CN110714002B - Plant nitrilase mutant, coding gene and application thereof - Google Patents

Plant nitrilase mutant, coding gene and application thereof Download PDF

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CN110714002B
CN110714002B CN201810765047.5A CN201810765047A CN110714002B CN 110714002 B CN110714002 B CN 110714002B CN 201810765047 A CN201810765047 A CN 201810765047A CN 110714002 B CN110714002 B CN 110714002B
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郑仁朝
郑裕国
张琴
汤晓玲
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses a crucifer nitrilase mutant, belonging to the technical field of biological engineering. The method comprises the steps of embedding a 225-position 285-position peptide segment of an amino acid sequence of the Arabidopsis thaliana nitrilase into the Brassica rapa nitrilase with a deleted corresponding peptide segment to obtain a Brassica rapa/Arabidopsis thaliana nitrilase chimera, and further carrying out site-directed saturation mutation on a coding gene of the chimera BanIT to obtain a plant nitrilase mutant, wherein the plant nitrilase mutant is one or more than two of the following: (1) the mutation of L at the 223 th position is Q; (2) the 263 th H mutation is D; (3) the Q mutation at position 279 is E. The catalytic activity of the plant nitrilase mutant provided by the invention is improved by 2.23 times, the solubility of recombinant protein is greatly improved, the value of the enantioselectivity E is kept above 400, and the mutant has a good application prospect in the synthesis of (S) -3-cyano-5-methylhexanoic acid by efficiently catalyzing racemic isobutyl succinonitrile.

Description

Plant nitrilase mutant, coding gene and application thereof
Technical Field
The invention relates to the technical field of bioengineering, and particularly relates to a crucifer nitrilase mutant, a coding gene and application thereof in preparing pregabalin key chiral intermediate (S) -3-cyano-5-methylhexanoic acid by hydrolyzing racemic isobutylsuccinonitrile.
Background
Nitrile compounds are important intermediates for organic synthesis, can be used for synthesizing chemicals such as amide, carboxylic acid, hydroxamic acid and the like with higher added value and wider application range, and are widely applied to the industrial fields such as chemical industry, pesticides, medicines and the like. However, the chemical hydrolysis of nitrile usually requires strong acid (or strong base), high temperature, high pressure and other reaction conditions, and the environmental pollution is serious. The nitrilase biocatalysis has high chemical, regional and stereo selectivity, mild reaction conditions and little environmental pollution, meets the requirement of green sustainable development, and has wide application prospect in the field of organic chemical industry. Nitrilase has been successfully applied to the industrial production of fine and medicinal chemicals such as nicotinic acid, (R) -mandelic acid, 1, 5-dimethyl-2-piperidone and the like at present.
With the development of modern molecular biology technology and the demand of industrial production environment for biocatalysts, protein molecular modification has become a hot spot of current enzyme engineering research. The molecular modification technology plays an important role in the modification of application attributes such as nitrilase catalytic activity, substrate specificity, thermal stability and stereoselectivity.
Schreiner et al molecularly modify Alcaligenes faecalis nitrilase to obtain a mutant capable of efficiently catalyzing hydrolysis of (R) -2-chloro-mandelonitrile to synthesize (R) -2-chloro-mandelic acid (Enzyme Microb. Tech.,2010,47, 140-146). DeSantis et al, which have been used to modify nitrilase using DNA shuffling to obtain nitrilase mutants capable of catalyzing 3-hydroxyglutaronitrile to synthesize S-type and R-type products, respectively, have ee values greater than 95% and yields up to 98% (J.Am.chem.Soc.,2003,125, 11476-11477).
One challenge of exogenous gene expression in E.coli is that target proteins often form inactive inclusion bodies, which seriously affect the catalytic performance of the enzyme. The molecular modification technology can reduce the formation of inclusion bodies and improve the soluble expression of protein by replacing one or more amino acids. Xie et al successfully constructed a double mutant C40A/C60N (Biotechnol. Bioeng.,2009,102,20-28) with improved catalytic activity and protein soluble expression by 50% by replacing cysteine residues of the Lov D amino acid sequence.
Pregabalin (Pregabalin, PGB for short), chemically named (S) -3-aminomethyl-5-methylhexanoic acid, is an isobutyl substituent at position 3 of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) (angew. chem. int. ed.,2008,47,3500-3504), and is a main drug for treating diseases such as spinal cord injury, anxiety and epilepsy. The route for synthesizing pregabalin key chiral intermediate (S) -3-cyano-5-methylhexanoic acid ((S) -CMHA) through hydrolyzing racemic isobutyl succinonitrile (IBSN) in a nitrilase region in a stereoselective manner has the remarkable advantages of cheap raw materials, simple process, high atom economy and the like. However, the catalytic activity and stereoselectivity of nitrilase reported at present are low, and the requirement of industrial production cannot be met. Therefore, it is important to develop a novel and highly efficient nitrilase capable of efficiently separating IBSN.
Disclosure of Invention
The invention aims to provide a crucifer nitrilase mutant with improved solubility and catalytic activity, which meets the requirement of industrial production.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention embeds the 225-plus 285-bit peptide segment of the amino acid sequence of the Arabidopsis thaliana nitrilase (AaNIT) into the Brassica rapa nitrilase (BrNIT) lacking the corresponding peptide segment to obtain the Brassica rapa/Arabidopsis thaliana nitrilase chimera (BaNIT), and the amino acid sequence is shown as SEQ ID NO. 2. And further carrying out site-directed saturation mutagenesis on the encoding gene of the chimera BanIT to obtain the plant nitrilase mutant.
The plant nitrilase mutant is one or more than two of the following: (1) the mutation of L at the 223 th position is Q; (2) the 263 th H mutation is D; (3) the Q mutation at position 279 is E.
In particular, the amount of the solvent to be used,
the amino acid sequence of the BanIT-L223Q (L at the 223 th position is mutated into Q) is shown as SEQ ID NO. 4;
the amino acid sequence of the BanIT-H263D (H at the 263 th position is mutated into D) is shown as SEQ ID NO. 6;
the amino acid sequence of the BanIT-Q279E (Q at position 279 is mutated into E) is shown as SEQ ID NO. 8;
the amino acid sequence of the BaNIT-L223Q/H263D (L at the 223 position is mutated into Q, H at the 263 position is mutated into D) is shown as SEQ ID NO. 10;
the amino acid sequence of the BaNIT-L223Q/Q279E (the mutation of L at the 223 position is Q, and the mutation of Q at the 279 position is E) is shown as SEQ ID NO. 12;
the amino acid sequence of the BanIT-H263D/Q279E (H at the 263 position is mutated into D, Q at the 279 position is mutated into E) is shown as SEQ ID NO. 14;
the amino acid sequence of the BANIT-L223Q/H263D/Q279E (the mutation of L at position 223 to Q, the mutation of H at position 263 to D and the mutation of Q at position 279 to E) is shown as SEQ ID NO. 16.
Research shows that compared with wild nitrilase, the catalytic activity and stereoselectivity of the chimera (BanIT) to the substrate racemic IBSN are obviously improved. The solubility and catalytic activity of the plant nitrilase mutant are further improved compared with those of a chimera (BanIT).
Conservative substitution patterns for other amino acid positions of the plant nitrilase mutant, addition or deletion of one or more amino acids, amino terminal truncation, and carboxy terminal truncation are also included in the scope of the present invention.
The invention also provides a coding gene for coding the plant nitrilase mutant, and the nucleotide sequence of the coding gene is shown as SEQ ID NO.3 or SEQ ID NO.5 or SEQ ID NO.7 or SEQ ID NO.9 or SEQ ID NO.11 or SEQ ID NO.13 or SEQ ID NO. 15.
The invention also provides a recombinant vector containing the coding gene. Preferably, the original vector is pET28 b.
The invention also provides a recombinant gene engineering bacterium containing the recombinant vector. The recombinant vector transforms host cells to obtain recombinant genetic engineering bacteria, the host cells can be various conventional host cells in the field, and preferably, the host cells are Escherichia coli E.coli BL 21.
The invention also provides a preparation method for constructing the plant nitrilase mutant, which comprises the following steps:
(1) designing a PCR primer aiming at a sequence of a turnip nitrilase gene, and amplifying by using the PCR primer to obtain a DNA fragment I containing the nucleotide sequence 675-855 bit of the Arabidopsis thaliana nitrilase by using the cDNA of the Arabidopsis thaliana as a template;
(2) using a recombinant plasmid carrying a turnip nitrilase gene as a template, and obtaining a BrNIT plasmid fragment with the turnip nitrilase nucleotide sequence 678-858 bit deletion by utilizing reverse PCR amplification;
(3) recombining the DNA fragment I and the BrNIT plasmid fragment, then transforming the recombined product to host bacteria, and screening to obtain a recombined parent nitrilase expression strain, wherein the nucleotide sequence of the parent nitrilase is shown as SEQ ID NO. 1;
(4) designing a site-directed mutagenesis primer, and carrying out overlap extension PCR by using the recombinant plasmid carrying the parent nitrilase gene obtained in the step (3) as a template to obtain a single-site mutagenesis product of which the L at the 223 rd position is mutated into Q or the H at the 263 th position is mutated into D or the Q at the 279 th position is mutated into E in the parent nitrilase;
(5) performing overlap extension PCR by using the site-specific mutation primer by using the single-site mutation product as a template to obtain a double-site mutation product; then, taking the double-site mutation product as a template, and carrying out overlap extension PCR by using the fixed-point primer to obtain a three-site mutation product;
(6) and respectively transforming the single-site mutation product, the double-site mutation product and the three-site mutation product into host bacteria, screening to obtain a nitrilase mutant expression strain, and performing induced expression to obtain the plant nitrilase mutant.
In the steps (1) - (3), a one-step cloning method is adopted to embed the nucleotide sequence corresponding to the 225-285 th peptide segment of the Arabidopsis thaliana nitrilase (AaNIT) into the plasmid fragment of the Brassica rapa nitrilase (BrNIT) lacking the corresponding peptide segment, so as to obtain the parent nitrilase (BaNIT) engineering bacterium.
Wherein the primers required for amplifying the DNA fragment I:
an upstream primer: 5'-GAATGGCAGTCTTCTATGATGCACATCGC-3' (SEQ ID NO. 17);
a downstream primer: 5'-GAAGTTCGGACCAGCCAGAACCTGACCC-3' (SEQ ID NO. 18).
Primers required for amplification of BrNIT plasmid fragment:
an upstream primer: 5'-GCGATGTGCATCATAGAAGACTGCCATTC-3' (SEQ ID NO. 19);
a downstream primer: 5'-GGGTCAGGTTCTGGCTGGTCCGAACTTC-3' (SEQ ID NO. 20).
In steps (4) to (5), saturation site-directed mutagenesis was performed on the parent nitrilase gene.
Wherein Leu mutation at position 223 is a primer required by Gln:
an upstream primer: 5'-CAGTCTTCTATGCTGCACATCGCTCTGGAAGG-3' (SEQ ID NO. 21);
a downstream primer: 5'-CCTTCCAGAGCGATGTGCAGCATAGAAGACTG-3' (SEQ ID NO. 22).
His 263 position mutated to Asp required primer:
an upstream primer: 5'-CAACCAGGAAGACGACGCTATCGTTTCTCAGGG-3' (SEQ ID NO. 23);
a downstream primer: 5'-CCCTGAGAAACGATAGCGTCGTCTTCCTGGTTG-3' (SEQ ID NO. 24).
Primer required for mutation of Gln at position 279 to Glu:
an upstream primer: 5'-CATCTCTCCGCTGGGTCAGGTTCTGGCTGG-3' (SEQ ID NO. 25);
a downstream primer: 5'-CCAGCCAGAACCTGACCCAGCGGAGAGATG-3' (SEQ ID NO. 26).
Preferably, the original vector of the recombinant plasmid is pET28 b. The host bacterium is Escherichia coli E.coli BL 21.
Another purpose of the invention is to provide an application of the plant nitrilase mutant in preparing (S) -3-cyano-5-methylhexanoic acid by catalyzing racemic isobutyl succinonitrile.
The application is that wet thalli, wet thalli immobilized cells and enzyme or immobilized enzyme extracted after ultrasonic disruption of the wet thalli, which are obtained by carrying out fermentation culture on engineering bacteria containing plant nitrilase mutant coding genes, are used as catalysts, racemic isobutyl succinonitrile is used as a substrate, a buffer solution with the pH value of 6-10 is used as a reaction medium, water bath reaction is carried out at the temperature of 20-50 ℃ and the speed of 200-400rpm, and after the reaction is finished, the reaction liquid is separated and purified to obtain (S) -3-cyano-5-methylhexanoic acid.
The nitrilase mutant provided by the invention can be used in the form of whole cells of engineering bacteria, crude enzyme without purification, partially purified enzyme or completely purified enzyme. The nitrilase mutants of the invention may also be prepared as biocatalysts in the form of immobilized enzymes or immobilized cells using immobilization techniques known in the art.
Preferably, in the reaction system, the concentration of the substrate is 100-150g/L, and the dosage of the catalyst is 5-20g/L based on the weight of wet cells, wherein the water content of the wet cells is 70-90%.
Preferably, the reaction medium is Tris-HCl buffer solution with the pH value of 8.0, and the catalytic reaction temperature is 35 ℃.
Preferably, the wet thalli is recombinant engineering bacteria E.coli BL21(DE3)/pET28b-BaNIT-L223 2, E.coli BL21(DE3)/pET28b-BaNIT-H263D, E.coli BL21(DE3)/pET28b-BaNIT-Q279E, E.coli BL E (DE E)/pET 28E-BaNIT-L223E/H263E, E.coli BL21(DE E)/pET 28E-BaNIT-L223E/Q3669572, E.coli BL E (DE E)/BaNIT-L223E/Q279, E.coli BL E (DE E)/pET 28E-BaNIT-H263E/Q279, E.coli BL 72 (DE E)/pET 28-BaNIT-H263E/Q279/E.coli BL E/E.3672/E.coli BL E/279.
The fermentation culture method comprises the following steps: inoculating the recombinant engineering bacteria into LB liquid culture medium containing kanamycin (the final concentration is 50mg/L), and performing shake culture at 37 ℃ and 200rpm for 8 hours; 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 150rpm 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 induction culture at 28 ℃ and 150rpm for 10h, and centrifuging at 4 ℃ and 9000rpm for 10min to collect thalli cells. Washing twice with normal saline, and storing the thallus obtained by centrifugation in a refrigerator at-20 ℃.
The invention has the following beneficial effects:
compared with a parent nitrilase chimera (BanIT), the catalytic activity of the plant nitrilase mutant provided by the invention is improved by more than 1.2 times, and the plant nitrilase mutant has good application prospect in synthesizing (S) -3-cyano-5-methylhexanoic acid by efficiently catalyzing racemic isobutyl butanedinitrile. Especially triple mutant BanIT-L223Q/H263D/Q279E, the catalytic activity is improved by 2.23 times, the solubility of the recombinant protein is greatly improved, and the E value of the enantioselectivity is kept above 400.
The plant nitrilase mutant provided by the invention can be hydrolyzed by catalyzing high-concentration IBSN (100g/L) with a small amount of cells, the conversion rate can reach more than 48.0 percent (ee is more than 98.5 percent), the industrial production cost is greatly reduced, and the industrial production requirement of the pregabalin key chiral intermediate is met.
Drawings
FIG. 1 shows soluble expression of nitrilase mutants (SDS-PAGE gel electrophoresis), where M is marker with band positions representing 50kD, Lane 1 BanIT, Lane 2 BanIT-L223Q, Lane 3 BanIT-H263D, Lane 4 BanIT-Q279E, and Lane 5 BanIT-L223Q/H263D/Q279E.
FIG. 2 is a graph comparing nitrilase mutant L223Q/H263D/Q279E with parent BanIT and wild type BrNIT whole cell catalytic IBSN.
FIG. 3 is a diagram showing the reaction progress of nitrilase mutant L223Q/H263D/Q279E whole cell (5g/L) catalysis of IBSN (100g/L) for preparing (S) -CMHA.
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
Construction of parent nitrilase Gene
The key peptide segment 225-285 region is determined by comparing and analyzing nucleotide and amino acid sequences of nitrilase of cruciferous plants. The wild type turnip nitrilase (BrNIT) sequence is shown in GenBank accession number: ABM 55734.1; the wild type arabidopsis thaliana nitrilase (AaNIT) sequence is found in GenBank accession no: KFK 44999.
The nucleotide sequence corresponding to the peptide fragment of 225-285 bit of Arabidopsis thaliana nitrilase (AaNIT) is embedded into the plasmid fragment of the turnip nitrilase (BrNIT) with the deletion of the corresponding peptide fragment by adopting a one-step cloning method. Primers were designed as shown in Table 1.
Table 1: design table of primers for BanIT chimeric enzyme
Figure BDA0001728834670000051
The DNA fragment of the 225-285-bit peptide segment was cloned using the AaNIT nucleotide sequence as a template. PCR reaction System (50. mu.L) Template DNA<1μg，
Figure BDA0001728834670000052
Master Mix, upstream and downstream primers 0.2. mu.M each, remaining ddH2O was made up to total volume. PCR reaction parameters: (1) pre-denaturation at 94 ℃ for 5 min; (2) denaturation at 94 ℃ for 30 s; (3) annealing at 58 ℃ for 30 s; (4) extension at 72 ℃ for 10s, and 30 cycles of steps (2) - (4); (5) re-extension at 72 deg.C for 10min, and storage at 4 deg.C. And (3) carrying out agarose gel electrophoresis analysis on the PCR product, cutting and recovering the gel, inactivating the gel for 10min at 65 ℃, and standing the gel for later use at 4 ℃.
Meanwhile, the recombinant plasmid containing the BrNIT nucleotide sequence is used as a template to design a primer to amplify the BrNIT plasmid fragment lacking the 226-position 286-position peptide segment.
The vector linearization is obtained by adopting a reverse PCR amplification mode. PCR reaction system (50. mu.L) template DNA 0.1ng-1ng, 2 XPphanta Max Buffer, dNTPs (10mM each)0.2mM, upstream and downstream primers 0.2. mu.M, Phanta Max Super-Fidelity DNA Polymerase 1U, and remaining ddH2O was made up to total volume. PCR reaction parameters: (1) pre-denaturation at 95 ℃ for 30 s; (2) denaturation at 95 ℃ for 15 s; (3) annealing at 63 ℃ for 15 s; (4) extension at 72 ℃ for 6.0min, and circulation of steps (2) - (4) for 30 times; (5) completely extending at 72 deg.C for 5min, and storing at 4 deg.C. After the PCR product is analyzed by agarose gel electrophoresis, endonuclease Dpn I is added to digest for 3h at 37 ℃, and then the product is inactivated for 10min at 65 ℃.
Linearizing the BrNIT vector sequence with the deleted corresponding peptide fragment gene fragment, and introducing the terminal sequence of the linearized vector into the 5 ' end of the insert forward/reverse PCR primer, so that the 5 ' and 3 ' extreme ends of the PCR product respectively have sequences consistent with the two terminals of the linearized vector.
The insert and linearized vector obtained above were used with NanoDropTMAnd measuring the gene concentration by using an One/OneC ultramicro ultraviolet spectrophotometer, and calculating the addition amount of the linearized vector of each inserted peptide segment and the corresponding deletion peptide segment. Connecting a reaction system: linearized vector 0.03pmol, insert 0.06pmol, 5 × CE II Buffer4 μ L, Exnase II2 μ L, ddH2Oto 20 μ L. After mixing the PCR samples, the mixture was left at 37 ℃ and kept at temperature for 30min, and then cooled to 4 ℃.
The ligated PCR products were heat shock transformed into e.coli BL21(DH5 α) competent cells, and plated on solid LB plates containing kanamycin resistance after recovery. And (4) selecting a single colony, inoculating the single colony into an LB liquid culture medium for incubation, and extracting plasmid for sequencing. The correct sequencing result is the nucleotide sequence of the parent nitrilase gene, namely SEQ ID NO.1 (the amino acid sequence is SEQ ID NO.2) in the sequence table. The parent nitrilase gene is transformed into competent cells of Escherichia coli E.coli BL21(DE3), and the competent cells are spread on an LB plate containing kanamycin and cultured overnight to obtain the parent nitrilase engineering bacteria E.coli BL21(DE3)/pET28 b-BanIT.
Example 2
Site-directed saturation mutagenesis of nitrilase sites 223, 263 and 279
Corresponding primers were designed for saturation mutagenesis of leu (l) at position 223, his (h) at position 263 and gln (q) at position 279 in the parent amino acid sequence, as shown in table 2.
Table 2: primer design sheet
Figure BDA0001728834670000061
Note: n is A/G/C/T, K is G/T, and M is A/C.
The recombinant plasmid pET28b-BanIT containing the target gene fragment is used as a template, and the whole plasmid amplification is carried out on the template according to the method of overlap extension PCR.
The PCR amplification system was (50. mu.L): template DNA 0.1ng-1ng, 2X Phanta Max Buffer 25. mu.L, dNTPs (10mM each) 1. mu.L, mutation primer upstream and downstream 1. mu.L each, Phanta Max Super-Fidelity DNA Polymerase 1U, and the rest ddH2O was made up to total volume.
PCR reaction parameters: (1) pre-denaturation at 95 ℃ for 30 s; (2) denaturation at 95 ℃ for 15 s; (3) annealing at 63 ℃ for 15 s; (4) extending for 6min at 72 ℃, and circulating the steps (2) to (4) for 30 times; (5) completely extending at 72 deg.C for 5min, and storing at 4 deg.C.
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 endonuclease Dpn I is added to carry out enzyme digestion at 37 ℃ for 3h to remove the template plasmid DNA, and inactivation is carried out at 65 ℃ for 10 min. Coli bl21(DE3) competent cells were transformed by heat shock, plated on LB plates containing kanamycin after recovery and cultured overnight, and a library of about 300 clones of each plate was obtained.
Picking single colony to 96-hole culture plate with LB culture medium, culturing at 37 deg.C to thallus OD600When the concentration is about 0.6 to 0.8, IPTG (final concentration: 0.1mM) is added to the above LB liquid medium, and induction culture is carried out at 28 ℃ and 150rpm for 10 to 12 hours. Centrifuging with 96-well plate centrifuge at 4 deg.C and 3000rpm for 30min, discarding supernatant, adding 600 μ L sodium phosphate buffer (50mM, pH 7.4) into the collected thallus, and mixing.
200 μ L of the bacterial suspension was mixed with 10 μ L of IBSN (100mg/mL in N, N-dimethyl sulfoxide), reacted at 37 ℃ with shaking for 1h, and 30 μ L of 2M HCl was added to each well to stop the reaction. Sucking 10 μ L of reaction solution, mixing with 150 μ L of the composition of o-phthalaldehyde and mercaptoethanol, and incubating in a thermostat at 37 ℃ for 30 min; the fluorescence intensity of each sample was measured under a microplate reader, with the excitation wavelength set at 412nm and the emission wavelength set at 467 nm. According to the change of the fluorescence intensity, the catalytic activity of the mutant is judged, and then the clone with the change intensity far larger than that of a control strain (starting strain) is screened out.
After the screened positive clones are subjected to secondary screening verification, whole plasmids of mutant strains are extracted, introduced point mutation is determined through DNA sequencing, and the mutant strain DNA sequencing result with the highest activity of each site shows that Leu at position 223 is mutated into Gln (L223Q), His at position 263 is mutated into Asp (H263D) and Gln at position 279 is mutated into Glu (Q279E), so that nitrilase mutant engineering bacteria E.coli BL21(DE3)/pET28b-BanIT-L223Q, E.coli BL21(DE3)/pET28b-BanIT-H263D and E.coli BL21(DE3)/pET28b-BanIT-Q279E are obtained. The nucleotide sequences of mutants L223Q, H263D and Q279E are SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7, respectively (the corresponding amino acid sequences are SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO. 8).
Example 3
Construction of nitrilase combination mutants
Site-directed mutagenesis was performed by whole plasmid amplification using expression plasmid pET28b-BanIT-L223Q or pET28b-BanIT-H263D as template.
The PCR amplification system was (50. mu.L):template DNA 0.1ng-1ng, 2X Phanta Max Buffer 25. mu.L, dNTPs (10mM each) 1. mu.L, mutation primer upstream and downstream 1. mu.L each, Phanta Max Super-Fidelity DNA Polymerase 1. mu.L, and the rest ddH2O was made up to total volume.
PCR reaction parameters: (1) pre-denaturation at 95 ℃ for 30 s; (2) denaturation at 95 ℃ for 15 s; (3) annealing at 60 ℃ for 15 s; (4) extending for 6min at 72 ℃, and circulating the steps (2) to (4) for 30 times; (5) completely extending at 72 deg.C for 5min, and storing at 4 deg.C.
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 endonuclease Dpn I is added to carry out enzyme digestion at 37 ℃ for 3h to remove the template plasmid DNA, and inactivation is carried out at 65 ℃ for 10 min. The plasmid was transformed into E.coli BL21(DE3) competent cells by heat shock, recovered, plated on LB plate containing kanamycin and cultured overnight, and single colonies were picked up and cultured in LB liquid medium containing kanamycin resistance (final concentration: 50mg/L) and extracted for plasmid sequencing.
The correct sequencing result is nitrilase combined mutant engineering strain E.coli BL21(DE3)/pET28b-BaNIT-L223Q/H263D, E.coli BL21(DE3)/pET28b-BaNIT-L223Q/Q279E, E.coli BL21(DE3)/pET28b-BaNIT-H263D/Q279E, E.coli BL21(DE3)/pET28b-BaNIT-L223Q/H263D/Q279E, and the corresponding nucleotide sequences of the combined mutant engineering strain are respectively SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.15 (the corresponding amino acid sequences are SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14 and SEQ ID NO. 16).
Example 4
Expression of nitrilase mutants
The mutants of BaNIT-L223Q, BaNIT-H263D, BaNIT-Q279E obtained in examples 2 and 3, the combination mutant of BaNIT-L223Q/H263D, BaNIT-L223Q/Q279E, BaNIT-H263D/Q279E and BaNIT-L223Q/H263D/Q279E, and the parent BaNIT and wild type BrNIT were inoculated into LB medium containing kanamycin (50 mg/L final concentration), cultured at 37 ℃ for 6-8H, inoculated at 2% (v/v) into fresh LB liquid medium containing kanamycin (50 mg/L final concentration) for expansion culture, cultured at 37 ℃, and cultured at 150rpm until the OD of the bacterial cell body is reached600When the concentration is about 0.6 to 0.8, IPTG (final concentration: 0.1mM) is added to the LB liquid medium, and induction is carried out at 28 ℃ and 150rpmCulturing for 10-12h, centrifuging at 9000rpm at 4 deg.C for 10min, and collecting thallus cells. Washing twice with normal saline, and storing the thallus obtained by centrifugation in a refrigerator at-20 ℃.
Example 5
Soluble expression study of nitrilase mutants
The bacterial cells collected in example 4 (equivalent amount) were dissolved in Tris-HCl (50mM) buffer at pH 8.0, and after resuspending the cells, the cells were disrupted by sonication (400W, 5min, 1s disruption and 1s pause). After the crushed product is centrifuged (12000rpm, 5min), respectively taking supernatant (crude enzyme liquid) for denaturation treatment, and verifying the soluble expression level of the protein by SDS-PAGE gel electrophoresis.
As shown by SDS-PAGE gel electrophoresis results (FIG. 1), the soluble expression levels of the mutants of BanIT-L223Q, BanIT-H263D and BanIT-Q279E are slightly improved compared with the parent BanIT, and the soluble expression levels of the triple mutant of BanIT-L223Q/H263D/Q279E are greatly improved. Therefore, the solubility of the target protein is improved by modifying the parent BanIT.
Example 6
Determination of Activity of recombinant Escherichia coli containing nitrile hydrolase mutant
The recombinant E.coli obtained in example 4 was subjected to viability assay. The reaction system comprises the following components: 1mL of Tris-HCl buffer (50mM, pH 8.0), IBSN 20mM, wet cells 0.2 mg. The reaction solution was preheated at 40 ℃ for 2min and then reacted at 600rpm for 10 min. 500. mu.L of the reaction mixture was sampled, quenched by addition of 200. mu.L of 2M HCl, extracted with ethyl acetate, and the upper organic phase was dried over anhydrous sodium sulfate and then subjected to gas chromatography to determine the conversion of the substrate and the enantiomeric excess (ee) of the product.
Enantiomeric excess values of the substrate IBSN and the product CMHA were determined by gas chromatography. The gas chromatography model is 7890N (Agilent) and the capillary column model is BGB-174(BGB Analytik Switzerland). The chromatographic conditions are as follows: the sample introduction amount is 1.0 mu L, the temperature of the sample inlet and the detector is 250 ℃, the column temperature is 120 ℃, the temperature is kept for 15min, the temperature is raised to 170 ℃ at the speed of 10 ℃/min, and the temperature is kept for 9 min. The carrier gas is high-purity helium, the flow rate is 1.0mL/min, and the split ratio is 50: 1. For the calculation of the enantiomeric excess (ee) and the conversion (c), reference is made to the method of Rakels et al (Enzyme Microb. Technol.,1993,15: 1051).
The results of the activity measurement of the recombinant Escherichia coli containing the nitrilase mutant are shown in tables 3 and 4, the catalytic activity of the combined mutant is obviously improved compared with that of the parent, wherein the activity of the triple mutant, namely BanIT-L223Q/H263D/Q279E, is 2.2 times that of the parent, and the E value of all mutants is kept above 400.
Table 3: comparison of the Activity of nitrilases
Figure BDA0001728834670000091
Table 4: comparison of the Activity of nitrilases
Figure BDA0001728834670000092
Example 7
Application of recombinant escherichia coli containing nitrile hydrolase in preparation of (S) -CMHA (chitosan-binding protein)
The transformation system composition and transformation operation were as follows: 1L of Tris-HCl buffer solution (50mM, pH 8.0) was added to the recombinant nitrilase mutant E.coli BL21(DE3)/pET28b-BanIT-L223Q/H263D/Q279E, the parent E.coli BL21(DE3)/pET28b-BanIT and the wild type E.coli BL21(DE3)/pET28b-BrNIT (15 g/L of addition), respectively, and the substrate content was 100 g/L. Reaction conditions are as follows: the reaction progress was checked by hand gas chromatography at 35 ℃ and 400rpm during the reaction, and the conditions for gas chromatography were as shown in example 6.
As can be seen from FIG. 2, the conversion rates of the mutant BanIT-L223Q/H263D/Q279E and the parent BanIT reach 48.1% and 40.4% respectively after reaction for 10H, and the E values of the product (S) -CMHA are both kept above 400; while the conversion rate of wild-type BrNIT is 35.1%, and the E value of the product (S) -CMHA is about 150.
After 10 hours of reaction, the reaction was terminated, E.coli cells were removed by centrifugation, and the reaction mixture was distilled under reduced pressure to 1/3 volumes. The mixture was extracted by adding 2 times the volume of ethyl acetate (sample after vacuum distillation), and the lower aqueous phase was collected. The pH of the lower aqueous phase pool was adjusted to 4.0 with 2M HCl. 2 volumes of ethyl acetate were again added for extraction and the lower aqueous phase was discarded. The upper organic phase was collected and subjected to rotary evaporation to remove ethyl acetate to give (S) -3-cyano-5-methylhexanoic acid as an oil (ee > 99.5%).
Example 8
Application of recombinant escherichia coli containing nitrile hydrolase in preparation of (S) -CMHA (second)
The composition and catalytic operation of the catalytic system are as follows: 100mL of Tris-HCl buffer (50mM, pH 8.0), 150g/L of substrate IBSN was added, and the recombinant nitrilase mutant E.coli BL21(DE3)/pET28b-BanIT-L223Q/H263D/Q279E (20g/L) obtained in example 4 was added; reaction conditions are as follows: the progress of the reaction was checked by gas chromatography at 30 ℃ and 400rpm, the conditions of which were as shown in example 6.
The reaction is carried out for 10 hours, the conversion rate reaches 40.1 percent, and the ee of the product is more than 99.6 percent.
Example 9
Application of recombinant escherichia coli containing nitrile hydrolase in preparation of (S) -CMHA (C)
0.1g of E.coli BL21(DE3)/pET28b-BanIT-L223Q/H263D, E.coli BL21(DE3)/pET28b-BanIT-L223Q/Q279E, E.coli BL21(DE3)/pET28b-BanIT-H263D/Q279 and parent strain E.coli BL21(DE3)/pET28b-BanIT wet cells obtained in example 4 were weighed out and suspended in 10mL of Tris-HCl buffer (50mM, pH 8.0). 100g/L of IBSN was added thereto, and the mixture was reacted in a water bath at 30 ℃ and 200 rpm. Samples were taken at different times to examine the progress of the reaction, and the sample treatment and examination were performed as described in example 6.
The reaction time is 10H, the conversion rates of E.coli BL21(DE3)/pET28b-BanIT-L223Q/H263D, E.coli BL21(DE3)/pET28b-BanIT-L223Q/Q279E, E.coli BL21(DE3)/pET28b-BanIT-H263D/Q279 and parent strain E.coli BL21(DE3)/pET28b-BanIT reach 43.6%, 41.9%, 42.7% and 30.2% respectively.
Example 10
Application of recombinant escherichia coli containing nitrile hydrolase in preparation of (S) -CMHA (IV)
0.05g of E.coli BL21(DE3)/pET28b-BanIT-L223Q/H263D/Q279E wet cells obtained in example 4 were weighed out and suspended in 10mL of Tris-HCl buffer (50mM, pH 8.0). 1.0g of IBSN was added thereto, and the mixture was reacted in a water bath at 35 ℃ and 200 rpm. Samples were taken at different times to examine the progress of the reaction, and the sample treatment and examination were performed as described in example 6. As shown in FIG. 3, the reaction time was 18 hours, the conversion rate was 43.5%, and the ee of the product was > 99.3%.
The invention is not limited by the foregoing detailed description, and various modifications can be made within the scope of the invention as outlined by the claims.
Sequence listing
<110> Zhejiang industrial university
<120> plant nitrilase mutant, coding gene and application thereof
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1050
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgtctggct ctgaagaaat gtccaaagct ctgaatgcta ccactccagg tttcccggac 60
atccctagca ccatcgttcg cgccacgatc gttcaggctt ccactgtata caacgacact 120
cctaaaacca tcgaaaaagc tgaaaaattc atcgcggaag ctgctagcga cggtgcgcag 180
ctggtggtct ttccggaagc tttcatcgct ggttacccgc gtggctatcg tttcggcatc 240
ggtgtaggtg tgcacaacga ggcgggccgt gattgtttcc gccgctatca tgctagcgcg 300
atcgttgtcc cgggtccgga ggttgataaa ctggcagaaa ttgctcgtaa atacaaagtc 360
tacctggtaa tgggtgccat ggagaaagat ggttataccc tgtactgtac tgcgctgttt 420
ttcagctctg aaggtcgttt cctgggcaag caccgcaaag tcatgccgac gtctctggaa 480
cgttgcatct ggggcttcgg tgatggttct actatcccgg tctacgacac cccgctgggc 540
aagctgggcg ccgcaatctg ttgggaaaac cgcatgccgc tgtaccgtac tagcctgtac 600
ggcaaaggta tcgagctgta ttgcgctccg actgccgatg gctctaaaga atggcagtct 660
tctatgctgc acatcgctct ggaaggtggt tgcttcgttc tgtctgcttg ccagttctgc 720
cgtcgtaaag acttcccgga ccacccggac tacctgttca ccgactggga cgacaaccag 780
gaagaccacg ctatcgtttc tcagggtggt tctgttatca tctctccgct gggtcaggtt 840
ctggctggtc cgaacttcga gtctgagggc ctgatcactg cagatctgga tctgggcgat 900
gtagcgcgtg caaaactgta tttcgatgtt gttggtcact actcccgccc tgagattttt 960
aatctgacgg ttaacgagac tccgaagaaa ccggttactt tcgtttccaa gtccgtaaaa 1020
gctgaggacg actctgagcc gcaggacaaa 1050
<210> 2
<211> 350
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Met Ser Gly Ser Glu Glu Met Ser Lys Ala Leu Asn Ala Thr Thr Pro
1 5 10 15
Gly Phe Pro Asp Ile Pro Ser Thr Ile Val Arg Ala Thr Ile Val Gln
20 25 30
Ala Ser Thr Val Tyr Asn Asp Thr Pro Lys Thr Ile Glu Lys Ala Glu
35 40 45
Lys Phe Ile Ala Glu Ala Ala Ser Asp Gly Ala Gln Leu Val Val Phe
50 55 60
Pro Glu Ala Phe Ile Ala Gly Tyr Pro Arg Gly Tyr Arg Phe Gly Ile
65 70 75 80
Gly Val Gly Val His Asn Glu Ala Gly Arg Asp Cys Phe Arg Arg Tyr
85 90 95
His Ala Ser Ala Ile Val Val Pro Gly Pro Glu Val Asp Lys Leu Ala
100 105 110
Glu Ile Ala Arg Lys Tyr Lys Val Tyr Leu Val Met Gly Ala Met Glu
115 120 125
Lys Asp Gly Tyr Thr Leu Tyr Cys Thr Ala Leu Phe Phe Ser Ser Glu
130 135 140
Gly Arg Phe Leu Gly Lys His Arg Lys Val Met Pro Thr Ser Leu Glu
145 150 155 160
Arg Cys Ile Trp Gly Phe Gly Asp Gly Ser Thr Ile Pro Val Tyr Asp
165 170 175
Thr Pro Leu Gly Lys Leu Gly Ala Ala Ile Cys Trp Glu Asn Arg Met
180 185 190
Pro Leu Tyr Arg Thr Ser Leu Tyr Gly Lys Gly Ile Glu Leu Tyr Cys
195 200 205
Ala Pro Thr Ala Asp Gly Ser Lys Glu Trp Gln Ser Ser Met Leu His
210 215 220
Ile Ala Leu Glu Gly Gly Cys Phe Val Leu Ser Ala Cys Gln Phe Cys
225 230 235 240
Arg Arg Lys Asp Phe Pro Asp His Pro Asp Tyr Leu Phe Thr Asp Trp
245 250 255
Asp Asp Asn Gln Glu Asp His Ala Ile Val Ser Gln Gly Gly Ser Val
260 265 270
Ile Ile Ser Pro Leu Gly Gln Val Leu Ala Gly Pro Asn Phe Glu Ser
275 280 285
Glu Gly Leu Ile Thr Ala Asp Leu Asp Leu Gly Asp Val Ala Arg Ala
290 295 300
Lys Leu Tyr Phe Asp Val Val Gly His Tyr Ser Arg Pro Glu Ile Phe
305 310 315 320
Asn Leu Thr Val Asn Glu Thr Pro Lys Lys Pro Val Thr Phe Val Ser
325 330 335
Lys Ser Val Lys Ala Glu Asp Asp Ser Glu Pro Gln Asp Lys
340 345 350
<210> 3
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<213> Artificial Sequence (Artificial Sequence)
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atgtctggct ctgaagaaat gtccaaagct ctgaatgcta ccactccagg tttcccggac 60
atccctagca ccatcgttcg cgccacgatc gttcaggctt ccactgtata caacgacact 120
cctaaaacca tcgaaaaagc tgaaaaattc atcgcggaag ctgctagcga cggtgcgcag 180
ctggtggtct ttccggaagc tttcatcgct ggttacccgc gtggctatcg tttcggcatc 240
ggtgtaggtg tgcacaacga ggcgggccgt gattgtttcc gccgctatca tgctagcgcg 300
atcgttgtcc cgggtccgga ggttgataaa ctggcagaaa ttgctcgtaa atacaaagtc 360
tacctggtaa tgggtgccat ggagaaagat ggttataccc tgtactgtac tgcgctgttt 420
ttcagctctg aaggtcgttt cctgggcaag caccgcaaag tcatgccgac gtctctggaa 480
cgttgcatct ggggcttcgg tgatggttct actatcccgg tctacgacac cccgctgggc 540
aagctgggcg ccgcaatctg ttgggaaaac cgcatgccgc tgtaccgtac tagcctgtac 600
ggcaaaggta tcgagctgta ttgcgctccg actgccgatg gctctaaaga atggcagtct 660
tctatgcagc acatcgctct ggaaggtggt tgcttcgttc tgtctgcttg ccagttctgc 720
cgtcgtaaag acttcccgga ccacccggac tacctgttca ccgactggga cgacaaccag 780
gaagaccacg ctatcgtttc tcagggtggt tctgttatca tctctccgct gggtcaggtt 840
ctggctggtc cgaacttcga gtctgagggc ctgatcactg cagatctgga tctgggcgat 900
gtagcgcgtg caaaactgta tttcgatgtt gttggtcact actcccgccc tgagattttt 960
aatctgacgg ttaacgagac tccgaagaaa ccggttactt tcgtttccaa gtccgtaaaa 1020
gctgaggacg actctgagcc gcaggacaaa 1050
<210> 4
<211> 350
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Ser Gly Ser Glu Glu Met Ser Lys Ala Leu Asn Ala Thr Thr Pro
1 5 10 15
Gly Phe Pro Asp Ile Pro Ser Thr Ile Val Arg Ala Thr Ile Val Gln
20 25 30
Ala Ser Thr Val Tyr Asn Asp Thr Pro Lys Thr Ile Glu Lys Ala Glu
35 40 45
Lys Phe Ile Ala Glu Ala Ala Ser Asp Gly Ala Gln Leu Val Val Phe
50 55 60
Pro Glu Ala Phe Ile Ala Gly Tyr Pro Arg Gly Tyr Arg Phe Gly Ile
65 70 75 80
Gly Val Gly Val His Asn Glu Ala Gly Arg Asp Cys Phe Arg Arg Tyr
85 90 95
His Ala Ser Ala Ile Val Val Pro Gly Pro Glu Val Asp Lys Leu Ala
100 105 110
Glu Ile Ala Arg Lys Tyr Lys Val Tyr Leu Val Met Gly Ala Met Glu
115 120 125
Lys Asp Gly Tyr Thr Leu Tyr Cys Thr Ala Leu Phe Phe Ser Ser Glu
130 135 140
Gly Arg Phe Leu Gly Lys His Arg Lys Val Met Pro Thr Ser Leu Glu
145 150 155 160
Arg Cys Ile Trp Gly Phe Gly Asp Gly Ser Thr Ile Pro Val Tyr Asp
165 170 175
Thr Pro Leu Gly Lys Leu Gly Ala Ala Ile Cys Trp Glu Asn Arg Met
180 185 190
Pro Leu Tyr Arg Thr Ser Leu Tyr Gly Lys Gly Ile Glu Leu Tyr Cys
195 200 205
Ala Pro Thr Ala Asp Gly Ser Lys Glu Trp Gln Ser Ser Met Gln His
210 215 220
Ile Ala Leu Glu Gly Gly Cys Phe Val Leu Ser Ala Cys Gln Phe Cys
225 230 235 240
Arg Arg Lys Asp Phe Pro Asp His Pro Asp Tyr Leu Phe Thr Asp Trp
245 250 255
Asp Asp Asn Gln Glu Asp His Ala Ile Val Ser Gln Gly Gly Ser Val
260 265 270
Ile Ile Ser Pro Leu Gly Gln Val Leu Ala Gly Pro Asn Phe Glu Ser
275 280 285
Glu Gly Leu Ile Thr Ala Asp Leu Asp Leu Gly Asp Val Ala Arg Ala
290 295 300
Lys Leu Tyr Phe Asp Val Val Gly His Tyr Ser Arg Pro Glu Ile Phe
305 310 315 320
Asn Leu Thr Val Asn Glu Thr Pro Lys Lys Pro Val Thr Phe Val Ser
325 330 335
Lys Ser Val Lys Ala Glu Asp Asp Ser Glu Pro Gln Asp Lys
340 345 350
<210> 5
<211> 1050
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgtctggct ctgaagaaat gtccaaagct ctgaatgcta ccactccagg tttcccggac 60
atccctagca ccatcgttcg cgccacgatc gttcaggctt ccactgtata caacgacact 120
cctaaaacca tcgaaaaagc tgaaaaattc atcgcggaag ctgctagcga cggtgcgcag 180
ctggtggtct ttccggaagc tttcatcgct ggttacccgc gtggctatcg tttcggcatc 240
ggtgtaggtg tgcacaacga ggcgggccgt gattgtttcc gccgctatca tgctagcgcg 300
atcgttgtcc cgggtccgga ggttgataaa ctggcagaaa ttgctcgtaa atacaaagtc 360
tacctggtaa tgggtgccat ggagaaagat ggttataccc tgtactgtac tgcgctgttt 420
ttcagctctg aaggtcgttt cctgggcaag caccgcaaag tcatgccgac gtctctggaa 480
cgttgcatct ggggcttcgg tgatggttct actatcccgg tctacgacac cccgctgggc 540
aagctgggcg ccgcaatctg ttgggaaaac cgcatgccgc tgtaccgtac tagcctgtac 600
ggcaaaggta tcgagctgta ttgcgctccg actgccgatg gctctaaaga atggcagtct 660
tctatgctgc acatcgctct ggaaggtggt tgcttcgttc tgtctgcttg ccagttctgc 720
cgtcgtaaag acttcccgga ccacccggac tacctgttca ccgactggga cgacaaccag 780
gaagacgacg ctatcgtttc tcagggtggt tctgttatca tctctccgct gggtcaggtt 840
ctggctggtc cgaacttcga gtctgagggc ctgatcactg cagatctgga tctgggcgat 900
gtagcgcgtg caaaactgta tttcgatgtt gttggtcact actcccgccc tgagattttt 960
aatctgacgg ttaacgagac tccgaagaaa ccggttactt tcgtttccaa gtccgtaaaa 1020
gctgaggacg actctgagcc gcaggacaaa 1050
<210> 6
<211> 350
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Ser Gly Ser Glu Glu Met Ser Lys Ala Leu Asn Ala Thr Thr Pro
1 5 10 15
Gly Phe Pro Asp Ile Pro Ser Thr Ile Val Arg Ala Thr Ile Val Gln
20 25 30
Ala Ser Thr Val Tyr Asn Asp Thr Pro Lys Thr Ile Glu Lys Ala Glu
35 40 45
Lys Phe Ile Ala Glu Ala Ala Ser Asp Gly Ala Gln Leu Val Val Phe
50 55 60
Pro Glu Ala Phe Ile Ala Gly Tyr Pro Arg Gly Tyr Arg Phe Gly Ile
65 70 75 80
Gly Val Gly Val His Asn Glu Ala Gly Arg Asp Cys Phe Arg Arg Tyr
85 90 95
His Ala Ser Ala Ile Val Val Pro Gly Pro Glu Val Asp Lys Leu Ala
100 105 110
Glu Ile Ala Arg Lys Tyr Lys Val Tyr Leu Val Met Gly Ala Met Glu
115 120 125
Lys Asp Gly Tyr Thr Leu Tyr Cys Thr Ala Leu Phe Phe Ser Ser Glu
130 135 140
Gly Arg Phe Leu Gly Lys His Arg Lys Val Met Pro Thr Ser Leu Glu
145 150 155 160
Arg Cys Ile Trp Gly Phe Gly Asp Gly Ser Thr Ile Pro Val Tyr Asp
165 170 175
Thr Pro Leu Gly Lys Leu Gly Ala Ala Ile Cys Trp Glu Asn Arg Met
180 185 190
Pro Leu Tyr Arg Thr Ser Leu Tyr Gly Lys Gly Ile Glu Leu Tyr Cys
195 200 205
Ala Pro Thr Ala Asp Gly Ser Lys Glu Trp Gln Ser Ser Met Leu His
210 215 220
Ile Ala Leu Glu Gly Gly Cys Phe Val Leu Ser Ala Cys Gln Phe Cys
225 230 235 240
Arg Arg Lys Asp Phe Pro Asp His Pro Asp Tyr Leu Phe Thr Asp Trp
245 250 255
Asp Asp Asn Gln Glu Asp Asp Ala Ile Val Ser Gln Gly Gly Ser Val
260 265 270
Ile Ile Ser Pro Leu Gly Gln Val Leu Ala Gly Pro Asn Phe Glu Ser
275 280 285
Glu Gly Leu Ile Thr Ala Asp Leu Asp Leu Gly Asp Val Ala Arg Ala
290 295 300
Lys Leu Tyr Phe Asp Val Val Gly His Tyr Ser Arg Pro Glu Ile Phe
305 310 315 320
Asn Leu Thr Val Asn Glu Thr Pro Lys Lys Pro Val Thr Phe Val Ser
325 330 335
Lys Ser Val Lys Ala Glu Asp Asp Ser Glu Pro Gln Asp Lys
340 345 350
<210> 7
<211> 1050
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgtctggct ctgaagaaat gtccaaagct ctgaatgcta ccactccagg tttcccggac 60
atccctagca ccatcgttcg cgccacgatc gttcaggctt ccactgtata caacgacact 120
cctaaaacca tcgaaaaagc tgaaaaattc atcgcggaag ctgctagcga cggtgcgcag 180
ctggtggtct ttccggaagc tttcatcgct ggttacccgc gtggctatcg tttcggcatc 240
ggtgtaggtg tgcacaacga ggcgggccgt gattgtttcc gccgctatca tgctagcgcg 300
atcgttgtcc cgggtccgga ggttgataaa ctggcagaaa ttgctcgtaa atacaaagtc 360
tacctggtaa tgggtgccat ggagaaagat ggttataccc tgtactgtac tgcgctgttt 420
ttcagctctg aaggtcgttt cctgggcaag caccgcaaag tcatgccgac gtctctggaa 480
cgttgcatct ggggcttcgg tgatggttct actatcccgg tctacgacac cccgctgggc 540
aagctgggcg ccgcaatctg ttgggaaaac cgcatgccgc tgtaccgtac tagcctgtac 600
ggcaaaggta tcgagctgta ttgcgctccg actgccgatg gctctaaaga atggcagtct 660
tctatgctgc acatcgctct ggaaggtggt tgcttcgttc tgtctgcttg ccagttctgc 720
cgtcgtaaag acttcccgga ccacccggac tacctgttca ccgactggga cgacaaccag 780
gaagaccacg ctatcgtttc tcagggtggt tctgttatca tctctccgct gggtgaagtt 840
ctggctggtc cgaacttcga gtctgagggc ctgatcactg cagatctgga tctgggcgat 900
gtagcgcgtg caaaactgta tttcgatgtt gttggtcact actcccgccc tgagattttt 960
aatctgacgg ttaacgagac tccgaagaaa ccggttactt tcgtttccaa gtccgtaaaa 1020
gctgaggacg actctgagcc gcaggacaaa 1050
<210> 8
<211> 350
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Met Ser Gly Ser Glu Glu Met Ser Lys Ala Leu Asn Ala Thr Thr Pro
1 5 10 15
Gly Phe Pro Asp Ile Pro Ser Thr Ile Val Arg Ala Thr Ile Val Gln
20 25 30
Ala Ser Thr Val Tyr Asn Asp Thr Pro Lys Thr Ile Glu Lys Ala Glu
35 40 45
Lys Phe Ile Ala Glu Ala Ala Ser Asp Gly Ala Gln Leu Val Val Phe
50 55 60
Pro Glu Ala Phe Ile Ala Gly Tyr Pro Arg Gly Tyr Arg Phe Gly Ile
65 70 75 80
Gly Val Gly Val His Asn Glu Ala Gly Arg Asp Cys Phe Arg Arg Tyr
85 90 95
His Ala Ser Ala Ile Val Val Pro Gly Pro Glu Val Asp Lys Leu Ala
100 105 110
Glu Ile Ala Arg Lys Tyr Lys Val Tyr Leu Val Met Gly Ala Met Glu
115 120 125
Lys Asp Gly Tyr Thr Leu Tyr Cys Thr Ala Leu Phe Phe Ser Ser Glu
130 135 140
Gly Arg Phe Leu Gly Lys His Arg Lys Val Met Pro Thr Ser Leu Glu
145 150 155 160
Arg Cys Ile Trp Gly Phe Gly Asp Gly Ser Thr Ile Pro Val Tyr Asp
165 170 175
Thr Pro Leu Gly Lys Leu Gly Ala Ala Ile Cys Trp Glu Asn Arg Met
180 185 190
Pro Leu Tyr Arg Thr Ser Leu Tyr Gly Lys Gly Ile Glu Leu Tyr Cys
195 200 205
Ala Pro Thr Ala Asp Gly Ser Lys Glu Trp Gln Ser Ser Met Leu His
210 215 220
Ile Ala Leu Glu Gly Gly Cys Phe Val Leu Ser Ala Cys Gln Phe Cys
225 230 235 240
Arg Arg Lys Asp Phe Pro Asp His Pro Asp Tyr Leu Phe Thr Asp Trp
245 250 255
Asp Asp Asn Gln Glu Asp His Ala Ile Val Ser Gln Gly Gly Ser Val
260 265 270
Ile Ile Ser Pro Leu Gly Glu Val Leu Ala Gly Pro Asn Phe Glu Ser
275 280 285
Glu Gly Leu Ile Thr Ala Asp Leu Asp Leu Gly Asp Val Ala Arg Ala
290 295 300
Lys Leu Tyr Phe Asp Val Val Gly His Tyr Ser Arg Pro Glu Ile Phe
305 310 315 320
Asn Leu Thr Val Asn Glu Thr Pro Lys Lys Pro Val Thr Phe Val Ser
325 330 335
Lys Ser Val Lys Ala Glu Asp Asp Ser Glu Pro Gln Asp Lys
340 345 350
<210> 9
<211> 1050
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atgtctggct ctgaagaaat gtccaaagct ctgaatgcta ccactccagg tttcccggac 60
atccctagca ccatcgttcg cgccacgatc gttcaggctt ccactgtata caacgacact 120
cctaaaacca tcgaaaaagc tgaaaaattc atcgcggaag ctgctagcga cggtgcgcag 180
ctggtggtct ttccggaagc tttcatcgct ggttacccgc gtggctatcg tttcggcatc 240
ggtgtaggtg tgcacaacga ggcgggccgt gattgtttcc gccgctatca tgctagcgcg 300
atcgttgtcc cgggtccgga ggttgataaa ctggcagaaa ttgctcgtaa atacaaagtc 360
tacctggtaa tgggtgccat ggagaaagat ggttataccc tgtactgtac tgcgctgttt 420
ttcagctctg aaggtcgttt cctgggcaag caccgcaaag tcatgccgac gtctctggaa 480
cgttgcatct ggggcttcgg tgatggttct actatcccgg tctacgacac cccgctgggc 540
aagctgggcg ccgcaatctg ttgggaaaac cgcatgccgc tgtaccgtac tagcctgtac 600
ggcaaaggta tcgagctgta ttgcgctccg actgccgatg gctctaaaga atggcagtct 660
tctatgcagc acatcgctct ggaaggtggt tgcttcgttc tgtctgcttg ccagttctgc 720
cgtcgtaaag acttcccgga ccacccggac tacctgttca ccgactggga cgacaaccag 780
gaagacgacg ctatcgtttc tcagggtggt tctgttatca tctctccgct gggtcaggtt 840
ctggctggtc cgaacttcga gtctgagggc ctgatcactg cagatctgga tctgggcgat 900
gtagcgcgtg caaaactgta tttcgatgtt gttggtcact actcccgccc tgagattttt 960
aatctgacgg ttaacgagac tccgaagaaa ccggttactt tcgtttccaa gtccgtaaaa 1020
gctgaggacg actctgagcc gcaggacaaa 1050
<210> 10
<211> 350
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Met Ser Gly Ser Glu Glu Met Ser Lys Ala Leu Asn Ala Thr Thr Pro
1 5 10 15
Gly Phe Pro Asp Ile Pro Ser Thr Ile Val Arg Ala Thr Ile Val Gln
20 25 30
Ala Ser Thr Val Tyr Asn Asp Thr Pro Lys Thr Ile Glu Lys Ala Glu
35 40 45
Lys Phe Ile Ala Glu Ala Ala Ser Asp Gly Ala Gln Leu Val Val Phe
50 55 60
Pro Glu Ala Phe Ile Ala Gly Tyr Pro Arg Gly Tyr Arg Phe Gly Ile
65 70 75 80
Gly Val Gly Val His Asn Glu Ala Gly Arg Asp Cys Phe Arg Arg Tyr
85 90 95
His Ala Ser Ala Ile Val Val Pro Gly Pro Glu Val Asp Lys Leu Ala
100 105 110
Glu Ile Ala Arg Lys Tyr Lys Val Tyr Leu Val Met Gly Ala Met Glu
115 120 125
Lys Asp Gly Tyr Thr Leu Tyr Cys Thr Ala Leu Phe Phe Ser Ser Glu
130 135 140
Gly Arg Phe Leu Gly Lys His Arg Lys Val Met Pro Thr Ser Leu Glu
145 150 155 160
Arg Cys Ile Trp Gly Phe Gly Asp Gly Ser Thr Ile Pro Val Tyr Asp
165 170 175
Thr Pro Leu Gly Lys Leu Gly Ala Ala Ile Cys Trp Glu Asn Arg Met
180 185 190
Pro Leu Tyr Arg Thr Ser Leu Tyr Gly Lys Gly Ile Glu Leu Tyr Cys
195 200 205
Ala Pro Thr Ala Asp Gly Ser Lys Glu Trp Gln Ser Ser Met Gln His
210 215 220
Ile Ala Leu Glu Gly Gly Cys Phe Val Leu Ser Ala Cys Gln Phe Cys
225 230 235 240
Arg Arg Lys Asp Phe Pro Asp His Pro Asp Tyr Leu Phe Thr Asp Trp
245 250 255
Asp Asp Asn Gln Glu Asp Asp Ala Ile Val Ser Gln Gly Gly Ser Val
260 265 270
Ile Ile Ser Pro Leu Gly Gln Val Leu Ala Gly Pro Asn Phe Glu Ser
275 280 285
Glu Gly Leu Ile Thr Ala Asp Leu Asp Leu Gly Asp Val Ala Arg Ala
290 295 300
Lys Leu Tyr Phe Asp Val Val Gly His Tyr Ser Arg Pro Glu Ile Phe
305 310 315 320
Asn Leu Thr Val Asn Glu Thr Pro Lys Lys Pro Val Thr Phe Val Ser
325 330 335
Lys Ser Val Lys Ala Glu Asp Asp Ser Glu Pro Gln Asp Lys
340 345 350
<210> 11
<211> 1050
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atgtctggct ctgaagaaat gtccaaagct ctgaatgcta ccactccagg tttcccggac 60
atccctagca ccatcgttcg cgccacgatc gttcaggctt ccactgtata caacgacact 120
cctaaaacca tcgaaaaagc tgaaaaattc atcgcggaag ctgctagcga cggtgcgcag 180
ctggtggtct ttccggaagc tttcatcgct ggttacccgc gtggctatcg tttcggcatc 240
ggtgtaggtg tgcacaacga ggcgggccgt gattgtttcc gccgctatca tgctagcgcg 300
atcgttgtcc cgggtccgga ggttgataaa ctggcagaaa ttgctcgtaa atacaaagtc 360
tacctggtaa tgggtgccat ggagaaagat ggttataccc tgtactgtac tgcgctgttt 420
ttcagctctg aaggtcgttt cctgggcaag caccgcaaag tcatgccgac gtctctggaa 480
cgttgcatct ggggcttcgg tgatggttct actatcccgg tctacgacac cccgctgggc 540
aagctgggcg ccgcaatctg ttgggaaaac cgcatgccgc tgtaccgtac tagcctgtac 600
ggcaaaggta tcgagctgta ttgcgctccg actgccgatg gctctaaaga atggcagtct 660
tctatgcagc acatcgctct ggaaggtggt tgcttcgttc tgtctgcttg ccagttctgc 720
cgtcgtaaag acttcccgga ccacccggac tacctgttca ccgactggga cgacaaccag 780
gaagaccacg ctatcgtttc tcagggtggt tctgttatca tctctccgct gggtgaagtt 840
ctggctggtc cgaacttcga gtctgagggc ctgatcactg cagatctgga tctgggcgat 900
gtagcgcgtg caaaactgta tttcgatgtt gttggtcact actcccgccc tgagattttt 960
aatctgacgg ttaacgagac tccgaagaaa ccggttactt tcgtttccaa gtccgtaaaa 1020
gctgaggacg actctgagcc gcaggacaaa 1050
<210> 12
<211> 350
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Met Ser Gly Ser Glu Glu Met Ser Lys Ala Leu Asn Ala Thr Thr Pro
1 5 10 15
Gly Phe Pro Asp Ile Pro Ser Thr Ile Val Arg Ala Thr Ile Val Gln
20 25 30
Ala Ser Thr Val Tyr Asn Asp Thr Pro Lys Thr Ile Glu Lys Ala Glu
35 40 45
Lys Phe Ile Ala Glu Ala Ala Ser Asp Gly Ala Gln Leu Val Val Phe
50 55 60
Pro Glu Ala Phe Ile Ala Gly Tyr Pro Arg Gly Tyr Arg Phe Gly Ile
65 70 75 80
Gly Val Gly Val His Asn Glu Ala Gly Arg Asp Cys Phe Arg Arg Tyr
85 90 95
His Ala Ser Ala Ile Val Val Pro Gly Pro Glu Val Asp Lys Leu Ala
100 105 110
Glu Ile Ala Arg Lys Tyr Lys Val Tyr Leu Val Met Gly Ala Met Glu
115 120 125
Lys Asp Gly Tyr Thr Leu Tyr Cys Thr Ala Leu Phe Phe Ser Ser Glu
130 135 140
Gly Arg Phe Leu Gly Lys His Arg Lys Val Met Pro Thr Ser Leu Glu
145 150 155 160
Arg Cys Ile Trp Gly Phe Gly Asp Gly Ser Thr Ile Pro Val Tyr Asp
165 170 175
Thr Pro Leu Gly Lys Leu Gly Ala Ala Ile Cys Trp Glu Asn Arg Met
180 185 190
Pro Leu Tyr Arg Thr Ser Leu Tyr Gly Lys Gly Ile Glu Leu Tyr Cys
195 200 205
Ala Pro Thr Ala Asp Gly Ser Lys Glu Trp Gln Ser Ser Met Gln His
210 215 220
Ile Ala Leu Glu Gly Gly Cys Phe Val Leu Ser Ala Cys Gln Phe Cys
225 230 235 240
Arg Arg Lys Asp Phe Pro Asp His Pro Asp Tyr Leu Phe Thr Asp Trp
245 250 255
Asp Asp Asn Gln Glu Asp His Ala Ile Val Ser Gln Gly Gly Ser Val
260 265 270
Ile Ile Ser Pro Leu Gly Glu Val Leu Ala Gly Pro Asn Phe Glu Ser
275 280 285
Glu Gly Leu Ile Thr Ala Asp Leu Asp Leu Gly Asp Val Ala Arg Ala
290 295 300
Lys Leu Tyr Phe Asp Val Val Gly His Tyr Ser Arg Pro Glu Ile Phe
305 310 315 320
Asn Leu Thr Val Asn Glu Thr Pro Lys Lys Pro Val Thr Phe Val Ser
325 330 335
Lys Ser Val Lys Ala Glu Asp Asp Ser Glu Pro Gln Asp Lys
340 345 350
<210> 13
<211> 1050
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atgtctggct ctgaagaaat gtccaaagct ctgaatgcta ccactccagg tttcccggac 60
atccctagca ccatcgttcg cgccacgatc gttcaggctt ccactgtata caacgacact 120
cctaaaacca tcgaaaaagc tgaaaaattc atcgcggaag ctgctagcga cggtgcgcag 180
ctggtggtct ttccggaagc tttcatcgct ggttacccgc gtggctatcg tttcggcatc 240
ggtgtaggtg tgcacaacga ggcgggccgt gattgtttcc gccgctatca tgctagcgcg 300
atcgttgtcc cgggtccgga ggttgataaa ctggcagaaa ttgctcgtaa atacaaagtc 360
tacctggtaa tgggtgccat ggagaaagat ggttataccc tgtactgtac tgcgctgttt 420
ttcagctctg aaggtcgttt cctgggcaag caccgcaaag tcatgccgac gtctctggaa 480
cgttgcatct ggggcttcgg tgatggttct actatcccgg tctacgacac cccgctgggc 540
aagctgggcg ccgcaatctg ttgggaaaac cgcatgccgc tgtaccgtac tagcctgtac 600
ggcaaaggta tcgagctgta ttgcgctccg actgccgatg gctctaaaga atggcagtct 660
tctatgctgc acatcgctct ggaaggtggt tgcttcgttc tgtctgcttg ccagttctgc 720
cgtcgtaaag acttcccgga ccacccggac tacctgttca ccgactggga cgacaaccag 780
gaagacgacg ctatcgtttc tcagggtggt tctgttatca tctctccgct gggtgaagtt 840
ctggctggtc cgaacttcga gtctgagggc ctgatcactg cagatctgga tctgggcgat 900
gtagcgcgtg caaaactgta tttcgatgtt gttggtcact actcccgccc tgagattttt 960
aatctgacgg ttaacgagac tccgaagaaa ccggttactt tcgtttccaa gtccgtaaaa 1020
gctgaggacg actctgagcc gcaggacaaa 1050
<210> 14
<211> 350
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Met Ser Gly Ser Glu Glu Met Ser Lys Ala Leu Asn Ala Thr Thr Pro
1 5 10 15
Gly Phe Pro Asp Ile Pro Ser Thr Ile Val Arg Ala Thr Ile Val Gln
20 25 30
Ala Ser Thr Val Tyr Asn Asp Thr Pro Lys Thr Ile Glu Lys Ala Glu
35 40 45
Lys Phe Ile Ala Glu Ala Ala Ser Asp Gly Ala Gln Leu Val Val Phe
50 55 60
Pro Glu Ala Phe Ile Ala Gly Tyr Pro Arg Gly Tyr Arg Phe Gly Ile
65 70 75 80
Gly Val Gly Val His Asn Glu Ala Gly Arg Asp Cys Phe Arg Arg Tyr
85 90 95
His Ala Ser Ala Ile Val Val Pro Gly Pro Glu Val Asp Lys Leu Ala
100 105 110
Glu Ile Ala Arg Lys Tyr Lys Val Tyr Leu Val Met Gly Ala Met Glu
115 120 125
Lys Asp Gly Tyr Thr Leu Tyr Cys Thr Ala Leu Phe Phe Ser Ser Glu
130 135 140
Gly Arg Phe Leu Gly Lys His Arg Lys Val Met Pro Thr Ser Leu Glu
145 150 155 160
Arg Cys Ile Trp Gly Phe Gly Asp Gly Ser Thr Ile Pro Val Tyr Asp
165 170 175
Thr Pro Leu Gly Lys Leu Gly Ala Ala Ile Cys Trp Glu Asn Arg Met
180 185 190
Pro Leu Tyr Arg Thr Ser Leu Tyr Gly Lys Gly Ile Glu Leu Tyr Cys
195 200 205
Ala Pro Thr Ala Asp Gly Ser Lys Glu Trp Gln Ser Ser Met Leu His
210 215 220
Ile Ala Leu Glu Gly Gly Cys Phe Val Leu Ser Ala Cys Gln Phe Cys
225 230 235 240
Arg Arg Lys Asp Phe Pro Asp His Pro Asp Tyr Leu Phe Thr Asp Trp
245 250 255
Asp Asp Asn Gln Glu Asp Asp Ala Ile Val Ser Gln Gly Gly Ser Val
260 265 270
Ile Ile Ser Pro Leu Gly Glu Val Leu Ala Gly Pro Asn Phe Glu Ser
275 280 285
Glu Gly Leu Ile Thr Ala Asp Leu Asp Leu Gly Asp Val Ala Arg Ala
290 295 300
Lys Leu Tyr Phe Asp Val Val Gly His Tyr Ser Arg Pro Glu Ile Phe
305 310 315 320
Asn Leu Thr Val Asn Glu Thr Pro Lys Lys Pro Val Thr Phe Val Ser
325 330 335
Lys Ser Val Lys Ala Glu Asp Asp Ser Glu Pro Gln Asp Lys
340 345 350
<210> 15
<211> 1050
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atgtctggct ctgaagaaat gtccaaagct ctgaatgcta ccactccagg tttcccggac 60
atccctagca ccatcgttcg cgccacgatc gttcaggctt ccactgtata caacgacact 120
cctaaaacca tcgaaaaagc tgaaaaattc atcgcggaag ctgctagcga cggtgcgcag 180
ctggtggtct ttccggaagc tttcatcgct ggttacccgc gtggctatcg tttcggcatc 240
ggtgtaggtg tgcacaacga ggcgggccgt gattgtttcc gccgctatca tgctagcgcg 300
atcgttgtcc cgggtccgga ggttgataaa ctggcagaaa ttgctcgtaa atacaaagtc 360
tacctggtaa tgggtgccat ggagaaagat ggttataccc tgtactgtac tgcgctgttt 420
ttcagctctg aaggtcgttt cctgggcaag caccgcaaag tcatgccgac gtctctggaa 480
cgttgcatct ggggcttcgg tgatggttct actatcccgg tctacgacac cccgctgggc 540
aagctgggcg ccgcaatctg ttgggaaaac cgcatgccgc tgtaccgtac tagcctgtac 600
ggcaaaggta tcgagctgta ttgcgctccg actgccgatg gctctaaaga atggcagtct 660
tctatgcagc acatcgctct ggaaggtggt tgcttcgttc tgtctgcttg ccagttctgc 720
cgtcgtaaag acttcccgga ccacccggac tacctgttca ccgactggga cgacaaccag 780
gaagacgacg ctatcgtttc tcagggtggt tctgttatca tctctccgct gggtgaagtt 840
ctggctggtc cgaacttcga gtctgagggc ctgatcactg cagatctgga tctgggcgat 900
gtagcgcgtg caaaactgta tttcgatgtt gttggtcact actcccgccc tgagattttt 960
aatctgacgg ttaacgagac tccgaagaaa ccggttactt tcgtttccaa gtccgtaaaa 1020
gctgaggacg actctgagcc gcaggacaaa 1050
<210> 16
<211> 350
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Met Ser Gly Ser Glu Glu Met Ser Lys Ala Leu Asn Ala Thr Thr Pro
1 5 10 15
Gly Phe Pro Asp Ile Pro Ser Thr Ile Val Arg Ala Thr Ile Val Gln
20 25 30
Ala Ser Thr Val Tyr Asn Asp Thr Pro Lys Thr Ile Glu Lys Ala Glu
35 40 45
Lys Phe Ile Ala Glu Ala Ala Ser Asp Gly Ala Gln Leu Val Val Phe
50 55 60
Pro Glu Ala Phe Ile Ala Gly Tyr Pro Arg Gly Tyr Arg Phe Gly Ile
65 70 75 80
Gly Val Gly Val His Asn Glu Ala Gly Arg Asp Cys Phe Arg Arg Tyr
85 90 95
His Ala Ser Ala Ile Val Val Pro Gly Pro Glu Val Asp Lys Leu Ala
100 105 110
Glu Ile Ala Arg Lys Tyr Lys Val Tyr Leu Val Met Gly Ala Met Glu
115 120 125
Lys Asp Gly Tyr Thr Leu Tyr Cys Thr Ala Leu Phe Phe Ser Ser Glu
130 135 140
Gly Arg Phe Leu Gly Lys His Arg Lys Val Met Pro Thr Ser Leu Glu
145 150 155 160
Arg Cys Ile Trp Gly Phe Gly Asp Gly Ser Thr Ile Pro Val Tyr Asp
165 170 175
Thr Pro Leu Gly Lys Leu Gly Ala Ala Ile Cys Trp Glu Asn Arg Met
180 185 190
Pro Leu Tyr Arg Thr Ser Leu Tyr Gly Lys Gly Ile Glu Leu Tyr Cys
195 200 205
Ala Pro Thr Ala Asp Gly Ser Lys Glu Trp Gln Ser Ser Met Gln His
210 215 220
Ile Ala Leu Glu Gly Gly Cys Phe Val Leu Ser Ala Cys Gln Phe Cys
225 230 235 240
Arg Arg Lys Asp Phe Pro Asp His Pro Asp Tyr Leu Phe Thr Asp Trp
245 250 255
Asp Asp Asn Gln Glu Asp Asp Ala Ile Val Ser Gln Gly Gly Ser Val
260 265 270
Ile Ile Ser Pro Leu Gly Glu Val Leu Ala Gly Pro Asn Phe Glu Ser
275 280 285
Glu Gly Leu Ile Thr Ala Asp Leu Asp Leu Gly Asp Val Ala Arg Ala
290 295 300
Lys Leu Tyr Phe Asp Val Val Gly His Tyr Ser Arg Pro Glu Ile Phe
305 310 315 320
Asn Leu Thr Val Asn Glu Thr Pro Lys Lys Pro Val Thr Phe Val Ser
325 330 335
Lys Ser Val Lys Ala Glu Asp Asp Ser Glu Pro Gln Asp Lys
340 345 350
<210> 17
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gaatggcagt cttctatgct gcacatcgc 29
<210> 18
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gaagttcgga ccagccagaa cctgaccc 28
<210> 19
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gcgatgtgca gcatagaaga ctgccattc 29
<210> 20
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gggtcaggtt ctggctggtc cgaacttc 28
<210> 21
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cagtcttcta tgctgcacat cgctctggaa gg 32
<210> 22
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ccttccagag cgatgtgcag catagaagac tg 32
<210> 23
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
caaccaggaa gacgacgcta tcgtttctca ggg 33
<210> 24
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ccctgagaaa cgatagcgtc gtcttcctgg ttg 33
<210> 25
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
catctctccg ctgggtcagg ttctggctgg 30
<210> 26
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ccagccagaa cctgacccag cggagagatg 30

Claims (9)

1. A plant nitrilase mutant characterized in that its amino acid sequence is as shown in SEQ ID No. 16.
2. A gene encoding the plant nitrilase mutant of claim 1, wherein the nucleotide sequence of the gene is as shown in SEQ ID No. 15.
3. A recombinant vector comprising the encoding gene of claim 2.
4. A recombinant genetically engineered bacterium comprising the recombinant vector of claim 3.
5. A method of producing a plant nitrilase mutant according to claim 1, characterised in that it comprises the following steps:
(1) designing a PCR primer aiming at a sequence of a turnip nitrilase gene, and amplifying by using the PCR primer to obtain a DNA fragment I containing the nucleotide sequence 675-855 bit of the Arabidopsis thaliana nitrilase by using the cDNA of the Arabidopsis thaliana as a template;
(2) using a recombinant plasmid carrying a turnip nitrilase gene as a template, and obtaining a BrNIT plasmid fragment with the turnip nitrilase nucleotide sequence 678-858 bit deletion by utilizing reverse PCR amplification;
(3) recombining the DNA fragment I and the BrNIT plasmid fragment, then transforming the recombined product to host bacteria, and screening to obtain a recombined parent nitrilase expression strain, wherein the nucleotide sequence of the parent nitrilase is shown as SEQ ID NO. 1;
(4) designing a site-directed mutagenesis primer, and carrying out overlap extension PCR by using the recombinant plasmid carrying the parent nitrilase gene obtained in the step (3) as a template to obtain a single-site mutagenesis product of which the L at the 223 rd position is mutated into Q or the H at the 263 th position is mutated into D or the Q at the 279 th position is mutated into E in the parent nitrilase;
(5) performing overlap extension PCR by using the site-specific mutation primer by using the single-site mutation product as a template to obtain a double-site mutation product; then, taking the double-site mutation product as a template, and carrying out overlap extension PCR by using the fixed-point primer to obtain a three-site mutation product;
(6) and transforming the three-site mutation product into host bacteria, screening to obtain a nitrilase mutant expression strain, and performing induced expression to obtain the plant nitrilase mutant.
6. Use of the plant nitrilase mutant of claim 1 for catalyzing racemic isobutyl succinonitrile to (S) -3-cyano-5-methylhexanoic acid.
7. The use of claim 6, wherein the use is to use wet thalli, wet thalli immobilized cells, enzyme extracted after ultrasonic disruption of the wet thalli or immobilized enzyme obtained by fermentation culture of engineering bacteria containing genes encoding plant nitrilase mutants as catalysts, racemic isobutyl butanedinitrile as a substrate, buffer solution with pH of 6-10 as a reaction medium, carry out water bath reaction at 20-50 ℃ and 200-400rpm, and after the reaction is finished, separate and purify the reaction solution to obtain (S) -3-cyano-5-methylhexanoic acid.
8. The method as claimed in claim 7, wherein the concentration of the substrate in the reaction system is 100-150g/L, and the amount of the catalyst is 5-20g/L based on the weight of wet cells, wherein the water content of the wet cells is 70-90%.
9. The use according to claim 7, wherein the reaction medium is a Tris-HCl buffer at pH 8.0 and the catalytic reaction temperature is 35 ℃.
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