阅读说明：本技术 一种植物腈水解酶突变体、编码基因及其应用 (Plant nitrilase mutant, coding gene and application thereof ) 是由 郑仁朝 郑裕国 张琴 汤晓玲 于 2018-07-12 设计创作，主要内容包括：本发明公开了一种十字花科植物腈水解酶突变体,属于生物工程技术领域。本发明将高山南芥腈水解酶氨基酸序列的225-285位肽段嵌入到缺失对应肽段的芜菁腈水解酶中,获得芜菁/高山南芥腈水解酶嵌合体,进而对嵌合体BaNIT的编码基因进行定点饱和突变,获得植物腈水解酶突变体,所述的植物腈水解酶突变体为下列之一或其中两种以上：(1)第223位的L突变为Q；(2)第263位的H突变为D；(3)第279位的Q突变为E。本发明提供的植物腈水解酶突变体的催化活力提高2.23倍,重组蛋白的可溶性大幅提高,对映体选择率E值保持在400以上,在高效催化外消旋异丁基丁二腈合成(S)-3-氰基-5-甲基己酸中具有良好的应用前景。(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.)

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 ℃.

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 th position is mutated into Q, H at the 263 th 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.

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 reached₆₀₀Adding isopropyl-beta-D-thiogalactopyranoside (IPTG) with final concentration of 0.1mM at 0.6-0.8, inducing and culturing at 28 deg.C and 150rpm for 10h, centrifuging at 4 deg.C and 9000rpm for 10min, and collecting thallusA cell. 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: