CN111321132B

CN111321132B - Nitrilase mutant with improved reaction specificity and application thereof

Info

Publication number: CN111321132B
Application number: CN202010098911.8A
Authority: CN
Inventors: 郑仁朝; 张焱; 闻鹏飞; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2020-02-18
Filing date: 2020-02-18
Publication date: 2021-08-24
Anticipated expiration: 2040-02-18
Also published as: CN111321132A

Abstract

The invention discloses a nitrilase mutant with improved reaction specificity and application thereof, belonging to the technical field of enzyme engineering. The mutant is obtained by carrying out single-site mutation or multi-site mutation on 77 th site, 200 th site, 224 th site or 226 th site of rice nitrilase with an amino acid sequence shown as SEQ ID NO. 1. The invention obtains the mutant with obviously improved reaction specificity by modifying the rice nitrilase molecule with non-specificity, and is beneficial to directionally generating the target product. The mutant Y77E/R224S/V226R constructed by the invention catalyzes phenylacetonitrile, and the main reaction product is phenylacetamide which accounts for 94.8 percent of the product content; the constructed mutant K200R/R224W catalyzes phenylacetonitrile, and the main reaction product is phenylacetic acid which accounts for 95.1 percent of the product content. The nitrilase mutant after reaction specificity regulation has important industrial application potential in green synthesis of carboxylic acid and amide compounds.

Description

Nitrilase mutant with improved reaction specificity and application thereof

Technical Field

The invention relates to the technical field of enzyme engineering, in particular to a nitrilase reaction specificity-changed mutant and application thereof.

Background

Nitrile compounds are important raw materials of organic chemical industry, are easy to be converted into high-value-added chemicals such as amides, carboxylic acids, hydroxamic acids, ketones and the like, and are widely applied to the fields of chemical industry, pesticides, medicines and the like. The enzymatic hydrolysis and hydration of nitrile has the obvious advantages of high process efficiency, environmental friendliness, high chemical, regio-and stereoselectivity and the like, and becomes an important method for industrial synthesis of carboxylic acid and amide.

It is generally accepted that nitrilases only catalyze the hydrolysis of nitrile compounds to carboxylic acids. However, some nitrilases have nitrile hydration activity and catalyze the formation of the corresponding amides from nitrile compounds, such as Bradyrhizobium japonicum nitrilase which catalyzes the hydrolysis of beta-aminopropionitrile with proportions of beta-aminopropionamide up to 33% (J.mol. Catal. B: enzyme, 2015,115,113); pseudomonas sp.UW4 nitrilase catalyzes the hydrolysis of indole-3-acetonitrile, and the content of indole-3-acetamide is 4.3 times that of indole-3-acetic acid (appl.Environ.Microbiol.,2014,80, 4640).

The nitrilase has both nitrile hydrolysis and nitrile hydration activities, namely reaction non-specificity, so that the nitrilase faces challenges in biological organic synthesis and has application potential. On one hand, amide is formed in the nitrile hydration side reaction of nitrilase, so that the yield of carboxylic acid which is a product of nitrile hydrolysis reaction is reduced, and pressure is brought to a subsequent separation process; on the other hand, the synthesis of α -amino (hydroxy) amides is one of the least atom economical and greenish reaction types in the pharmaceutical industry. Nitrile hydratase is a natural biocatalyst that catalyzes the synthesis of amides, but is subject to cyanide ion (CN)^-) Strongly inhibited. When it catalyzes alpha-amino (hydroxy) nitriles, the strong ligands CN are present in aqueous solution in equilibrium with the dissociation of the ketone (aldehyde) and HCN^-The non-heme iron atom or the non-corrinoid cobalt atom competing for the nitrile hydratase active center forms a low spin complex, irreversibly inactivating the enzyme. The nitrilase active center does not need metal auxiliary group, can tolerate high-concentration cyanide ions, improves the amide synthesis capacity, can create novel nitrile hydratase, and develops a brand new way for efficiently synthesizing alpha-amino (hydroxyl) amide. Therefore, the specific regulation and control of the nitrilase reaction are of great significance for developing new nitrilase biosynthesis functions.

Scholars at home and abroad have already conducted some studies on the modification of the specificity of nitrilase reaction. Kiziak et al found that the substitution of alanine at position 165 of P.fluoroscens EBC191 nitrilase with an aromatic amino acid reduced the proportion of mandelamide in the product; whereas the proportion of mandelamide increases from 0.7% to 70% after mutation of tryptophan at position 164 of Alcaligenes faecalis nitrilase to alanine (appl. environ. microbiol.,2009,75, 5592). After mutation of tryptophan to alanine at position 168 of the Neurospora crassa nitrilase was found by marti nkov et al, the proportion of amide in the product increased substantially (j.mol.cat.b enzyme, 2012,77, 74-80.). Synechocystis sp.PCC6803 nitrilase is transformed by Jiangshuin and the like, a mutant F193N with the amide proportion reaching 73% is obtained, the amide synthesis amount is 35 times of that of a wild type, and the catalytic activity is 50% of that of the wild type (Catal. Sci. technol.,2017,7, 1122-1128). The research provides a useful reference for the specific regulation and control of nitrilase reaction, but the mutant cannot be applied to the industrial biocatalysis process due to incomplete strengthening or elimination of nitrile hydration activity.

Disclosure of Invention

The invention aims to carry out molecular modification on rice Oryza sativa nitrilase (OsNIT) to obtain a mutant with effectively regulated and controlled nitrilase reaction specificity, thereby meeting the requirement of industrial biocatalysis application.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention realizes effective regulation and control of the reaction specificity of the enzyme by carrying out single-site mutation or multi-site mutation on the 77 th site, the 200 th site, the 224 th site or the 226 th site of the rice nitrilase OsNIT with an amino acid sequence shown as SEQ ID NO.1 through molecular modification. Wherein the tyrosine Tyr at the 77 th position is mutated into glutamic acid Glu, histidine His and leucine Leu, the lysine Lys at the 200 th position is mutated into methionine Met and arginine Arg, the arginine Arg at the 224 th position is mutated into tryptophan Trp and serine Ser, and the valine Val at the 226 th position is mutated into arginine Arg.

The research of the invention shows that the three sites of 77 th site, 200 th site and 224 th site have larger influence on the reaction specificity of nitrilase, and the mutation of 226 th site can simultaneously increase the proportion of amide and improve the conversion rate.

The mutant can be obtained by site-directed saturation mutagenesis and multiple rounds of site-directed mutagenesis construction.

Specifically, the invention provides a nitrilase mutant with improved nitrile hydration activity, and the amino acid sequence of the mutant is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 11.

The 3 mutants with 77 site mutation obtained by the invention catalyze the reaction of phenylacetonitrile, the main product is phenylacetamide, and the good activity is retained. The amide proportion of the mutant Y77E product reaches 84.1%, the amide proportion of the mutant Y77H product reaches 69.5%, and the amide proportion of the mutant Y77L product reaches 94.1%. The further constructed Y77E/R224S/V226R triple mutant has the amide content of 94.8 percent in the product.

The invention provides application of the nitrilase mutant with improved nitrile hydration reaction activity in catalyzing benzyl cyanide to synthesize phenylacetamide.

The application comprises the steps of taking wet thalli obtained after fermentation culture of engineering bacteria containing nitrilase mutant coding genes or enzyme extracted after the wet thalli is crushed as a biocatalyst, taking phenylacetonitrile as a substrate, taking a buffer solution with the pH of 7.5-8.5 as a reaction medium, carrying out conversion reaction at the temperature of 30-60 ℃ and under the condition of 150-500 r/min, and taking reaction liquid for separation and purification after the reaction is finished to obtain phenylacetamide.

Preferably, the concentration of the substrate in the reaction system is 30mM, and the amount of the catalyst is 10g/L based on the weight of wet cells, wherein the water content of the wet cells is 88-92%.

Preferably, a Tris-HCl buffer solution of pH 8.0 is used, and the reaction is carried out at 30 ℃ for 30 min.

The invention provides a nitrilase mutant with improved nitrile hydrolysis reaction activity, and the amino acid sequence of the mutant is shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO. 10.

The invention obtains mutants with 2 site mutations at 200 site and 2 site mutations at 224 site, the main product of the catalytic phenylacetonitrile reaction is phenylacetic acid, and high activity is retained even activity is improved. The proportion of carboxylic acids in the mutant K200M product reached 77.4% and the proportion of carboxylic acids in the mutant K200R product reached 68.3%. The proportion of carboxylic acids in the mutant R224W product reached 82.1% and the proportion of carboxylic acids in the mutant R224S product reached 61.0%. The proportion of carboxylic acid in the constructed double mutant K200R/R224W product reaches 95.1 percent.

The invention provides application of the nitrilase mutant with improved nitrile hydrolysis reaction activity in catalyzing benzyl cyanide to synthesize phenylacetic acid.

The application comprises the following steps: wet thalli obtained after fermentation culture of engineering bacteria containing nitrilase mutant coding genes or enzyme extracted after the wet thalli are crushed is used as a biocatalyst, phenylacetonitrile is used as a substrate, a buffer solution with the pH of 7.5-8.5 is used as a reaction medium, conversion reaction is carried out at the temperature of 30-60 ℃ and the speed of 150-500 r/min, and reaction liquid is taken for separation and purification after the reaction is finished, so that phenylacetic acid is obtained.

The host cell of the engineering bacteria can adopt Escherichia coli BL 21.

The preparation method of the wet thallus comprises the following steps: inoculating the genetically engineered bacteria to LB liquid culture medium containing kanamycin (the final concentration is 50mg/L), and performing shake culture at 37 ℃ and 200rpm for 6-8 h; the seed solution was inoculated into a fresh LB liquid medium containing 50mg/L kanamycin at a volume ratio of 2%, and cultured with shaking at 37 ℃ and 180rpm until the OD of the cells was reached₆₀₀0.6-0.8, adding isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.1mM, performing induced culture at 28 ℃ and 180rpm for 10-12h, and centrifuging at 4 ℃ and 8000rpm for 10min to collect thalli cells.

The invention has the beneficial effects that:

the invention obtains the mutant with obviously improved reaction specificity by modifying the molecule of reaction non-specificity rice nitrilase OsNIT, and is beneficial to directionally generating a target product. The mutant Y77E/R224S/V226R constructed by the invention catalyzes phenylacetonitrile, and the main reaction product is phenylacetamide which accounts for 94.8 percent of the product content; the constructed mutant K200R/R224W catalyzes phenylacetonitrile, and the main reaction product is phenylacetic acid which accounts for 95.1 percent of the product content. The nitrilase mutant after reaction specificity regulation has important industrial application potential in green synthesis of carboxylic acid and amide compounds.

Drawings

FIG. 1 shows the reaction process of wild rice nitrilase OsNIT catalyzing phenylacetonitrile.

FIG. 2 shows the reaction progress of the double mutant K200R/R224W catalyzing phenylacetonitrile.

FIG. 3 shows the reaction progress of triple mutant Y77E/R224S/V226R catalyzing phenylacetonitrile.

FIG. 4 shows the results of the determination of the amide ratio of the reaction products of wild-type and mutant nitrilases.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

Example 1

1. Selection of nitrilase mutation site

When rice nitrilase (OsNIT, GenBank accession number: AB027054, amino acid sequence SEQ ID NO.1, nucleotide sequence SEQ ID NO.2) catalyzes the reaction of phenylacetonitrile, the ratio of amide to carboxylic acid in the product is close to 1:1, as shown in FIG. 1. The invention determines that the catalytic triad is 196Cys-71Glu-162Lys through bioinformatics analysis, and further screens 18 amino acid residues possibly influencing reaction specificity near a catalytic pocket: a72, A167, F175, E198, Y77, S229, T166, E169, I195, R224, S230, W197, H159, N199, K200, T220, A221, and V226.

The amino acid residues are subjected to site-directed saturation mutation respectively to obtain 6 mutation sites Y77, I195, W197, N199, K200, T220, R224 and V226 which have large influence on the specificity of catalytic reaction, wherein the nitrilase mutant containing the mutations at the Y77, K200, R224 and V226 sites retains or improves the catalytic activity of the enzyme while the specificity of the reaction is improved.

The invention finally determines that the nitrilase mutant with high reaction specificity and higher catalytic activity is obtained by site-directed saturation mutation and combined mutation of tyrosine Tyr at position 77, lysine Lys at position 200, Arg at position 224 and valine Val at position 226.

2. Construction and expression of nitrilase mutant recombinant bacteria

Designing a primer, and adding a His-tag label at the tail end of the rice nitrilase through PCR amplification to purify the nitrilase for later use.

A plasmid containing a rice nitrilase (OsNIT) gene is used as a template, and saturated mutation primer sequences are designed at the 77 th, 200 th, 224 th and 226 th positions as follows:

TABLE 1

Note: n is A/G/C/T, K is G/T, and M is A/C.

The template was amplified whole plasmid by PCR, PCR reaction (50 μ L): template DNA 0.5. mu.l, Forward primer 1. mu.l, Reverse primer 1. mu.l, 2 × Phanta Max buffer 25. mu.l, dNTP Max (10mM reach) 1. mu.l, Phanta Max Super-Fidelity DNA Polymerase 0.5. mu.l, ddH₂O 21μl。

Setting a PCR program: pre-denaturation at 96 ℃ for 2 min; denaturation at 96 ℃ for 10s, annealing at Tm +3 ℃ for 5s, extension at 72 ℃ for 6.5min, and 30 cycles; final extension at 72 ℃ for 10 min. Storing at 4 ℃.

After the PCR product is analyzed to be positive by 0.9 percent agarose gel electrophoresis, 20 mu L of PCR reaction solution is taken, 1 mu L of restriction enzyme Dpn I is added to carry out enzyme digestion for 0.5 to 1 hour at 37 ℃ to remove the template plasmid DNA, and inactivation is carried out for 15min at 65 ℃. Coli BL21(DE3) competent cells were transformed by heat shock, recovered and plated on LB plates containing kanamycin for overnight culture, and a library of about 200 clones of each plate was obtained. Selecting single colony to culture in LB liquid culture medium containing kanamycin resistance (final concentration is 50mg/L), extracting plasmid to sequence, and screening out the required target mutant.

Inoculating correctly sequenced mutant into LB culture medium containing kanamycin (50 mg/L final concentration), culturing at 37 deg.C for 6-8h, transferring into fresh LB liquid culture medium containing kanamycin (50 mg/L final concentration) at 2% (v/v) for amplification culture, and culturing at 37 deg.C and 180rpm until thallus OD₆₀₀When the concentration is about 0.6-0.8, IPTG (final concentration of 0.1mM) is added to the LB liquid medium, induction culture is carried out at 28 ℃ and 180rpm for 10-12h, and the bacterial cells are collected by centrifugation at 4 ℃ and 8000rpm for 10 min.

3. Measurement of reaction specificity and Activity of nitrilase mutant

And (3) performing activity determination on the recombinant escherichia coli obtained in the step 2. The reaction system comprises the following components: 10mL of Tris-HCl buffer (50mM, pH 8.0), 0.1g of wet cells, and 30mM of phenylacetonitrile (dissolved in methanol). The reaction mixture was reacted at 30 ℃ and 180rpm for 30 min. 1mL of the sample was taken, and 20. mu.L of 2M HCl was added to terminate the reaction, and the mixture was centrifuged at 12,000rpm for 1min to obtain a supernatant. The concentration and the proportion of phenylacetic acid and phenylacetamide in the product are analyzed by High Performance Liquid Chromatography (HPLC), and the enzyme activity is calculated.

The liquid chromatography adopts a C18 column, the column temperature is 40 ℃, the detection wavelength is 210nm, and the mobile phase is methanol: water 40:60 (containing 0.1% H)₃PO₄) The flow rate was 1 ml/min.

The single-point mutation result is analyzed by the method, and the mutant at the 77 th site can change the reaction specificity to lead the mutant to tend to generate amide, so that the catalytic activity is better retained. As shown in Table 2, the amide proportion in the mutant Y77E reaction product is improved to 84.2%, and the catalytic activity is 39.2% of that of the parent; the proportion of amide in the reaction product of the mutant Y77H is increased to 69.5%, and the catalytic activity is 46.1% of that of the parent strain; the amide proportion in the mutant Y77L reaction product is improved to 94.1%, and the catalytic activity is 15.0% of that of the parent.

TABLE 2 comparison of the specificity and Activity of the wild-type nitrilase reaction with the 77 th mutant

The mutants at the 200 th site and the 224 th site can change the reaction specificity to ensure that the reaction tends to generate carboxylic acid, and meanwhile, the catalytic activity is well preserved and even improved, as shown in the table 3, the proportion of the carboxylic acid in the reaction product of the mutant K200M is improved to 77.4 percent, and the catalytic activity is 95.0 percent of that of the parent strain; the proportion of carboxylic acid in the reaction product of the mutant K200R is improved to 68.3 percent, and the catalytic activity is 113.2 percent of that of the parent strain; the proportion of carboxylic acid in the reaction product of the mutant R224W is improved to 82.1 percent, and the catalytic activity is 209.4 percent of that of the parent strain; the proportion of carboxylic acid in the reaction product of the mutant R224S is improved to 61.0%, and the catalytic activity is 187.1% of that of the parent.

TABLE 3 comparison of the specificity and Activity of the reactions of wild-type nitrilases with the mutants at positions 200 and 224

Example 2

1. Construction and expression of nitrilase multiple mutant

Using the mutant plasmid with excellent reaction specificity or high enzyme activity obtained in example 1 as a template, combining with excellent mutation of other sites, performing PCR amplification of the whole plasmid in the PCR system of example 1 to obtain multiple mutants, performing heat shock transformation to introduce the mutants into e.coli BL21(DE3) competent cells to construct multiple mutant engineering bacteria, and expressing the constructed multiple mutants.

2. Product ratio and activity determination of wild type strain and nitrile hydrolase-containing multiple mutant strain

And (3) carrying out activity determination on the obtained nitrilase multiple mutant bacteria. The reaction system comprises the following components: 10mL of Tris-HCl buffer (50mM, pH 8.0), 10g/L of wet cells, and 30mM of phenylacetonitrile (dissolved in methanol). The reaction mixture was reacted at 30 ℃ for 30min, and 20. mu.L of 2M HCl was added to terminate the reaction, followed by centrifugation at 12,000rpm for 1min to collect the supernatant. The product was analyzed for phenylacetic acid and phenylacetamide concentrations by HPLC as described in example 1. The reaction progress of the phenylacetonitrile catalyzed by the multiple mutant nitrilases is shown in FIGS. 2 and 3. The results of the amide ratios and viability assays for the wild type and mutant nitrilase reaction products are shown in Table 4 and FIG. 4.

TABLE 4 comparison of the specificity and Activity of the wild-type nitrilase with the multiple mutants

The reaction specificity of the multiple mutants is obviously improved compared with that of the parent, wherein the proportion of carboxylic acid in the reaction product of the mutant K200R/R224W is improved from 49.6% to 95.1%, and the catalytic activity is 261% of that of the parent; and the amide proportion in the reaction product of the mutant Y77E/R224S/V226R is improved from 50.4% to 94.8%, and the catalytic activity is 22.4% of that of the parent.

Sequence listing

<110> Zhejiang industrial university

<120> nitrilase mutant with improved reaction specificity and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 362

<212> PRT

<213> Rice (Oryza sativa)

<400> 1

Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala

1 5 10 15

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

20 25 30

Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr

35 40 45

Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser

50 55 60

Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Tyr Pro Arg Gly

65 70 75 80

Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp

85 90 95

Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val

100 105 110

Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys

115 120 125

Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr

130 135 140

Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His

145 150 155 160

Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly

165 170 175

Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly

180 185 190

Ala Leu Ile Cys Trp Glu Asn Lys Met Pro Leu Leu Arg Thr Ala Leu

195 200 205

Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Arg

210 215 220

Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys

225 230 235 240

Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro

245 250 255

Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp

260 265 270

Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu

275 280 285

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

290 295 300

Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val

305 310 315 320

Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln

325 330 335

Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala

340 345 350

Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr

355 360

<210> 2

<211> 1086

<212> DNA

<213> Rice (Oryza sativa)

<400> 2

atggctatgg ttccgtctgg ttctggtggt ggtccgccgg ttatcgctga agttgaaatg 60

aacggtggtg ctacctctgg tgctgctacc gttcgtgcta ccgttgttca ggcttctacc 120

gttttctacg acaccccggc taccctggac aaagctgaac gtctgatcga agaagctgct 180

ggttacggtt ctcagctggt tgttttcccg gaagctttcg ttggtggtta cccgcgtggt 240

tctaccttcg gtttcggtgc taacatctct atcggtaacc cgaaagacaa aggtaaagaa 300

gaattccgta aataccacgc tgctgctatc gaagttccgg gtccggaagt tacccgtctg 360

gctgctatgg ctggtaaata caaagttttc ctggttatgg gtgttatcga acgtgaaggt 420

tacaccctgt actgctctgt tctgttcttc gacccgctgg gtcgttacct gggtaaacac 480

cgtaaactga tgccgaccgc tctggaacgt atcatctggg gtttcggtga cggttcgact 540

atcccggtgt acgacacgcc gctgggtaaa atcggtgctc tgatctgctg ggaaaacaaa 600

atgccgctgc tgcgtaccgc tctgtacggt aaaggtatcg aaatctactg cgctccgacc 660

gcggattctc gtcaggtatg gcaggcatct atgacccaca tcgctctgga aggtggttgc 720

ttcgttctgt ctgctaacca gttctgccgt cgtaaagact acccgccgcc gccggaatac 780

gttttcaccg gtctgggtga agaaccgtct ccggacaccg ttgtttgccc gggtggttct 840

gttatcatct ctccgtctgg tgaagttctg gctggtccga actacgaagg tgaagctctg 900

atcaccgctg acctggacct gggtgaaatc gttcgtgcta aattcgactt cgacgttgtt 960

ggtcactacg ctcgtccgga agttctgtct ctggttgtta acgaccagcc gcacctccca 1020

gttagcttca cctctgctgc ggaaaaaacc accgctgcta aatctgactc taccgctaaa 1080

ccgtac 1086

<210> 3

<211> 362

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala

1 5 10 15

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

20 25 30

Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr

35 40 45

Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser

50 55 60

Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Glu Pro Arg Gly

65 70 75 80

Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp

85 90 95

Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val

100 105 110

Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys

115 120 125

Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr

130 135 140

Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His

145 150 155 160

Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly

165 170 175

Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly

180 185 190

Ala Leu Ile Cys Trp Glu Asn Lys Met Pro Leu Leu Arg Thr Ala Leu

195 200 205

Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Arg

210 215 220

Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys

225 230 235 240

Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro

245 250 255

Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp

260 265 270

Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu

275 280 285

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

290 295 300

Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val

305 310 315 320

Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln

325 330 335

Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala

340 345 350

Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr

355 360

<210> 4

<211> 362

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala

1 5 10 15

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

20 25 30

Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr

35 40 45

Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser

50 55 60

Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly His Pro Arg Gly

65 70 75 80

Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp

85 90 95

Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val

100 105 110

Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys

115 120 125

Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr

130 135 140

Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His

145 150 155 160

Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly

165 170 175

Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly

180 185 190

Ala Leu Ile Cys Trp Glu Asn Lys Met Pro Leu Leu Arg Thr Ala Leu

195 200 205

Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Arg

210 215 220

Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys

225 230 235 240

Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro

245 250 255

Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp

260 265 270

Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu

275 280 285

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

290 295 300

Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val

305 310 315 320

Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln

325 330 335

Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala

340 345 350

Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr

355 360

<210> 5

<211> 362

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala

1 5 10 15

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

20 25 30

Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr

35 40 45

Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser

50 55 60

Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Leu Pro Arg Gly

65 70 75 80

Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp

85 90 95

Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val

100 105 110

Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys

115 120 125

Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr

130 135 140

Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His

145 150 155 160

Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly

165 170 175

Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly

180 185 190

Ala Leu Ile Cys Trp Glu Asn Lys Met Pro Leu Leu Arg Thr Ala Leu

195 200 205

Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Arg

210 215 220

Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys

225 230 235 240

Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro

245 250 255

Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp

260 265 270

Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu

275 280 285

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

290 295 300

Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val

305 310 315 320

Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln

325 330 335

Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala

340 345 350

Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr

355 360

<210> 6

<211> 362

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala

1 5 10 15

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

20 25 30

Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr

35 40 45

Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser

50 55 60

Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Tyr Pro Arg Gly

65 70 75 80

Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp

85 90 95

Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val

100 105 110

Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys

115 120 125

Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr

130 135 140

Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His

145 150 155 160

Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly

165 170 175

Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly

180 185 190

Ala Leu Ile Cys Trp Glu Asn Met Met Pro Leu Leu Arg Thr Ala Leu

195 200 205

Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Arg

210 215 220

Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys

225 230 235 240

Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro

245 250 255

Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp

260 265 270

Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu

275 280 285

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

290 295 300

Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val

305 310 315 320

Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln

325 330 335

Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala

340 345 350

Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr

355 360

<210> 7

<211> 362

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala

1 5 10 15

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

20 25 30

Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr

35 40 45

Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser

50 55 60

Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Tyr Pro Arg Gly

65 70 75 80

Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp

85 90 95

Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val

100 105 110

Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys

115 120 125

Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr

130 135 140

Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His

145 150 155 160

Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly

165 170 175

Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly

180 185 190

Ala Leu Ile Cys Trp Glu Asn Arg Met Pro Leu Leu Arg Thr Ala Leu

195 200 205

Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Arg

210 215 220

Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys

225 230 235 240

Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro

245 250 255

Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp

260 265 270

Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu

275 280 285

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

290 295 300

Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val

305 310 315 320

Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln

325 330 335

Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala

340 345 350

Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr

355 360

<210> 8

<211> 362

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala

1 5 10 15

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

20 25 30

Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr

35 40 45

Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser

50 55 60

Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Tyr Pro Arg Gly

65 70 75 80

Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp

85 90 95

Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val

100 105 110

Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys

115 120 125

Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr

130 135 140

Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His

145 150 155 160

Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly

165 170 175

Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly

180 185 190

Ala Leu Ile Cys Trp Glu Asn Lys Met Pro Leu Leu Arg Thr Ala Leu

195 200 205

Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Trp

210 215 220

Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys

225 230 235 240

Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro

245 250 255

Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp

260 265 270

Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu

275 280 285

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

290 295 300

Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val

305 310 315 320

Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln

325 330 335

Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala

340 345 350

Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr

355 360

<210> 9

<211> 362

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala

1 5 10 15

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

20 25 30

Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr

35 40 45

Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser

50 55 60

Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Tyr Pro Arg Gly

65 70 75 80

Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp

85 90 95

Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val

100 105 110

Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys

115 120 125

Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr

130 135 140

Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His

145 150 155 160

Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly

165 170 175

Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly

180 185 190

Ala Leu Ile Cys Trp Glu Asn Lys Met Pro Leu Leu Arg Thr Ala Leu

195 200 205

Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Ser

210 215 220

Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys

225 230 235 240

Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro

245 250 255

Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp

260 265 270

Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu

275 280 285

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

290 295 300

Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val

305 310 315 320

Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln

325 330 335

Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala

340 345 350

Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr

355 360

<210> 10

<211> 362

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala

1 5 10 15

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

20 25 30

Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr

35 40 45

Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser

50 55 60

Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Tyr Pro Arg Gly

65 70 75 80

Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp

85 90 95

Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val

100 105 110

Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys

115 120 125

Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr

130 135 140

Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His

145 150 155 160

Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly

165 170 175

Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly

180 185 190

Ala Leu Ile Cys Trp Glu Asn Arg Met Pro Leu Leu Arg Thr Ala Leu

195 200 205

Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Trp

210 215 220

Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys

225 230 235 240

Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro

245 250 255

Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp

260 265 270

Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu

275 280 285

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

290 295 300

Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val

305 310 315 320

Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln

325 330 335

Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala

340 345 350

Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr

355 360

<210> 11

<211> 362

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala

1 5 10 15

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

20 25 30

Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr

35 40 45

Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser

50 55 60

Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Glu Pro Arg Gly

65 70 75 80

Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp

85 90 95

Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val

100 105 110

Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys

115 120 125

Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr

130 135 140

Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His

145 150 155 160

Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly

165 170 175

Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly

180 185 190

Ala Leu Ile Cys Trp Glu Asn Lys Met Pro Leu Leu Arg Thr Ala Leu

195 200 205

Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Ser

210 215 220

Gln Arg Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys

225 230 235 240

Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro

245 250 255

Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp

260 265 270

Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu

275 280 285

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

290 295 300

Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val

305 310 315 320

Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln

325 330 335

Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala

340 345 350

Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr

355 360

Claims

1. A nitrilase mutant with improved reaction specificity is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO. 11.

2. The use of a nitrilase mutant with improved reaction specificity for catalyzing the synthesis of phenylacetamide from phenylacetonitrile according to claim 1, wherein the amino acid sequence of the mutant is shown in SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 11.

3. The use of claim 2, comprising: wet thalli obtained after fermentation culture of engineering bacteria containing nitrilase mutant coding genes or enzyme extracted after the wet thalli are crushed is used as a biocatalyst, phenylacetonitrile is used as a substrate, a buffer solution with the pH of 7.5-8.5 is used as a reaction medium, conversion reaction is carried out at the temperature of 30-60 ℃ and the speed of 150-500 r/min, and reaction liquid is taken for separation and purification after the reaction is finished, so that phenylacetamide is obtained.

4. The use according to claim 3, wherein the concentration of the substrate in the reaction system is 30mM, and the amount of the catalyst is 10g/L based on the weight of wet cells, wherein the water content of the wet cells is 88-92%.

5. The use of a nitrilase mutant with improved reaction specificity for catalyzing the synthesis of phenylacetic acid from phenylacetonitrile according to claim 1, wherein the amino acid sequence of the mutant is shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.

6. The use of claim 5, comprising: wet thalli obtained after fermentation culture of engineering bacteria containing nitrilase mutant coding genes or enzyme extracted after the wet thalli are crushed is used as a biocatalyst, phenylacetonitrile is used as a substrate, a buffer solution with the pH of 7.5-8.5 is used as a reaction medium, conversion reaction is carried out at the temperature of 30-60 ℃ and the speed of 150-500 r/min, and reaction liquid is taken for separation and purification after the reaction is finished, so that phenylacetic acid is obtained.

7. The use according to claim 6, wherein the concentration of the substrate in the reaction system is 30mM, and the amount of the catalyst is 10g/L based on the weight of wet cells, wherein the water content of the wet cells is 88-92%.