln Pro

385

<210>4

<211>388

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Ala Ser Leu Arg Ala Asn Asp Ala Pro Ile Val Leu Leu His Gly Phe

1 5 10 15

Thr Gly Trp Gly Arg Glu Glu Met Phe Gly Phe Lys Tyr Trp Gly Gly

20 25 30

Val Arg Gly Asp Ile Glu Gln Trp Leu Asn Asp Asn Gly Tyr Arg Thr

3540 45

Tyr Thr Leu Ala Val Gly Pro Leu Ser Ser Asn Trp Asp Arg Ala Cys

50 55 60

Glu Ala Tyr Ala Gln Leu Val Gly Gly Thr Val Asp Tyr Gly Ala Ala

65 70 75 80

His Ala Ala Lys His Gly His Ala Arg Phe Gly Arg Thr Tyr Pro Gly

85 90 95

Leu Leu Pro Glu Leu Lys Arg Gly Gly Arg Ile His Ile Ile Ala His

100 105 110

Ser Gln Gly Gly Gln Thr Ala Arg Met Leu Val Ser Leu Leu Glu Asn

115 120 125

Gly Ser Gln Glu Glu Arg Glu Tyr Ala Lys Ala His Asn Val Ser Leu

130 135 140

Ser Pro Leu Phe Glu Gly Gly His His Phe Val Leu Ser Val Thr Thr

145 150 155 160

Ile Ala Thr Pro His Asp Gly Thr Thr Leu Val Asn Met Val Asp Phe

165 170 175

Thr Asp Arg Phe Phe Asp Leu Gln Lys Ala Val Leu Glu Ala Ala Ala

180 185 190

Val Ala Ser Asn Val Pro Tyr Thr Ser Gln Val Tyr Asp Phe Lys Leu

195 200 205

Asp Gln Trp Gly Leu Arg Arg Gln Pro Gly Glu Ser Phe Asp His Tyr

210 215 220

Phe Glu Arg Leu Lys Arg Ser Pro Val Trp Thr Ser Thr Asp Thr Ala

225 230 235 240

Arg Tyr Asp Leu Ser Val Ser Gly Ala Glu Lys Leu Asn Gln Trp Val

245 250 255

Gln Ala Ser Pro Asn Thr Tyr Tyr Leu Ser Phe Ser Thr Glu Arg Thr

260 265 270

Tyr Arg Gly Ala Leu Thr Gly Asn His Tyr Pro Glu Leu Gly Met Asn

275 280 285

Ala Phe Ser Ala Val Val Cys Ala Pro Phe Leu Gly Ser Tyr Arg Asn

290 295 300

Pro Thr Leu Gly Ile Asp Asp Arg Trp Leu Glu Asn Asp Gly Ile Val

305 310 315 320

Asn Thr Val Ser Met Asn Gly Pro Lys Arg Gly Ser Ser Asp Arg Ile

325 330 335

Val Pro Tyr Asp Gly Thr Leu Lys Lys Gly Val Trp Asn Asp Met Gly

340 345 350

Thr Tyr Asn Val Asp His Leu Glu Ile Ile Gly Asn Asp Pro Asn Pro

355 360 365

Ser Phe Asp Ile Arg Ala Phe Tyr Leu Arg Leu Ala Glu Gln Leu Ala

370 375 380

Ser Leu Gln Pro

385

<210>5

<211>388

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>5

Ala Ser Leu Arg Ala Asn Asp Ala Pro Ile Val Leu Leu His Gly Phe

1 5 10 15

Thr Gly Trp Gly Arg Glu Glu Met Phe Gly Phe Lys Tyr Trp Gly Gly

20 25 30

Val Arg Gly Asp Ile Glu Gln Trp Leu Asn Asp Asn Gly Tyr Arg Thr

35 40 45

Tyr Thr Leu Ala Val Gly Pro Leu Ser Ser Asn Trp Asp Arg Ala Cys

50 55 60

Glu Ala Tyr Ala Gln Leu Val Gly Gly Thr Val Asp Tyr Gly Ala Ala

65 70 75 80

His Ala Ala Lys His Gly His Ala Arg Phe Gly Arg Thr Tyr Pro Gly

85 90 95

Leu Leu Pro Glu Leu Lys Arg Gly Gly Arg Ile His Ile Ile Ala His

100 105 110

Ser Gln Gly Gly Gln Thr Ala Arg Met Leu Val Ser Leu Leu Glu Asn

115 120 125

Gly Ser Gln Glu Glu Arg Glu Tyr Ala Lys Ala His Asn Val Ser Leu

130 135 140

Ser Pro Leu Phe Glu Gly Gly His His Phe Val Leu Ser Val Thr Thr

145 150 155 160

Ile Ala Thr Pro His Asp Gly Thr Thr Leu Val Asn Met Val Asp Phe

165 170 175

Thr Asp Arg Phe Phe Asp Leu Gln Lys Ala Val Leu Glu Ala Ala Ala

180 185 190

Val Ala Ser Asn Val Pro Tyr Thr Ser Gln Val Tyr Asp Phe Lys Leu

195 200 205

Asp Gln Trp Gly Leu Arg Arg Gln Pro Gly Glu Ser Phe Asp His Tyr

210 215 220

Phe Glu Arg Leu Lys Arg Ser Pro Val Trp Thr Ser Thr Asp Thr Ala

225 230 235 240

Arg Tyr Asp Leu Ser Val Ser Gly Ala Glu Lys Leu Asn Gln Trp Val

245 250 255

Gln Ala Ser Pro Asn Thr Tyr Tyr Leu Ser Phe Ser Thr Glu Arg Thr

260 265 270

Tyr Arg Gly Ala Leu Thr Gly Asn His Tyr Pro Glu Leu Gly Met Glu

275 280 285

Phe Thr Lys Ala Val Val Cys Ala Pro Phe Leu Gly Ser Tyr Arg Asn

290 295 300

Pro Thr Leu Gly Ile Asp Asp Arg Trp Leu Glu Asn Asp Gly Ile Val

305 310 315 320

Asn Thr Val Ser Met Asn Gly Pro Lys Arg Gly Ser Ser Asp Arg Ile

325 330 335

Val Pro Tyr Asp Gly Thr Leu Lys Lys Gly Val Trp Asn Asp Met Gly

340 345 350

Thr Tyr Asn Val Asp His Leu Glu Ile Ile Gly Val Asp Pro Asn Pro

355 360 365

Ser Phe Asp Ile Arg Ala Phe Tyr Leu Arg Leu Ala Glu Gln Leu Ala

370 375 380

Ser Leu Gln Pro

385

<210>6

<211>388

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Ala Ser Leu Arg Ala Asn Asp Ala Pro Ile Val Leu Leu His Gly Phe

1 5 10 15

Thr Gly Trp Gly Arg Glu Glu Met Phe Gly Phe Lys Tyr Trp Gly Gly

20 25 30

Val Arg Gly Asp Ile Glu Gln Trp Leu Asn Asp Asn Gly Tyr Arg Thr

35 40 45

Tyr Thr Leu Ala Val Gly Pro Leu Ser Ser Asn Trp Asp Arg Ala Cys

50 55 60

Glu Ala Tyr Ala Gln Leu Val Gly Gly Thr Val Asp Tyr Gly Ala Ala

65 70 75 80

His Ala Ala Lys His Gly His Ala Arg Phe Gly Arg Thr Tyr Pro Gly

85 90 95

Leu Leu Pro Glu Leu Lys Arg Gly Gly Arg Ile His Ile Ile Ala His

100 105 110

Ser Gln Gly Gly Gln Thr Ala Arg Met Leu Val Ser Leu Leu Glu Asn

115 120 125

Gly Ser Gln Glu Glu Arg Glu Tyr Ala Lys Ala His Asn Val Ser Leu

130 135 140

Ser Pro Leu Phe Glu Gly Gly His His Phe Val Leu Ser Val Thr Thr

145 150 155 160

Ile Ala Thr Pro His Asp Gly Thr Thr Leu Val Asn Met Val Asp Phe

165 170 175

Thr Asp Arg Phe Phe Asp Leu Gln Lys Ala Val Leu Glu Ala Ala Ala

180 185 190

Val Ala Ser Asn Val Pro Tyr Thr Ser Gln Val Tyr Asp Phe Lys Leu

195 200 205

Asp Gln Trp Gly Leu Arg Arg Gln Pro Gly Glu Ser Phe Asp His Tyr

210 215 220

Phe Glu Arg Leu Lys Arg Ser Pro Val Trp Thr Ser Thr Asp Thr Ala

225 230 235 240

Arg Tyr Asp Leu Ser Val Ser Gly Ala Glu Lys Leu Asn Gln Trp Val

245 250 255

Gln Ala Ser Pro Asn Thr Tyr Tyr Leu Ser Phe Ser Thr Glu Arg Thr

260 265 270

Tyr Arg Gly Ala Leu Thr Gly Asn His Tyr Pro Glu Leu Gly Met Glu

275 280 285

Phe Thr Lys Ala Val Val Cys Ala Pro Phe Leu Gly Ser Tyr Arg Asn

290 295 300

Pro Thr Leu Gly Ile Asp Asp Arg Trp Leu Glu Asn Asp Gly Ile Val

305 310 315 320

Asn Thr Val Ser Met Asn Gly Pro Lys Arg Gly Ser Ser Asp Arg Ile

325 330 335

Val Pro Tyr Asp Gly Thr Leu Lys Lys Gly Val Trp Asn Asp Met Gly

340 345 350

Thr Tyr Asn Val Asp His Ser Glu Ile Ile Gly Val Asp Pro Asn Pro

355 360 365

Ser Phe Asp Ile Arg Ala Phe Tyr Leu Arg Leu Ala Glu Gln Leu Ala

370 375 380

Ser Leu Gln Pro

385

<210>7

<211>388

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>7

Ala Ser Leu Arg Ala Asn Asp Ala Pro Ile Val Leu Leu His Gly Phe

1 5 10 15

Thr Gly Trp Gly Arg Glu Glu Met Phe Gly Phe Lys Tyr Trp Gly Gly

20 25 30

Val Arg Gly Asp Ile Glu Gln Trp Leu Asn Asp Asn Gly Tyr Arg Thr

35 40 45

Tyr Thr Leu Ala Val GlyPro Leu Ser Ser Asn Trp Asp Arg Ala Cys

50 55 60

Glu Ala Tyr Ala Gln Leu Val Gly Gly Thr Val Asp Tyr Gly Ala Ala

65 70 75 80

His Ala Ala Lys His Gly His Ala Arg Phe Gly Arg Thr Tyr Pro Gly

85 90 95

Leu Leu Pro Glu Leu Lys Arg Gly Gly Arg Ile His Ile Ile Ala His

100 105 110

Ser Gln Gly Gly Gln Thr Ala Arg Met Leu Val Ser Leu Leu Glu Asn

115 120 125

Gly Ser Gln Glu Glu Arg Glu Tyr Ala Lys Ala His Asn Val Ser Leu

130 135 140

Ser Pro Leu Phe Glu Gly Gly His His Phe Val Leu Ser Val Thr Thr

145 150 155 160

Ile Ala Thr Pro His Asp Gly Thr Thr Leu Val Asn Met Val Asp Phe

165 170 175

Thr Asp Arg Phe Phe Asp Leu Gln Lys Ala Val Leu Glu Ala Ala Ala

180 185 190

Val Ala Ser Asn Val Pro Tyr Thr Ser Gln Val Tyr Asp Phe Lys Leu

195 200 205

Asp Gln Trp Gly Leu Arg Arg Gln Pro Gly Glu Ser Phe Asp His Tyr

210 215 220

Phe Glu Arg Leu Lys Arg Ser Pro Val Trp Thr Ser Thr Asp Thr Ala

225 230 235 240

Arg Tyr Asp Leu Ser Val Ser Gly Ala Glu Lys Leu Asn Gln Trp Val

245 250 255

Gln Ala Ser Pro Asn Thr Tyr Tyr Leu Ser Phe Ser Thr Glu Arg Thr

260 265 270

Tyr Arg Gly Ala Leu Thr Gly Asn His Tyr Pro Glu Leu Gly Met Glu

275 280 285

Phe Thr Lys Ala Val Val Cys Ala Pro Phe Leu Gly Ser Tyr Arg Asn

290 295 300

Pro Thr Leu Gly Ile Asp Asp Arg Trp Leu Glu Asn Asp Gly Ile Val

305 310 315 320

Asn Thr Val Ser Met Asn Gly Pro Lys Arg Gly Ser Ser Asp Arg Ile

325 330 335

Val Pro Tyr Asp Gly Thr Leu Lys Lys Gly Val Trp Asn Asp Met Gly

340 345 350

Thr Tyr Asn Val Asp His Ser Glu Ile Ile Gly Asn Asp Pro Asn Pro

355 360 365

Ser Phe Asp Ile Arg Ala Phe Tyr Leu Arg Leu Ala Glu Gln Leu Ala

370 375 380

Ser Leu Gln Pro

385

<210>8

<211>1164

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gcatccctac gcgccaatga tgcaccgatt gtgcttctcc atgggtttac cggatgggga 60

cgagaggaaa tgtttggatt caagtattgg ggcggcgtgc gcggcgatat cgaacaatgg 120

ctgaacgaca acggttatcg aacgtatacg ctggcggtcg gaccgctctc aagcaactgg 180

gaccgggcgt gtgaagcgta tgctcagctt gtcggcggga cggtcgatta tggggcagcc 240

catgcggcaa agcacggcca tgcgcggttt ggccgcactt atcccggcct gttgccggaa 300

ttgaaaaggg gtggccgcat ccatatcatc gcccacagcc aaggggggca gacggcccgc 360

atgcttgtct cgctcctaga gaacggaagc caagaagagc gggagtacgc caaggcgcat 420

aacgtgtcgt tgtcaccgtt gtttgaaggt ggacatcatt ttgtgttgag tgtgacgacc 480

atcgccactc ctcatgacgg gacgacgctt gtcaacatgg ttgatttcac cgatcgcttt 540

tttgacttgc aaaaagcggt gttggaagcg gcggctgtcg ccagcaacgt gccgtacacg 600

agtcaagtat acgattttaa gctcgaccaa tggggactgc gccgccagcc gggtgaatcg 660

ttcgaccatt attttgaacg gctcaagcgc tcccctgttt ggacgtccac agataccgcc 720

cgctacgatt tatccgtttc cggagctgag aagttgaatc aatgggtgca agcaagcccg 780

aatacgtatt atttgagttt ctctacagaa cggacgtatc gcggagcgct cacaggcaac 840

cattatcccg aactcggaat gaatgcattc agcgcggtcg tatgcgctcc gtttctcggt 900

tcgtaccgca atccgacgct cggcattgac gaccgatggt tggagaacga tggcattgtc 960

aatacggttt ccatgaacgg tccaaagcgt ggatcaagcg atcggatcgt gccgtatgac 1020

gggacgttga aaaaaggggt ttggaatgat atgggaacgt acaacgttga ccatttggaa 1080

atcatcggcg ttgacccgaa tccgtcattt gatattcgcg ccttttattt gcggcttgcc 1140

gagcagttgg cgagcttgca gcct 1164

<210>9

<211>1164

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gcatccctac gcgccaatga tgcaccgatt gtgcttctcc atgggtttac cggatgggga 60

cgagaggaaa tgtttggatt caagtattgg ggcggcgtgc gcggcgatat cgaacaatgg 120

ctgaacgaca acggttatcg aacgtatacg ctggcggtcg gaccgctctc aagcaactgg 180

gaccgggcgt gtgaagcgta tgctcagctt gtcggcggga cggtcgatta tggggcagcc 240

catgcggcaa agcacggcca tgcgcggttt ggccgcactt atcccggcct gttgccggaa 300

ttgaaaaggg gtggccgcat ccatatcatc gcccacagcc aaggggggca gacggcccgc 360

atgcttgtct cgctcctaga gaacggaagc caagaagagc gggagtacgc caaggcgcat 420

aacgtgtcgt tgtcaccgtt gtttgaaggt ggacatcatt ttgtgttgag tgtgacgacc 480

atcgccactc ctcatgacgg gacgacgctt gtcaacatgg ttgatttcac cgatcgcttt 540

tttgacttgc aaaaagcggt gttggaagcg gcggctgtcg ccagcaacgt gccgtacacg 600

agtcaagtat acgattttaa gctcgaccaa tggggactgc gccgccagcc gggtgaatcg 660

ttcgaccatt attttgaacg gctcaagcgc tcccctgttt ggacgtccac agataccgcc 720

cgctacgatt tatccgtttc cggagctgag aagttgaatc aatgggtgca agcaagcccg 780

aatacgtatt atttgagttt ctctacagaa cggacgtatc gcggagcgct cacaggcaac 840

cattatcccg aactcggaat gaatgcattc agcgcggtcg tatgcgctcc gtttctcggt 900

tcgtaccgca atccgacgct cggcattgac gaccgatggt tggagaacga tggcattgtc 960

aatacggttt ccatgaacgg tccaaagcgt ggatcaagcg atcggatcgt gccgtatgac 1020

gggacgttga aaaaaggggt ttggaatgat atgggaacgt acaacgttga ccattctgaa 1080

atcatcggcg ttgacccgaa tccgtcattt gatattcgcg ccttttattt gcggcttgcc 1140

gagcagttgg cgagcttgca gcct 1164

<210>10

<211>1164

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gcatccctac gcgccaatga tgcaccgatt gtgcttctcc atgggtttac cggatgggga 60

cgagaggaaa tgtttggatt caagtattgg ggcggcgtgc gcggcgatat cgaacaatgg 120

ctgaacgaca acggttatcg aacgtatacg ctggcggtcg gaccgctctc aagcaactgg 180

gaccgggcgt gtgaagcgta tgctcagctt gtcggcggga cggtcgatta tggggcagcc 240

catgcggcaa agcacggcca tgcgcggttt ggccgcactt atcccggcct gttgccggaa 300

ttgaaaaggg gtggccgcat ccatatcatc gcccacagcc aaggggggca gacggcccgc 360

atgcttgtct cgctcctaga gaacggaagc caagaagagc gggagtacgc caaggcgcat 420

aacgtgtcgt tgtcaccgtt gtttgaaggt ggacatcatt ttgtgttgag tgtgacgacc 480

atcgccactc ctcatgacgg gacgacgctt gtcaacatgg ttgatttcac cgatcgcttt 540

tttgacttgc aaaaagcggt gttggaagcg gcggctgtcg ccagcaacgt gccgtacacg 600

agtcaagtat acgattttaa gctcgaccaa tggggactgc gccgccagcc gggtgaatcg 660

ttcgaccatt attttgaacg gctcaagcgc tcccctgttt ggacgtccac agataccgcc 720

cgctacgatt tatccgtttc cggagctgag aagttgaatc aatgggtgca agcaagcccg 780

aatacgtatt atttgagttt ctctacagaa cggacgtatc gcggagcgct cacaggcaac 840

cattatcccg aactcggaat gaatgcattc agcgcggtcg tatgcgctcc gtttctcggt 900

tcgtaccgca atccgacgct cggcattgac gaccgatggt tggagaacga tggcattgtc 960

aatacggttt ccatgaacgg tccaaagcgt ggatcaagcg atcggatcgt gccgtatgac 1020

gggacgttga aaaaaggggt ttggaatgat atgggaacgt acaacgttga ccatttggaa 1080

atcgaaggcg ttgacccgaa tccgtcattt gatattcgcg ccttttattt gcggcttgcc 1140

gagcagttgg cgagcttgca gcct 1164

<210>11

<211>1164

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

gcatccctac gcgccaatga tgcaccgatt gtgcttctcc atgggtttac cggatgggga 60

cgagaggaaa tgtttggatt caagtattgg ggcggcgtgc gcggcgatat cgaacaatgg 120

ctgaacgaca acggttatcg aacgtatacg ctggcggtcg gaccgctctc aagcaactgg 180

gaccgggcgt gtgaagcgta tgctcagctt gtcggcggga cggtcgatta tggggcagcc 240

catgcggcaa agcacggcca tgcgcggttt ggccgcactt atcccggcct gttgccggaa 300

ttgaaaaggg gtggccgcat ccatatcatc gcccacagcc aaggggggca gacggcccgc 360

atgcttgtct cgctcctaga gaacggaagc caagaagagc gggagtacgc caaggcgcat 420

aacgtgtcgt tgtcaccgtt gtttgaaggt ggacatcatt ttgtgttgag tgtgacgacc 480

atcgccactc ctcatgacgg gacgacgctt gtcaacatgg ttgatttcac cgatcgcttt 540

tttgacttgc aaaaagcggt gttggaagcg gcggctgtcg ccagcaacgt gccgtacacg 600

agtcaagtat acgattttaa gctcgaccaa tggggactgc gccgccagcc gggtgaatcg 660

ttcgaccatt attttgaacg gctcaagcgc tcccctgttt ggacgtccac agataccgcc 720

cgctacgatt tatccgtttc cggagctgag aagttgaatc aatgggtgca agcaagcccg 780

aatacgtatt atttgagttt ctctacagaa cggacgtatc gcggagcgct cacaggcaac 840

cattatcccg aactcggaat gaatgcattc agcgcggtcg tatgcgctcc gtttctcggt 900

tcgtaccgca atccgacgct cggcattgac gaccgatggt tggagaacga tggcattgtc 960

aatacggttt ccatgaacgg tccaaagcgt ggatcaagcg atcggatcgt gccgtatgac 1020

gggacgttga aaaaaggggt ttggaatgat atgggaacgt acaacgttga ccatttggaa 1080

atcatcggca atgacccgaa tccgtcattt gatattcgcg ccttttattt gcggcttgcc 1140

gagcagttgg cgagcttgca gcct 1164

<210>12

<211>1164

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

gcatccctac gcgccaatga tgcaccgatt gtgcttctcc atgggtttac cggatgggga 60

cgagaggaaa tgtttggatt caagtattgg ggcggcgtgc gcggcgatat cgaacaatgg 120

ctgaacgaca acggttatcg aacgtatacg ctggcggtcg gaccgctctc aagcaactgg 180

gaccgggcgt gtgaagcgta tgctcagctt gtcggcggga cggtcgatta tggggcagcc 240

catgcggcaa agcacggcca tgcgcggttt ggccgcactt atcccggcct gttgccggaa 300

ttgaaaaggg gtggccgcat ccatatcatc gcccacagcc aaggggggca gacggcccgc 360

atgcttgtct cgctcctaga gaacggaagc caagaagagc gggagtacgc caaggcgcat 420

aacgtgtcgt tgtcaccgtt gtttgaaggt ggacatcatt ttgtgttgag tgtgacgacc 480

atcgccactc ctcatgacgg gacgacgctt gtcaacatgg ttgatttcac cgatcgcttt 540

tttgacttgc aaaaagcggt gttggaagcg gcggctgtcg ccagcaacgt gccgtacacg 600

agtcaagtat acgattttaa gctcgaccaa tggggactgc gccgccagcc gggtgaatcg 660

ttcgaccatt attttgaacg gctcaagcgc tcccctgttt ggacgtccac agataccgcc 720

cgctacgatt tatccgtttc cggagctgag aagttgaatc aatgggtgca agcaagcccg 780

aatacgtatt atttgagttt ctctacagaa cggacgtatc gcggagcgct cacaggcaac 840

cattatcccg aactcggaat ggaatttacc aaagcggtcg tatgcgctcc gtttctcggt 900

tcgtaccgca atccgacgct cggcattgac gaccgatggt tggagaacga tggcattgtc 960

aatacggttt ccatgaacgg tccaaagcgt ggatcaagcg atcggatcgt gccgtatgac 1020

gggacgttga aaaaaggggt ttggaatgat atgggaacgt acaacgttga ccatttggaa 1080

atcatcggcg ttgacccgaa tccgtcattt gatattcgcg ccttttattt gcggcttgcc 1140

gagcagttgg cgagcttgca gcct 1164

<210>13

<211>1164

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

gcatccctac gcgccaatga tgcaccgatt gtgcttctcc atgggtttac cggatgggga 60

cgagaggaaa tgtttggatt caagtattgg ggcggcgtgc gcggcgatat cgaacaatgg 120

ctgaacgaca acggttatcg aacgtatacg ctggcggtcg gaccgctctc aagcaactgg 180

gaccgggcgt gtgaagcgta tgctcagctt gtcggcggga cggtcgatta tggggcagcc 240

catgcggcaa agcacggcca tgcgcggttt ggccgcactt atcccggcct gttgccggaa 300

ttgaaaaggg gtggccgcat ccatatcatc gcccacagcc aaggggggca gacggcccgc 360

atgcttgtct cgctcctaga gaacggaagc caagaagagc gggagtacgc caaggcgcat 420

aacgtgtcgt tgtcaccgtt gtttgaaggt ggacatcatt ttgtgttgag tgtgacgacc 480

atcgccactc ctcatgacgg gacgacgctt gtcaacatgg ttgatttcac cgatcgcttt 540

tttgacttgc aaaaagcggt gttggaagcg gcggctgtcg ccagcaacgt gccgtacacg 600

agtcaagtat acgattttaa gctcgaccaa tggggactgc gccgccagcc gggtgaatcg 660

ttcgaccatt attttgaacg gctcaagcgc tcccctgttt ggacgtccac agataccgcc 720

cgctacgatt tatccgtttc cggagctgag aagttgaatc aatgggtgca agcaagcccg 780

aatacgtatt atttgagttt ctctacagaa cggacgtatc gcggagcgct cacaggcaac 840

cattatcccg aactcggaat ggaatttacc aaagcggtcg tatgcgctcc gtttctcggt 900

tcgtaccgca atccgacgct cggcattgac gaccgatggt tggagaacga tggcattgtc 960

aatacggttt ccatgaacgg tccaaagcgt ggatcaagcg atcggatcgt gccgtatgac 1020

gggacgttga aaaaaggggt ttggaatgat atgggaacgt acaacgttga ccattctgaa 1080

atcatcggcg ttgacccgaa tccgtcattt gatattcgcg ccttttattt gcggcttgcc 1140

gagcagttgg cgagcttgca gcct 1164

<210>14

<211>1164

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

gcatccctac gcgccaatga tgcaccgatt gtgcttctcc atgggtttac cggatgggga 60

cgagaggaaa tgtttggatt caagtattgg ggcggcgtgc gcggcgatat cgaacaatgg 120

ctgaacgaca acggttatcg aacgtatacg ctggcggtcg gaccgctctc aagcaactgg180

gaccgggcgt gtgaagcgta tgctcagctt gtcggcggga cggtcgatta tggggcagcc 240

catgcggcaa agcacggcca tgcgcggttt ggccgcactt atcccggcct gttgccggaa 300

ttgaaaaggg gtggccgcat ccatatcatc gcccacagcc aaggggggca gacggcccgc 360

atgcttgtct cgctcctaga gaacggaagc caagaagagc gggagtacgc caaggcgcat 420

aacgtgtcgt tgtcaccgtt gtttgaaggt ggacatcatt ttgtgttgag tgtgacgacc 480

atcgccactc ctcatgacgg gacgacgctt gtcaacatgg ttgatttcac cgatcgcttt 540

tttgacttgc aaaaagcggt gttggaagcg gcggctgtcg ccagcaacgt gccgtacacg 600

agtcaagtat acgattttaa gctcgaccaa tggggactgc gccgccagcc gggtgaatcg 660

ttcgaccatt attttgaacg gctcaagcgc tcccctgttt ggacgtccac agataccgcc 720

cgctacgatt tatccgtttc cggagctgag aagttgaatc aatgggtgca agcaagcccg 780

aatacgtatt atttgagttt ctctacagaa cggacgtatc gcggagcgct cacaggcaac 840

cattatcccg aactcggaat ggaatttacc aaagcggtcg tatgcgctcc gtttctcggt 900

tcgtaccgca atccgacgct cggcattgac gaccgatggt tggagaacga tggcattgtc 960

aatacggttt ccatgaacgg tccaaagcgt ggatcaagcg atcggatcgt gccgtatgac 1020

gggacgttga aaaaaggggt ttggaatgat atgggaacgt acaacgttga ccattctgaa 1080

atcatcggca atgacccgaa tccgtcattt gatattcgcg ccttttattt gcggcttgcc 1140

gagcagttgg cgagcttgca gcct 1164

Claims

The T1 lipase mutant is characterized in that the amino acid sequence of the mutant is SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO.5, SEQ NO.6 or SEQ NO. 7.
2. The gene encoding the T1 lipase mutant as claimed in claim 1, wherein the gene sequence of the mutant is SEQ No.9, SEQ No.10, SEQ No.11, SEQ No.12, SEQ No.13 or SEQ No. 14.
3. The use of the T1 lipase mutant as claimed in claim 1, wherein the lipase mutant is used in detergents, additives, foods, pharmaceuticals, paper making or bioenergy.
4. The use of the T1 lipase mutant as claimed in claim 3, wherein the lipase mutant is used for modification of refractory grease.