CN110714002A - A kind of plant nitrilase mutant, encoding gene and application thereof - Google Patents

Abstract

The invention discloses a cruciferous plant nitrilase mutant, which belongs to the technical field of biological engineering. In the present invention, the 225-285-position peptide segment of the amino acid sequence of Arabidopsis alpine nitrilase is embedded into the turnip nitrilase that lacks the corresponding peptide segment to obtain a turnip/Arabidopsis nitrilase chimera, and then the chimeric BaNIT The coding gene of the nitrile is subjected to site-directed saturation mutation to obtain a plant nitrilase mutant, wherein the plant nitrilase mutant is one of the following or more than two of them: (1) L mutation at position 223 is Q; (2) The H at position 263 is mutated to D; (3) the Q at position 279 is mutated to E. The catalytic activity of the plant nitrilase mutant provided by the present invention is increased by 2.23 times, the solubility of the recombinant protein is greatly improved, the enantiomeric selectivity E value is maintained above 400, and the synthesis of racemic isobutylsuccinonitrile ( S)-3-cyano-5-methylhexanoic acid has good application prospects.

Description

A kind of plant nitrilase mutant, encoding gene and application thereof

technical field

The invention relates to the technical field of bioengineering, in particular to a cruciferous plant nitrilase mutant, an encoding gene, and the preparation of a key chiral intermediate (S) of pregabalin by hydrolysis of racemic isobutyl succinonitrile - Use in 3-cyano-5-methylhexanoic acid.

Background technique

Nitrile compounds are important intermediates in organic synthesis. They can synthesize chemicals with higher added value and wider application range, such as amides, carboxylic acids and hydroxamic acids. They are widely used in chemical, pesticide and pharmaceutical industries. However, the chemical hydrolysis of nitrile often requires reaction conditions such as strong acid (or strong base), high temperature, and high pressure, resulting in serious environmental pollution. Nitrile hydrolase biocatalysis has high chemical, regio- and stereoselectivity, mild reaction conditions and low environmental pollution, which meets the requirements of green and sustainable development, and has broad application prospects in the field of organic chemicals. At present, nitrilase has been successfully used in the industrial production of fine and pharmaceutical chemicals such as niacin, (R)-mandelic acid and 1,5-dimethyl-2-piperidone.

With the development of modern molecular biology technology and the demand for biocatalysts in the industrial production environment, protein molecular modification has become a hot spot in current enzyme engineering research. Molecular modification techniques have played an important role in the modification of application properties such as nitrilase catalytic activity, substrate specificity, thermostability, and stereoselectivity.

Molecular modification of Alcaligenes faecalis nitrilase, Schreiner et al. obtained a mutant that can efficiently catalyze the hydrolysis of (R)-2-chloro-mandelonitrile to (R)-2-chloro-mandelic acid (Enzyme Microb.Tech. , 2010, 47, 140-146). DeSantis et al. used DNA shuffling technology to transform nitrilase and obtained nitrilase mutants that could catalyze the synthesis of S-type and R-type products from 3-hydroxyglutaronitrile, respectively, with an ee value greater than 95% and a yield of 98% (J . Am. Chem. Soc., 2003, 125, 11476-11477).

One challenge of exogenous gene expression in E. coli is that the target protein often forms inactive inclusion bodies, which seriously affects the catalytic performance of the enzyme. Molecular modification technology can reduce the formation of inclusion bodies and improve the soluble expression of proteins by replacing one or several amino acids. Xie et al. successfully constructed a double mutant C40A/C60N with a 50% increase in catalytic activity and protein soluble expression by replacing the cysteine residue in the Lov D amino acid sequence (Biotechnol. Bioeng., 2009, 102, 20-28 ).

Pregabalin (PGB for short), chemical name (S)-3-aminomethyl-5-methylhexanoic acid, is the 3-isobutyl group of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Substitutes (Angew.Chem.Int.Ed., 2008, 47, 3500-3504) are the main drugs for the treatment of spinal cord injury, anxiety and epilepsy. Regio- and stereoselective hydrolysis of racemic isobutylsuccinonitrile (IBSN) by nitrilase to synthesize key chiral intermediates of pregabalin (S)-3-cyano-5-methylhexanoic acid ((S)- The CMHA) route has the remarkable advantages of cheap raw materials, simple process and high atom economy. However, the reported nitrilases have low catalytic activity and stereoselectivity, which cannot meet the needs of industrial production. Therefore, it is of great significance to develop novel and efficient nitrilases that can efficiently resolve IBSNs.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a cruciferous plant nitrilase mutant with improved solubility and catalytic activity to meet the requirements of industrial production.

To achieve the above object, the present invention adopts the following technical solutions:

In the present invention, the 225-285 position peptide segment of the amino acid sequence of Arabidopsis nitrilase (AaNIT) is embedded into the turnip nitrilase (BrNIT) lacking the corresponding peptide segment to obtain the turnip/Arabidopsis nitrilase chimera (BaNIT), the amino acid sequence of which is shown in SEQ ID NO.2. Furthermore, site-directed saturation mutation was performed on the encoding gene of the chimeric BaNIT to obtain a plant nitrilase mutant.

The plant nitrilase mutant is one of the following or more than two of them: (1) L at position 223 is mutated to Q; (2) H at position 263 is mutated to D; (3) L at position 279 is mutated to D; Q is mutated to E.

specifically,

The amino acid sequence of BaNIT-L223Q (the L at position 223 is mutated to Q) is shown in SEQ ID NO.4;

The amino acid sequence of BaNIT-H263D (the H at position 263 is mutated to D) is shown in SEQ ID NO.6;

The amino acid sequence of BaNIT-Q279E (the Q at position 279 is mutated to E) is shown in SEQ ID NO.8;

The amino acid sequence of BaNIT-L223Q/H263D (L at position 223 is mutated to Q, and H at position 263 is mutated to D) is shown in SEQ ID NO.10;

The amino acid sequence of BaNIT-L223Q/Q279E (the L at position 223 is mutated to Q, and the Q at position 279 is mutated to E) is shown in SEQ ID NO.12;

The amino acid sequence of BaNIT-H263D/Q279E (H at position 263 is mutated to D, and Q at position 279 is mutated to E) is shown in SEQ ID NO.14;

The amino acid sequence of BaNIT-L223Q/H263D/Q279E (L at position 223 is mutated to Q, H at position 263 is mutated to D, and Q at position 279 is mutated to E) is shown in SEQ ID NO.16.

Studies have shown that the catalytic activity and stereoselectivity of the chimera (BaNIT) for the substrate racemic IBSN are significantly improved compared with the wild-type nitrilase. The solubility and catalytic activity of the above-mentioned plant nitrilase mutants were further improved than that of the chimera (BaNIT).

Conservative substitution forms, addition or deletion of one or several amino acids, amino-terminal truncated forms, and carboxyl-terminal truncated forms of the plant nitrilase mutants in other amino acid positions, these mutant forms are also included in the present invention. within the scope of the invention.

The present invention also provides an encoding gene encoding the plant nitrilase mutant, the nucleotide sequence of the encoding gene is 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 present invention also provides a recombinant vector comprising the encoding gene. Preferably, the original vector is pET28b.

The present invention also provides recombinant genetically engineered bacteria comprising the recombinant vector. The recombinant vector transforms host cells to obtain recombinant genetically engineered bacteria, and the host cells can be various conventional host cells in the art. Preferably, the host cells are Escherichia coli BL21.

The present invention also provides a preparation method for constructing the plant nitrilase mutant, the preparation method comprising the following steps:

(1) Designing PCR primers for the turnip nitrilase gene sequence, using the Arabidopsis alpine cDNA as a template, and using the PCR primers to amplify to obtain a DNA fragment containing the nucleotide sequence 675-855 of the Arabidopsis nitrilase nucleotide sequence I;

(2) using the recombinant plasmid carrying the turnip nitrile hydrolase gene as a template, using inverse PCR amplification to obtain the BrNIT plasmid fragment missing the nucleotide sequence 678-858 of the turnip nitrile hydrolase;

(3) Recombining the DNA fragment I and the BrNIT plasmid fragment, then transforming the recombined product into the host bacteria, and screening to obtain the recombinant parental nitrilase expression strain, wherein the nucleotide sequence of the parental nitrilase is as SEQ ID NO.1 shown;

(4) Designing site-directed mutagenesis primers, using the recombinant plasmid carrying the parental nitrilase gene obtained in step (3) as a template, performing overlap extension PCR to obtain the L mutation at position 223 in the parental nitrilase to be Q or 263 The single point mutation product of the H mutation at position 279 to D or the Q mutation at position 279 to E;

(5) Using the single-site mutation product as a template, using the site-directed mutagenesis primer to carry out overlapping extension PCR to obtain a double-site mutation product; then using the double-site mutation product as a template, using the site-directed primer to carry out overlapping extension PCR to obtain three-site mutation products point mutation product;

(6) transforming the single-site mutation product, double-site mutation product and triple-site mutation product into host bacteria respectively, screening to obtain a nitrilase mutant expression strain, inducing expression, and obtaining the plant nitrilase mutation body.

In steps (1)-(3), the nucleotide sequence corresponding to the 225-285 peptide of Arabidopsis alpine nitrilase (AaNIT) was inserted into the turnip nitrilase ( BrNIT) plasmid fragment, the parental nitrilase (BaNIT) engineered bacteria were obtained.

The primers required to amplify DNA fragment I:

Upstream primer: 5'-GAATGGCAGTCTTCTATGATGCACATCGC-3' (SEQ ID NO. 17);

Downstream primer: 5'-GAAGTTCGGACCAGCCAGAACCTGACCC-3' (SEQ ID NO. 18).

Primers required to amplify the BrNIT plasmid fragment:

Upstream primer: 5'-GCGATGTGCATCATAGAAGACTGCCATTC-3' (SEQ ID NO. 19);

Downstream primer: 5'-GGGTCAGGTTCTGGCTGGTCCGAACTTC-3' (SEQ ID NO. 20).

In steps (4)-(5), saturation site-directed mutagenesis is performed on the parental nitrilase gene.

The primers required for the mutation of Leu at position 223 to Gln:

Upstream primer: 5'-CAGTCTTCTATGCTGCACATCGCTCTGGAAGG-3' (SEQ ID NO.21);

Downstream primer: 5'-CCTTCCAGAGCGATGTGCAGCATAGAAGACTG-3' (SEQ ID NO. 22).

Primers required for the mutation of His at position 263 to Asp:

Upstream primer: 5'-CAACCAGGAAGACGACGCTATCGTTTCTCAGGG-3' (SEQ ID NO.23);

Downstream primer: 5'-CCCTGAGAAACGATAGCGTCGTCTTCCTGGTTG-3' (SEQ ID NO. 24).

Primer required for mutation of Gln at position 279 to Glu:

Upstream primer: 5'-CATCTCTCCGCTGGGTCAGGTTCTGGCTGG-3' (SEQ ID NO. 25);

Downstream primer: 5'-CCAGCCAGAACCTGACCCAGCGGAGAGATG-3' (SEQ ID NO. 26).

Preferably, the original vector of the recombinant plasmid is pET28b. The host bacteria is Escherichia coli E.coliBL21.

Another object of the present invention is to provide the application of the plant nitrilase mutant in catalyzing racemic isobutylsuccinonitrile to prepare (S)-3-cyano-5-methylhexanoic acid.

The described application is to use the wet cell body, the wet cell body immobilized cell, the enzyme extracted after the wet cell body is ultrasonically broken, or the immobilized enzyme obtained by centrifuging the engineered bacteria containing the plant nitrilase mutant encoding gene after fermentation and culture as The catalyst uses racemic isobutyl succinonitrile as the substrate, and the buffer solution with pH of 6-10 is used as the reaction medium, and the water bath reaction is carried out under the conditions of 20-50 ° C and 200-400 rpm. After the reaction is completed, the reaction solution is separated. Purification gave (S)-3-cyano-5-methylhexanoic acid.

The nitrilase mutants of the present invention can be used in the form of whole cells of engineered bacteria, or in the form of unpurified crude enzymes, or in the form of partially purified or completely purified enzymes. The nitrilase mutants of the invention can also be made into 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 100-150 g/L, and the amount of the catalyst is 5-20 g/L based on the weight of the wet cells, wherein the moisture content of the wet cells is 70-90%.

Preferably, the reaction medium is Tris-HCl buffer with pH value of 8.0, and the catalytic reaction temperature is 35°C.

Preferably, the wet cells are recombinant engineering bacteria E.coliBL21(DE3)/pET28b-BaNIT-L223Q, E.coli BL21(DE3)/pET28b-BaNIT-H263D, E.coliBL21(DE3)/pET28b-BaNIT-Q279E, E.coli BL21(DE3)/pET28b-BaNIT-L223Q/H263D, E.coliBL21(DE3)/pET28b-BaNIT-L223Q/Q279E, E.coli BL21(DE3 )/pET28b-BaNIT-H263D/Q279E, E. coli BL21(DE3)/pET28b-BaNIT-L223Q/H263D/Q279E.

The fermentation culture method is as follows: the recombinant engineered bacteria are inoculated into the LB liquid medium containing kanamycin (final concentration is 50 mg/L), and shaken for 8 hours under the conditions of 37 ° C and 200 rpm; In the LB liquid medium containing kanamycin (final concentration 50 mg/L), shake cultured at 37 °C and 150 rpm until the OD ₆₀₀ of the cells is 0.6-0.8, add isopropyl- β-D-thiogalactopyranoside (IPTG) was induced and cultured at 28°C and 150rpm for 10h, and the cells were collected by centrifugation at 4°C and 9000rpm for 10min. The cells were washed twice with physiological saline, and the cells obtained by centrifugation were stored in a -20°C refrigerator.

The beneficial effects that the present invention has:

Compared with the parental nitrilase chimera (BaNIT), the plant nitrilase mutant provided by the present invention has a catalytic activity more than 1.2 times higher, and can efficiently catalyze the synthesis of racemic isobutylsuccinonitrile (S)-3-cyano- 5-methylhexanoic acid has good application prospects. Especially the triple mutant BaNIT-L223Q/H263D/Q279E, the catalytic activity was increased by 2.23 times, the solubility of the recombinant protein was greatly improved, and the enantioselectivity E value remained above 400.

The plant nitrilase mutant provided by the invention can utilize a small amount of cells to catalyze the hydrolysis of high-concentration IBSN (100 g/L), the conversion rate can reach more than 48.0% (ee>98.5%), greatly reduce the cost of industrialized production, and meet the requirements of pregabalin Industrial production requirements for key chiral intermediates.

Description of drawings

Figure 1 shows the soluble expression of nitrilase mutants (SDS-PAGE gel electrophoresis), where M is marker, and its band position represents 50kD, lane 1 is BaNIT, lane 2 is BaNIT-L223Q, and lane 3 is BaNIT- H263D, lane 4 is BaNIT-Q279E, lane 5 is BaNIT-L223Q/H263D/Q279E.

Figure 2 shows the comparison of nitrilase mutant L223Q/H263D/Q279E with the parental BaNIT and wild-type BrNIT in whole cell catalytic IBSN.

Fig. 3 is a graph showing the reaction progress of nitrilase mutant L223Q/H263D/Q279E whole cell (5g/L) catalyzing IBSN (100g/L) to prepare (S)-CMHA.

Detailed ways

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

Example 1

Construction of the parental nitrilase gene

The key peptide segment 225-285 region was determined by aligning and analyzing the nucleotide and amino acid sequences of cruciferous nitrilase. The sequence of wild-type turnip nitrilase (BrNIT) can be found in GenBank accession number: ABM55734.1; the sequence of wild-type Arabidopsis nitrilase (AaNIT) can be found in GenBank accession number: KFK44999.

The nucleotide sequence corresponding to the 225-285 peptide fragment of Arabidopsis alpine nitrilase (AaNIT) was inserted into the turnip nitrilase (BrNIT) plasmid fragment lacking the corresponding peptide by a one-step cloning method. The designed primers are shown in Table 1.

Table 1: BaNIT Chimerase Primer Design Table

Master Mix，上下游引物各0.2μM，其余ddH₂O补充至总体积。PCR反应参数：(1)94℃预变性5min；(2)94℃变性30s；(3)58℃退火30s；(4)72℃延伸10s，步骤(2)-(4)循环30次；(5)72℃再次延伸10min，4℃保存。PCR产物经过琼脂糖凝胶电泳分析后切胶回收，65℃灭活10min，放置4℃备用。Using the AaNIT nucleotide sequence as a template, the DNA fragment of the 225-285 peptide was cloned. PCR reaction system (50μL): Template DNA<1μg,

Master Mix, upstream and downstream primers are 0.2 μM each, and the rest of ddH ₂ O is supplemented to the total volume. PCR reaction parameters: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30s; (3) annealing at 58°C for 30s; (4) extension at 72°C for 10s, steps (2)-(4) were cycled 30 times; ( 5) Extend again at 72°C for 10min, and store at 4°C. The PCR product was analyzed by agarose gel electrophoresis and then recovered by cutting the gel, inactivated at 65°C for 10 min, and placed at 4°C for use.

At the same time, using the recombinant plasmid containing the BrNIT nucleotide sequence as a template, primers were designed to amplify the BrNIT plasmid fragment lacking the 226-286 peptide segment.

Vector linearization was obtained by inverse PCR amplification. PCR reaction system (50μL): template DNA 0.1ng-1ng, 2×Phanta Max Buffer, dNTPs (10mM each) 0.2mM, upstream and downstream primers 0.2μM each, Phanta MaxSuper-Fidelity DNA Polymerase 1U, the rest ddH ₂ O supplemented to total volume. PCR reaction parameters: (1) pre-denaturation at 95°C for 30s; (2) denaturation at 95°C for 15s; (3) annealing at 63°C for 15s; (4) extension at 72°C for 6.0 min, steps (2)-(4) were cycled 30 times; (5) Thoroughly extend at 72°C for 5 min, and store at 4°C. After the PCR product was analyzed by agarose gel electrophoresis, the endonuclease Dpn I was added to digest at 37°C for 3h, and then inactivated at 65°C for 10min.

Linearize the BrNIT vector sequence that lacks the corresponding peptide gene fragment, and introduce the end sequence of the linearized vector at the 5' end of the forward/reverse PCR primer of the insert, so that the 5' and 3' ends of the PCR product have and linear Sequences that are identical at both ends of the vector.

The gene concentration of the inserted fragment and linearized vector obtained above was determined by NanoDrop ^TM One/OneC ultra-micro UV spectrophotometer, and the added amount of each inserted peptide segment and the linearized vector corresponding to the deleted peptide segment was calculated. Ligation reaction system: linearized vector 0.03 pmol, insert 0.06 pmol, 5×CE II Buffer 4 μL, Exnase II 2 μL, ddH ₂ Oto 20 μL. After mixing the PCR samples, they were placed at 37°C for 30 min, and then lowered to 4°C.

The ligated PCR product was heat-shock transformed into E. coli BL21 (DH5α) competent cells, and after recovery, it was spread on a solid LB plate containing kanamycin resistance for culture. Pick a single colony, incubate it in LB liquid medium, and extract the plasmid for sequencing. The correct sequencing result is the nucleotide sequence of the parental nitrilase gene, that is, SEQ ID NO. 1 (the amino acid sequence is SEQ ID NO. 2) in the sequence listing. The parental nitrilase gene was transformed into Escherichia coli E.coli BL21 (DE3) competent cells, spread on the LB plate containing kanamycin and cultured overnight to obtain the parental nitrilase engineering bacteria E.coliBL21 ( DE3)/pET28b-BaNIT.

Example 2

Site-directed saturation mutagenesis at nitrilase sites 223, 263 and 279

In order to carry out saturation mutation of Leu (L) at position 223, His (H) at position 263 and Gln (Q) at position 279 in the parental amino acid sequence, corresponding primers were designed, as shown in Table 2. Show.

Table 2: Primer Design Table

Note: N=A/G/C/T, K=G/T, M=A/C.

Using the recombinant plasmid pET28b-BaNIT containing the target gene fragment as the template, the whole plasmid was amplified according to the method of overlap extension PCR.

PCR amplification system (50μL): template DNA 0.1ng-1ng, 2×Phanta Max Buffer 25μL, dNTPs (10mM each) 1μL, mutation primer upstream and downstream each 1μL, Phanta Max Super-Fidelity DNAPolymerase 1U, the rest ddH ₂ O Make up to total volume.

PCR reaction parameters: (1) pre-denaturation at 95°C for 30s; (2) denaturation at 95°C for 15s; (3) annealing at 63°C for 15s; (4) extension at 72°C for 6 min, steps (2)-(4) were cycled 30 times; ( 5) Thoroughly extend at 72°C for 5min, and store at 4°C.

After the PCR product was analyzed as positive by 0.9% agarose gel electrophoresis, 20 μL of PCR reaction solution was taken, 1 μL of endonuclease Dpn I was added, and the template plasmid DNA was digested at 37°C for 3 hours to remove the template plasmid DNA, and inactivated at 65°C for 10 minutes. The cells were transformed into E. coliBL21 (DE3) competent cells by heat shock, and after recovery, they were spread on LB plates containing kanamycin and cultured overnight. Each plate obtained a mutant pool of about 300 clones.

Pick a single colony into a 96-well culture plate with LB medium, and cultivate at 37°C until the bacterial OD ₆₀₀ is about 0.6-0.8, add IPTG (final concentration: 0.1mM) to the above LB liquid medium, 28°C , 150rpm induced culture for 10-12h. Centrifuge in a 96-well plate centrifuge at 4°C, 3000 rpm for 30 min, discard the supernatant, add 600 μL of sodium phosphate buffer (50 mM, pH 7.4) to the collected cells and mix well.

Take 200 μL of bacterial suspension and mix with 10 μL of IBSN (100 mg/mL dissolved in N,N-dimethyl sulfoxide), shake at 37°C for 1 h, and add 30 μL of 2M HCl to each well to stop the reaction. Pipette 10 μL of the reaction solution and mix it with 150 μL of the combination of o-phthalaldehyde and mercaptoethanol, and incubate at 37 °C for 30 min; detect the fluorescence intensity of each sample under a microplate reader, where the excitation wavelength is set to 412 nm, and the emission wavelength is set to 467 nm . According to the change of the fluorescence intensity, the catalytic activity of the mutant was judged, and the clones whose change intensity was much larger than that of the control strain (starting strain) were screened out.

After re-screening and verification of the screened positive clones, the whole plasmid of the mutant strain was extracted, and the introduced point mutation was confirmed by DNA sequencing. The DNA sequencing results of the mutant strain with the highest activity at each site showed that the Leu mutation at position 223 was Gln (L223Q). , His at position 263 was mutated into Asp (H263D) and Gln at position 279 was mutated into Glu (Q279E), thereby obtaining 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. The nucleotide sequences of mutants L223Q, H263D and Q279E are respectively SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7 (the corresponding amino acid sequences are SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.7). NO.8).

Example 3

Construction of nitrilase combinatorial mutants

Using the expression plasmids pET28b-BaNIT-L223Q or pET28b-BaNIT-H263D as templates, site-directed mutagenesis was performed by whole plasmid amplification.

PCR amplification system (50μL): template DNA 0.1ng-1ng, 2×Phanta Max Buffer 25μL, dNTPs (10mM each) 1μL, mutation primer upstream and downstream each 1μL, Phanta Max Super-Fidelity DNAPolymerase 1μL, the rest ddH ₂ O Make up to total volume.

PCR reaction parameters: (1) pre-denaturation at 95°C for 30s; (2) denaturation at 95°C for 15s; (3) annealing at 60°C for 15s; (4) extension at 72°C for 6 min, steps (2)-(4) were cycled 30 times; ( 5) Thoroughly extend at 72°C for 5min, and store at 4°C.

After the PCR product was analyzed as positive by 0.9% agarose gel electrophoresis, 20 μL of PCR reaction solution was taken, 1 μL of endonuclease Dpn I was added, and the template plasmid DNA was digested at 37°C for 3 hours to remove the template plasmid DNA, and inactivated at 65°C for 10 minutes. Heat shock transformed into E.coli BL21(DE3) competent cells, after recovery, spread on LB plate containing kanamycin and culture overnight, pick a single colony and culture it on kanamycin-resistant (final concentration of 50mg/L) of LB liquid medium, extract the plasmid for sequencing.

The correct sequencing results are the nitrilase combination mutant engineering strains 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, the nucleotide sequences corresponding to the combined mutant engineering strains are 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

Mutants BaNIT-L223Q, BaNIT-H263D, BaNIT-Q279E obtained in Example 2 and Example 3, combined mutants BaNIT-L223Q/H263D, BaNIT-L223Q/Q279E, BaNIT-H263D/Q279E and BaNIT-L223Q/H263D /Q279E, as well as parental BaNIT and wild-type BrNIT were inoculated into LB medium containing kanamycin (final concentration of 50 mg/L), cultured at 37°C for 6-8 h, and transferred at 2% (v/v) inoculum Expand the culture in fresh LB liquid medium containing kanamycin (final concentration of 50mg/L), cultivate at 37°C and 150rpm until the bacterial OD ₆₀₀ is about 0.6-0.8, add to the above LB liquid medium IPTG (final concentration: 0.1 mM) was added, induced and cultured for 10-12 h at 28°C and 150 rpm, and the cells were collected by centrifugation at 4°C and 9000 rpm for 10 min. The cells were washed twice with physiological saline, and the cells obtained by centrifugation were stored in a -20°C refrigerator.

Example 5

Soluble expression studies of nitrilase mutants

The bacterial cells (equivalent) collected in Example 4 were dissolved in Tris-HCl (50 mM) buffer at pH 8.0, and the bacterial cells were resuspended and disrupted by ultrasonic waves (400 W, 5 min, 1 s disruption and 1 s pause). The crushed products were centrifuged (12000 rpm, 5 min) and the supernatant (crude enzyme solution) was taken for denaturation treatment, and the soluble expression level of the protein was verified by SDS-PAGE gel electrophoresis.

From the results of SDS-PAGE gel electrophoresis (Fig. 1), the soluble expression levels of the mutants BaNIT-L223Q, BaNIT-H263D, and BaNIT-Q279E were slightly higher than those of the parental BaNIT, and the triple mutant BaNIT-L223Q/H263D/Q279E had higher soluble expression levels. The level of soluble expression was greatly improved. It can be seen that the solubility of the target protein is improved by modifying the parent BaNIT.

Example 6

Determination of Viability of Recombinant Escherichia coli Containing Nitrilase Mutants

The recombinant E. coli obtained in Example 4 was subjected to viability assay. The composition of the reaction system: 1 mL of Tris-HCl buffer solution (50 mM, pH 8.0), 20 mM of IBSN, and 0.2 mg of wet cells. The reaction solution was preheated at 40°C for 2 min, and then reacted at 600 rpm for 10 min. Sampling 500 μL, adding 200 μL 2M HCl to stop the reaction and extracting with ethyl acetate. After drying the upper organic phase with anhydrous sodium sulfate, the conversion rate of the substrate and the enantiomeric excess (ee) of the product were determined by gas chromatography.

The enantiomeric excess of substrate IBSN and product CMHA was determined by gas chromatography. Gas chromatograph model 7890N (Agilent) and capillary column model BGB-174 (BGB Analytik Switzerland). The chromatographic conditions were as follows: the injection volume was 1.0 μL, the inlet and detector temperatures were both 250 °C, the column temperature was 120 °C for 15 min, and the temperature was raised to 170 °C at 10 °C/min for 9 min. The carrier gas was high-purity helium with a flow rate of 1.0 mL/min and a split ratio of 50:1. The calculation of enantiomeric excess (ee) and conversion (c) refers to the calculation method of Rakels et al. (Enzyme Microb. Technol., 1993, 15:1051).

The results of the determination of the activity of the recombinant E. coli containing the nitrilase mutant are shown in Table 3 and Table 4. The catalytic activity of the combined mutant is significantly higher than that of the parent. Among them, the activity of the triple mutant BaNIT-L223Q/H263D/Q279E is 2.2 times that of the parent, and The E value of all mutants remained above 400.

Table 3: Comparison of nitrilase activity

Table 4: Comparison of nitrilase activity

Example 7

Application of Recombinant Escherichia coli Containing Nitrilase in the Preparation of (S)-CMHA (1)

The transformation system composition and transformation operation are as follows: In 1L of Tris-HCl buffer solution (50mM, pH 8.0), the recombinant nitrilase mutant E.coli BL21(DE3)/pET28b-BaNIT-L223Q obtained in Example 4 was respectively added /H263D/Q279E, parental E.coli BL21(DE3)/pET28b-BaNIT and wild-type E.coli BL21(DE3)/pET28b-BrNIT (addition amount is 15g/L respectively), the content of substrate is 100g/L. Reaction conditions: 35° C., 400 rpm, the reaction progress was detected by chiral gas chromatography during the reaction, and the gas chromatography detection conditions were shown in Example 6.

It can be seen from Figure 2 that the conversion rates of the mutant BaNIT-L223Q/H263D/Q279E and the parent BaNIT reached 48.1% and 40.4%, respectively, and the E value of the product (S)-CMHA remained above 400 for 10 h; The conversion rate of BrNIT was 35.1%, and the E value of the product (S)-CMHA was about 150.

After the reaction for 10 hours, the above reaction was terminated, the E. coli cells were removed by centrifugation, and the reaction solution was distilled under reduced pressure to 1/3 volume. Add 2 volumes of ethyl acetate (sample after distillation under reduced pressure) for extraction, and collect the lower aqueous phase. The pH of the lower aqueous phase pool was adjusted to 4.0 with 2M HCl. 2 volumes of ethyl acetate were added again for extraction, and the lower aqueous phase was discarded. The upper organic phase was collected and rotary evaporated to remove the ethyl acetate to give (S)-3-cyano-5-methylhexanoic acid as an oil (ee>99.5%).

Example 8

Application of Recombinant Escherichia coli Containing Nitrilase in the Preparation of (S)-CMHA (2)

Catalytic system composition and catalytic operation are as follows: in 100mL Tris-HCl buffer system (50mM, pH 8.0), substrate IBSN addition amount is 150g/L, adds the recombinant nitrilase mutant E.coli BL21 ( DE3)/pET28b-BaNIT-L223Q/H263D/Q279E (20g/L); Reaction conditions: 30 ℃, 400rpm, reaction progress is detected by gas chromatography, and gas chromatography detection conditions are shown in Example 6.

After 10h of reaction, the conversion rate reached 40.1%, and the product ee>99.6%.

Example 9

Application of Recombinant Escherichia coli Containing Nitrilase in the Preparation of (S)-CMHA(Ⅲ)

Weigh 0.1 g 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 the parental strain E. coli BL21(DE3)/pET28b-BaNIT wet cells were suspended in 10 mL of Tris-HCl buffer system (50 mM, pH 8.0). 100g/L of IBSN was added, and the reaction was carried out in a water bath at 30°C and 200rpm. Samples were taken at different times to detect the reaction progress, and the sample processing methods and detection methods were shown in Example 6.

Reaction for 10h, E.coli BL21(DE3)/pET28b-BaNIT-L223Q/H263D, E.coli BL21(DE3)/pET28b-BaNIT-L223Q/Q279E, E.coli BL21(DE3)/pET28b-BaNIT-H263D/Q279 The transformation rates of the parent strain E.coli BL21(DE3)/pET28b-BaNIT reached 43.6%, 41.9%, 42.7% and 30.2%, respectively.

Example 10

Application of Recombinant Escherichia coli Containing Nitrilase in the Preparation of (S)-CMHA(4)

0.05 g of E. coli BL21(DE3)/pET28b-BaNIT-L223Q/H263D/Q279E wet cells obtained in Example 4 were weighed and suspended in 10 mL of Tris-HCl buffer system (50 mM, pH 8.0). 1.0 g of IBSN was added, and the reaction was carried out in a water bath at 35° C. and 200 rpm. Samples were taken at different times to detect the reaction progress, and the sample processing methods and detection methods were shown in Example 6. It can be seen from Figure 3 that the conversion rate reached 43.5% after the reaction for 18h, and the product ee was >99.3%.

The present invention is not limited by the above-mentioned specific text description, and various changes can be made in the present invention within the scope of the claims, and these changes are all within the scope of the present invention.

sequence listing

<110> Zhejiang University of Technology

<120> A kind of plant nitrilase mutant, encoding gene and application thereof

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1050

<212> DNA

<213> 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

<400> 2

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

<211> 1050

<212> DNA

<213> Artificial Sequence

<400> 3

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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<400> 17

gaatggcagt cttctatgct gcacatcgc 29

<210> 18

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 18

gaagttcgga ccagccagaa cctgaccc 28

<210> 19

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 19

gcgatgtgca gcatagaaga ctgccattc 29

<210> 20

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 20

gggtcaggtt ctggctggtc cgaacttc 28

<210> 21

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 21

cagtcttcta tgctgcacat cgctctggaa gg 32

<210> 22

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 22

ccttccagag cgatgtgcag catagaagac tg 32

<210> 23

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 23

caaccaggaa gacgacgcta tcgtttctca ggg 33

<210> 24

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 24

ccctgagaaa cgatagcgtc gtcttcctgg ttg 33

<210> 25

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 25

catctctccg ctgggtcagg ttctggctgg 30

<210> 26

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 26

ccagccagaa cctgacccag cggagagatg 30

Claims (9)

1. A plant nitrilase mutant, characterised in that its amino acid sequence is as shown in SEQ ID NO.4 or SEQ ID NO.6 or SEQ ID NO.8 or SEQ ID NO.10 or SEQ ID NO.12 or SEQ ID NO.14 or SEQ ID NO. 16.

2. A gene encoding a plant nitrilase mutant according to claim 1 characterised in that the nucleotide sequence of the gene is as shown in 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.

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 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.

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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