Nitrilase mutant with improved nitrile hydrolysis activity specificity and application thereof
阅读说明：本技术 一种腈水解活性专一性提高的腈水解酶突变体及其应用 (Nitrilase mutant with improved nitrile hydrolysis activity specificity and application thereof ) 是由 郑仁朝 汤晓玲 闻鹏飞 郑裕国 于 2020-12-30 设计创作，主要内容包括：本发明公开了一种腈水解活性专一性提高的腈水解酶突变体及其应用,所述突变体是将SEQ ID NO.1所示氨基酸序列第200位、第224位、第229位、第246位进行单突变或多突变获得的。本发明构建的突变体C145N、K200R/R224W/A229P、K200R/R224W/N246V催化苯乙腈产生的主产物为苯乙酸,分别占产物含量的96.5％、96.3％和98.8％,腈水合活性比K200R/R224W分别下降88.3％、82.2％和94.3％,有利于定向生成目的产物苯乙酸。对腈水解酶的反应专一性调控使其能够应用于酰胺和羧酸的绿色工业催化合成,具有重要意义。(The invention discloses a nitrilase mutant with improved nitrile hydrolysis activity specificity and application thereof, wherein the mutant is obtained by carrying out single mutation or multiple mutation on the 200 th site, the 224 th site, the 229 th site and the 246 th site of an amino acid sequence shown in SEQ ID NO. 1. The mutants C145N, K200R/R224W/A229P and K200R/R224W/N246V constructed by the invention catalyze phenylacetonitrile to generate main products, namely phenylacetic acid which respectively account for 96.5%, 96.3% and 98.8% of the product content, and the nitrile hydration activity is respectively reduced by 88.3%, 82.2% and 94.3% compared with K200R/R224W, so that the targeted production of the target product phenylacetic acid is facilitated. The method has important significance for the green industrial catalytic synthesis of amide and carboxylic acid by specifically regulating and controlling the reaction of nitrilase.)
1. A nitrilase mutant having an improved nitrile hydrolysis activity specificity, characterized in that the mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO.1 to single mutation or multiple mutations at the 200 th, 224 th, 229 th and 246 th positions.
2. A nitrilase mutant as claimed in claim 1 characterised in that the mutant is one of: (1) mutating the 145 th cysteine of the amino acid sequence shown in SEQ ID NO.1 into asparagine; (3) the amino acid sequence shown in SEQ ID NO.1 is mutated from lysine at the 200 th position to arginine, from arginine at the 224 th position to tryptophan and from alanine at the 229 th position to proline; (4) the amino acid sequence shown in SEQ ID NO.1 has the mutation of lysine at the 200 th position into arginine, the mutation of arginine at the 224 th position into tryptophan and the mutation of asparagine at the 246 th position into valine.
3. A gene encoding the nitrilase mutant of claim 1.
4. A recombinant genetically engineered bacterium constructed from the coding gene of claim 3.
5. Use of the nitrilase mutant of claim 1 for catalyzing the synthesis of phenylacetic acid from phenylacetonitrile.
6. The use according to claim 5, characterized in that said use is: wet thalli obtained after fermentation culture of engineering bacteria containing nitrile hydrolase mutant coding genes or enzyme extracted after the wet thalli is crushed is used as a biocatalyst, phenylacetonitrile is used as a substrate, methanol is used as an auxiliary agent, a buffer solution with the pH of 7.5-8.5 is used as a reaction medium to form a conversion system, conversion reaction is carried out at the temperature of 30-60 ℃ and the speed of 150-500 rpm/min, and reaction liquid is taken for separation and purification after the reaction is finished, so that phenylacetic acid is obtained.
7. The use according to claim 6, wherein the substrate is added to the conversion system at a final concentration of 20 to 100mM by volume of buffer, the catalyst is used in an amount of 5 to 20g/L by weight of wet cells; the volume concentration of the added methanol is 2-8%.
8. Use according to claim 6, characterized in that the buffer is a Tris-HCl buffer solution at pH 8.0.
9. The use according to claim 6, wherein the wet biomass is prepared by a method comprising: inoculating engineering bacteria containing nitrilase mutant coding genes into an LB liquid culture medium containing 50mg/L kanamycin at the final concentration, and performing shake culture at 37 ℃ and 200rpm for 8-10h to obtain a seed solution; inoculating the seed solution into fresh LB liquid medium containing 50mg/L kanamycin at a final concentration of 2% by volume, and shake-culturing at 37 deg.C and 180rpm until the thallus OD6000.6-0.8, adding isopropyl-beta-D-thiogalactopyranoside with final concentration of 0.1mM, performing induced culture at 28 ℃ and 180rpm for 10-12h, centrifuging at 4 ℃ and 8000rpm for 10min, and collecting thallus cells, namely wet thallus.
(I) technical field
The invention relates to a nitrilase mutant with improved nitrile hydrolysis activity specificity and application thereof in synthesis of phenylacetic acid.
(II) background of the invention
Nitrile compounds are a kind of organic chemical raw materials containing cyano groups, and are widely used in chemical industry, medicine, pesticide and material industry. Nitrilase can catalyze nitrile compounds to hydrolyze to generate carboxylic acid, has the advantages of unique chemoselectivity, stereoselectivity, regioselectivity and the like, and plays an important role in nitrile compound conversion. Some nitrilases have nitrile hydration activity in addition to catalyzing the hydrolysis of nitrile compounds to carboxylic acids. For example, Piotrowski et al have found that Arabidopsis nitrilase has both nitrile hydrolysis and nitrile hydration activities and is capable of catalyzing the conversion of beta-cyano-L-alanine to asparagine and aspartic acid, with asparagine levels as high as 60% or more (J.biol.chem.,2001,276, 2616-one 2621). In addition, Bradyrhizobium japonicum nitrilase catalyzes the hydrolysis of beta-aminopropionitrile, with a proportion of beta-aminopropionamide of up to 33% (J.mol. Catal. B: enzyme, 2015,115,113, 118).
The nitrilase has both nitrile hydrolysis and hydration activity, so that the nitrilase has great potential in biological organic synthesis. The hydrolytic activity of the nitrilase is improved, the carboxylic acid generated specifically is beneficial to the improvement of the yield and the separation and purification of the later-period product, and convenience is brought to the industrial production of the nitrilase. Therefore, the regulation and control of the reaction specificity of the nitrilase have important significance for the industrial application of the nitrilase.
With the rapid development of genetic engineering technology, the catalytic performances such as the activity, the stability, the substrate specificity and the like of nitrilase can be obviously improved through molecular modification. In recent years, the regulation and control of the reaction specificity of nitrilase has also become a research hotspot. Gong et al constructed the double mutant I128L-N161Q by site-directed mutagenesis, which catalyzed 3-cyanopyridine, increased the carboxylic acid content to 99.1% and the catalytic activity to 1.98 fold (Catalysis Science & Technology,2016,6(12): 4134-4141). DeSantis et al, performed a saturation mutagenesis of the entire gene sequence of nitrilase, obtained a mutant that was able to efficiently catalyze 3-hydroxyglutaronitrile at a substrate concentration of 3M, the product (R) -4-cyano-3-hydroxybutyric acid being the key chiral intermediate of atorvastatin (Journal of the American Chemical Society,2003,125(38): 11476-. The Yan et al modified rice (Oryza sativa) nitrilase OsNIT, when the mutant K200R/R224W takes benzyl cyanide as a substrate, the content of carboxylic acid reaches 95.1 percent (CN111321132A), but the hydration activity of the mutant K200R/R224W nitrile is still 22.74 percent of that of the wild type.
Disclosure of the invention
The invention aims to provide a nitrilase mutant for high-yield carboxylic acid, an editing gene, an engineering bacterium and application thereof, wherein OsNIT is subjected to reaction specificity regulation, the hydrolysis activity is maintained, the carboxylic acid content is increased, and the hydration activity is reduced so as to reduce the amide content.
The technical scheme adopted by the invention is as follows:
the invention provides a nitrilase mutant with improved nitrile hydrolysis activity specificity, which is obtained by performing single mutation or multiple mutations on the 200 th position, the 224 th position, the 229 th position and the 246 th position of an amino acid sequence shown in SEQ ID NO. 1. The mutant can be constructed by error-prone PCR, site-directed saturation mutagenesis and combined mutagenesis. The nucleotide sequence of the amino acid sequence coding gene shown in SEQ ID NO.1 is shown in SEQ ID NO. 2.
Further, the mutant is one of the following: (1) mutating the 145 th cysteine of the amino acid sequence shown in SEQ ID NO.1 into asparagine (C145N, shown in the amino acid sequence SEQ ID NO. 4); (3) the amino acid sequence shown in SEQ ID NO.1 is mutated from lysine at the 200 th position to arginine, from arginine at the 224 th position to tryptophan and from alanine at the 229 th position to proline (K200R/R224W/A229P and is shown in SEQ ID NO. 5); (4) the amino acid sequence shown in SEQ ID NO.1 has the advantages that the lysine at the 200 th position is mutated into arginine, the arginine at the 224 th position is mutated into tryptophan, and the asparagine at the 246 th position is mutated into valine (K200R/R224W/N246V and shown in the amino acid sequence SEQ ID NO. 6).
The invention also provides a coding gene of the nitrilase mutant, a recombinant vector constructed by the coding gene and a recombinant gene engineering bacterium, wherein the recombinant vector is constructed according to the following method: the nitrilase mutant gene is inserted between BamH I and Hind III sites of pET-28b vector to construct a recombinant plasmid containing nitrilase mutant gene. The engineering bacteria are prepared by the following method: and transferring the constructed recombinant plasmid into host bacteria to obtain recombinant engineering bacteria. The host bacterium can be Escherichia coli BL 21.
The invention provides an application of the nitrilase mutant in catalyzing benzyl cyanide to synthesize phenylacetic acid, which comprises the following steps: wet thalli obtained after fermentation culture of engineering bacteria containing nitrile hydrolase mutant coding genes or enzyme extracted after the wet thalli is crushed is used as a biocatalyst, phenylacetonitrile is used as a substrate, methanol is used as an auxiliary agent, a buffer solution with the pH of 7.5-8.5 is used as a reaction medium to form a conversion system, conversion reaction is carried out at the temperature of 30-60 ℃ and the speed of 150-500 rpm/min, and reaction liquid is taken for separation and purification after the reaction is finished, so that phenylacetic acid is obtained.
Further, the final concentration of the added substrate in the conversion system is 20-100mM by volume of buffer solution, the dosage of the catalyst is 5-20g/L by volume of the buffer solution, and the water content of the wet bacteria is 88-92%; the methanol is added in a concentration of 2-8% by volume, preferably 4.5%.
Further, the substrate concentration is preferably 100 mM.
Further, the amount of wet cells added is preferably 5 g/L.
Further, the buffer was Tris-HCl buffer solution at pH 8.0.
Further, the reaction conditions are preferably 30 ℃ and 180rpm reaction for 30 min.
Further, the preparation method of the wet thallus comprises the following steps: inoculating engineering bacteria containing nitrilase mutant coding genes into an LB liquid culture medium containing 50mg/L kanamycin at the final concentration, and performing shake culture at 37 ℃ and 200rpm for 8-10h to obtain a seed solution; inoculating the seed solution into fresh LB liquid medium containing 50mg/L kanamycin at a final concentration of 2% by volume, and shake-culturing at 37 deg.C and 180rpm until the thallus OD600Adding isopropyl-beta-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1mM, performing induction culture at 28 deg.C and 180rpm for 10-12h, and culturing at room temperature and humidity of 0.6-0.8Centrifuging at 4 deg.C and 8000rpm for 10min, and collecting thallus cells to obtain wet thallus.
Compared with the prior art, the invention has the following beneficial effects:
the invention carries out molecular modification on reaction non-specific nitrilase OsNIT through non-rational and rational design to obtain mutant C145N, mutant K200R/R224W/A229P and mutant K200R/R224W/N246V with obviously improved reaction specificity. When wild-type OsNIT catalyzes phenylacetonitrile, the phenylacetic acid content is 49.38%, and the hydrolysis activity of the OsNIT nitrile is recorded as 100%. The content of carboxylic acid in the catalytic product of the mutant C145N reaches 96.5 percent, and the relative nitrile hydrolysis activity is 39.3 percent; the content of carboxylic acid in the catalytic product of the mutant K200R/R224W/A229P is 96.3 percent, and the relative hydrolysis activity is 280.9 percent; the mutant K200R/R224W/N246V has the catalytic product with the content of carboxylic acid of 98.8 percent and the relative hydrolytic activity of 124.23 percent. The constructed mutants C145N, K200R/R224W/A229P and K200R/R224W/N246V catalyze phenylacetonitrile to generate a main product, namely phenylacetic acid which respectively accounts for 96.5%, 96.3% and 98.8% of the product content, and the nitrile hydration activity is reduced by 88.3%, 82.2% and 94.3% compared with K200R/R224W, so that the targeted production of the phenylacetic acid is facilitated. The method has important significance for the green industrial catalytic synthesis of amide and carboxylic acid by specifically regulating and controlling the reaction of nitrilase.
(IV) description of the drawings
FIG. 1 shows the comparison of the performances of wild OsNIT, mutant C145N, K200R/R224W/A229P and K200R/R224W/N246V in catalyzing phenylacetonitrile.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
composition of LB liquid medium: 10g/L peptone, 10g/L NaCl, 5g/L yeast extract powder, deionized water as solvent and natural pH value. Peptone and yeast extract powder were purchased from Saimer Feishale scientific Co., Ltd., and NaCl was purchased from national drug group reagents Ltd. And (3) sterilization conditions: sterilizing at 121 deg.C for 20min with high pressure steam sterilizing kettle. Kanamycin was added to the sterilized medium to a final concentration of 50. mu.g/mL, and the medium was used for culturing cells.
Composition of LB plate medium: the solid culture medium is prepared by adding 20g/L agar powder based on the components of the LB liquid culture medium, and sterilizing under the same conditions.
EXAMPLE 1 construction of nitrilase mutants
1. Construction of mutants
When rice nitrilase (OsNIT, GenBank accession number: AB027054, amino acid sequence SEQ ID NO.1) catalyzes benzyl cyanide reaction, the ratio of amide to carboxylic acid in the product is close to 1: 1. Through bioinformatics analysis, the catalytic triad is 196Cys-71Glu-162Lys, and then through rational design analysis, the 145 th cysteine, the 200 th lysine, the 224 th arginine, the 229 th alanine and the 246 th asparagine are subjected to site-directed saturated mutation to construct a mutant with obviously improved reaction specificity, specifically:
rice nitrilase (OsNIT, GenBank accession number: AB027054) gene (amino acid sequence SEQ ID NO.1, nucleotide sequence SEQ ID NO.2) is inserted between pET-28b vector BamH I and Hind III sites to construct recombinant plasmid. Using the recombinant plasmid as a template and the primers in Table 1, whole plasmid PCR amplification was performed in the reaction system in Table 2, and single-site-directed saturation mutagenesis was performed on the gene at positions 145, 200, 224, 229 and 246.
TABLE 1 primers
Note: n is A/G/C/T, K is G/T, and M is A/C.
TABLE 2 reaction System
Setting a PCR program: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at Tm +3 ℃ for 30s, extension at 72 ℃ for 4.5min, and 30 cycles; final extension at 72 deg.C for 10min, and storage at 4 deg.C.
And after the PCR product is analyzed and determined to be positive through 0.9% agarose gel electrophoresis, adding restriction enzyme Dpn I into the PCR product, carrying out enzyme digestion at 37 ℃ for 0.5-1h to remove the template plasmid DNA, and inactivating at 65 ℃ for 15min to obtain the PCR product without the template, namely the plasmid containing different mutants.
mu.L of template-depleted PCR product was added to 100. mu.L of E.coli BL21(DE3) competent cells, placed on ice for 30min, heat-shocked at 42 ℃ for 90s, added with 600. mu.L of LB liquid medium, thawed at 37 ℃ for 1h, spread on LB plates containing a final concentration of 50mg/L kanamycin, and cultured overnight at 37 ℃ to obtain a library of about 200 clones per plate. And selecting a single colony, inoculating the single colony in an LB liquid culture medium containing 50mg/L kanamycin resistance at a final concentration, culturing for 8 hours at 37 ℃, extracting plasmid for sequencing, obtaining a saturated mutant library containing 20 different amino acids at each site, namely engineering bacteria containing different mutants, and performing next activity determination and screening processes.
3. Preparation of mutant recombinant gene engineering bacterium wet thallus
Inoculating correctly sequenced mutant recombinant genetic engineering bacteria into LB liquid culture medium containing 50mg/L kanamycin at the final concentration, culturing for 8-10h at 37 ℃, transferring the bacteria into fresh LB liquid culture medium containing 50mg/L kanamycin at the final concentration by 2 percent (v/v) inoculation amount, and culturing at 37 ℃ and 180rpm until the bacteria OD is up to600When the concentration is about 0.6-0.8, adding IPTG with final concentration of 0.1mM, inducing culture at 28 deg.C and 180rpm for 10-12h, centrifuging at 4 deg.C and 8000rpm for 10min, and collecting wet thallus cells. Stored at-20 ℃ for later use (i.e., resting cells for the hydrolysis of phenylacetonitrile).
4. Determination of viability
The reaction system comprises the following components: 20mL Tris-HCl buffer (50mM, pH 8.0), wet cell 0.1g, methanol 0.9mL, benzyl cyanide 100mM (benzyl cyanide first dissolved in methanol). The reaction mixture was reacted at 30 ℃ and 180rpm for 30 min. 1mL of the sample was taken, and 20. mu.L of 2M HCl was added to terminate the reaction, and the mixture was centrifuged at 12,000rpm for 1min to obtain a supernatant. The concentration and the proportion of phenylacetic acid and phenylacetamide in the product are analyzed by High Performance Liquid Chromatography (HPLC), and the enzyme activity is calculated.
The nitrilase cell enzyme activity is defined as that the cell quantity required for generating 1 mu mol of phenylacetic acid per minute under the standard reaction condition of 30 ℃ and 180rpm is one enzyme activity unit (1U);
the liquid chromatography adopts a C18 column, the column temperature is 40 ℃, the detection wavelength is 210nm, and the mobile phase is methanol: water 30:70 (containing 0.1% H)3PO4) The flow rate was 1 ml/min.
The result of single point mutation is analyzed by the method, and the fact that the 145 th site is mutated from cysteine to asparagine is found to change the reaction specificity, so that the 145 th site tends to generate carboxylic acid, the hydrolysis activity is better reserved or improved, and the hydration activity is inhibited. 145 site saturation mutation results show that except for C145G, C145K, C145R, C145S, C145W, C145Y, C145E, C145F, C145H, C145Q and C145D, the carboxylic acid content and the enzyme activity of other mutants are calculated according to the steps, and the results are shown in Table 3. Therefore, the dominant mutant C145N is selected, the carboxylic acid content is increased, the hydrolysis activity is better kept, and the hydration activity is reduced. When the 229 th site was mutated from alanine to 19 other amino acids, the contents of the remaining mutant carboxylic acids and the enzyme activities were found to be shown in Table 4, except that A229W was inactive. Thereby selecting the dominant mutant A229P, which can improve the carboxylic acid content, reduce the hydration activity and improve the hydrolysis activity. When the 246 th site is mutated from asparagine to other 19 amino acids, the contents of the remaining mutant carboxylic acids and the enzyme activities are shown in table 5 except that N246E, N246P, N246F, N246R, N246M, N246Y, N246H, N246K and N246W are not active. Thereby selecting the dominant mutant N246V, which can improve the carboxylic acid content, reduce the hydration activity and improve the hydrolysis activity. Meanwhile, the dominant mutant at position 200 was selected as K200R, and the dominant mutant at position 224 was selected as R224W.
Finally, plasmids containing nitrilase mutants C145N, K200R, R224W, A229P and N246V are obtained by screening, and corresponding wet thalli are prepared by adopting the method of step 3.
TABLE 3 comparison of the specificity and Activity of wild-type OsNIT with 145-site mutants
TABLE 4 comparison of the specificity and Activity of wild-type OsNIT with 229-site mutants
TABLE 5 comparison of the specificity and Activity of wild-type OsNIT with 246-site mutant reactions
Example 2 measurement of the reaction specificity and Activity of nitrilase mutants C145N, K200R/R224W, A229P, N246V
Using the plasmid of K200R mutant obtained in example 1as a template, whole plasmid PCR amplification was performed using R224W site-directed mutagenesis primers (Lys 200-For, Lys200-Rev in Table 1), the PCR system and reaction conditions were the same as those in step 1, and the method of example 1 was used to construct a plasmid of mutant K200R/R224W (amino acid sequence shown in SEQ ID NO. 3), and wet cells were prepared according to the method of example 1.
The reaction system (20mL) consisted of: 20mL of Tris-HCl buffer (50mM, pH 8.0), 0.1g of wet nitrilase mutant cells prepared in example 1, 0.9mL of methanol, and 100mM of phenylacetonitrile (which was first dissolved in methanol). The reaction mixture was reacted at 30 ℃ and 180rpm for 30 min. 1mL of the sample was taken, and 20. mu.L of 2M HCl was added to terminate the reaction, and the mixture was centrifuged at 12,000rpm for 1min to obtain a supernatant. The concentration and ratio of phenylacetic acid and phenylacetamide in the product were analyzed by HPLC as described in example 1, and the enzyme activity was calculated, and the results are shown in Table 6.
TABLE 6 comparison of the specificity and Activity of wild-type OsNIT with mutants C145N, K200R/R224W, A229P, N246V
As shown in table 6, the content of carboxylic acid in the mutant C145N product reached 96.5%, the hydrolytic activity was 39.3% of the parent, and the hydrolytic activity was 1.4% of the parent. The mutant K200R/R224W had a carboxylic acid ratio of 95.1%, but the hydration activity decreased only to 22.74%. Mutant a229P increased the carboxylic acid ratio to 71.53 and had 145.3% hydrolytic activity as the parent. The carboxylic acid proportion of the mutant N246V was increased to 80.17%, and the hydrolytic activity thereof was maintained while the hydration activity was decreased.
Example 3 construction of the nitrilase multiple mutant K200R/R224W/A229P.
Analysis of the single-point mutation results in example 1 shows that the 200 th, 224 th and 229 th site mutations can change the reaction specificity to lead the reaction to tend to generate carboxylic acid, the hydrolysis activity is better retained or improved, the hydration activity is inhibited, and the mutant K200R/R224W/A229P is obtained by overlapping mutations, specifically:
the K200R/R224W mutant plasmid constructed in example 2 was used as a template, 229-site primers (Ala 229-For, Ala229-Rev in Table 1) were used to perform whole plasmid PCR amplification, the PCR system and reaction conditions were the same as those in step 1, and the plasmid containing the nitrilase mutant K200R/R224W/A229P was constructed in the method of example 1.
Wet cells were cultured in the same manner as in example 1 to obtain wet cells which were then assayed for the proportion of carboxylic acid and the viability.
Example 4 construction of the nitrilase multiple mutant K200R/R224W/N246V.
Analysis of the single-point mutation results in example 1 shows that the 200 th, 224 th and 246 th site mutations can change the reaction specificity to lead the reaction to tend to generate carboxylic acid, the hydrolysis activity is better retained or improved, the hydration activity is inhibited, and the mutant K200R/R224W/N246V is obtained by overlapping mutations, specifically:
the plasmid containing the nitrilase mutant K200R/R224W/N246V was constructed by the method of example 1, using the K200R/R224W mutant plasmid constructed in example 2 as a template, and using 246-site primers (Asn 246-For, Asn246-Rev in Table 1) to perform whole plasmid PCR, using the same PCR system and reaction conditions as in step 1.
Wet cells were cultured in the same manner as in example 1 to obtain wet cells which were then assayed for the proportion of carboxylic acid and the viability.
Example 5 nitrilase mutant K200R/R224W/A229P reaction specificity and Activity assay
The reaction system comprises the following components: 20 mM Tris-HCl buffer (50mM, pH 8.0), 0.1g of wet nitrilase mutant K200R/R224W/A229P prepared by the method of example 3, 0.9mL of methanol, and 100mM benzyl cyanide (dissolved in methanol). The reaction mixture was reacted at 30 ℃ and 180rpm for 30 min. 1mL of the sample was taken, and 20. mu.L of 2M HCl was added to terminate the reaction, and the mixture was centrifuged at 12,000rpm for 1min to obtain a supernatant. The concentration and ratio of phenylacetic acid and phenylacetamide in the product were analyzed by HPLC as described in example 1, and the enzyme activity was calculated.
As shown in Table 7, the mutant K200R/R224W/A229P catalyzes and produces 96.3% of phenylacetic acid, the hydrolysis activity of the mutant is 280.9% of that of the parent, and the hydration activity of the mutant is 3.6% of that of the parent.
TABLE 7 comparison of specificity and Activity of wild-type OsNIT reaction with mutant K200R/R224W/A229P
Example 6 nitrilase mutant K200R/R224W/N246V reaction specificity and Activity assay
The reaction system comprises the following components: 20 mM Tris-HCl buffer (50mM, pH 8.0), 0.1g of wet nitrilase mutant K200R/R224W/N246V prepared by the method of example 4, 0.9mL of methanol, and 100mM benzyl cyanide (dissolved in methanol). The reaction mixture was reacted at 30 ℃ and 180rpm for 30 min. 1mL of the sample was taken, and 20. mu.L of 2M HCl was added to terminate the reaction, and the mixture was centrifuged at 12,000rpm for 1min to obtain a supernatant. The concentration and the proportion of phenylacetic acid and phenylacetamide in the product are analyzed by High Performance Liquid Chromatography (HPLC), and the enzyme activity is calculated.
As shown in Table 8, the mutant K200R/R224W/N246V catalyzes phenylacetonitrile to generate phenylacetic acid with the content of 98.8 percent, the hydrolysis activity of 124.2 percent of that of the parent and the hydration activity of 1.7 percent of that of the parent.
TABLE 8 comparison of the specificity and Activity of the wild-type OsNIT reaction with the mutant K200R/R224W/N246V
Sequence listing
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gcggattctc gtcaggtatg gcaggcatct atgacccaca tcgctctgga aggtggttgc 720
ttcgttctgt ctgctaacca gttctgccgt cgtaaagact acccgccgcc gccggaatac 780
gttttcaccg gtctgggtga agaaccgtct ccggacaccg ttgtttgccc gggtggttct 840
gttatcatct ctccgtctgg tgaagttctg gctggtccga actacgaagg tgaagctctg 900
atcaccgctg acctggacct gggtgaaatc gttcgtgcta aattcgactt cgacgttgtt 960
ggtcactacg ctcgtccgga agttctgtct ctggttgtta acgaccagcc gcacctccca 1020
gttagcttca cctctgctgc ggaaaaaacc accgctgcta aatctgactc taccgctaaa 1080
ccgtacctcg agcaccacca ccaccaccac tga 1113
<210> 3
<211> 370
<212> PRT
<213> Unknown (Unknown)
<400> 3
Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala
1 5 10 15
Glu Val Glu Met Asn Gly Gly Ala Thr Ser Gly Ala Ala Thr Val Arg
20 25 30
Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr
35 40 45
Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser
50 55 60
Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Tyr Pro Arg Gly
65 70 75 80
Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp
85 90 95
Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val
100 105 110
Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys
115 120 125
Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr
130 135 140
Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His
145 150 155 160
Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly
165 170 175
Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly
180 185 190
Ala Leu Ile Cys Trp Glu Asn Arg Met Pro Leu Leu Arg Thr Ala Leu
195 200 205
Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Trp
210 215 220
Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys
225 230 235 240
Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro
245 250 255
Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp
260 265 270
Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu
275 280 285
Val Leu Ala Gly Pro Asn Tyr Glu Gly Glu Ala Leu Ile Thr Ala Asp
290 295 300
Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val
305 310 315 320
Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln
325 330 335
Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala
340 345 350
Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr Leu Glu His His His His
355 360 365
His His
370
<210> 4
<211> 370
<212> PRT
<213> Unknown (Unknown)
<400> 4
Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala
1 5 10 15
Glu Val Glu Met Asn Gly Gly Ala Thr Ser Gly Ala Ala Thr Val Arg
20 25 30
Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr
35 40 45
Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser
50 55 60
Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Tyr Pro Arg Gly
65 70 75 80
Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp
85 90 95
Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val
100 105 110
Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys
115 120 125
Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr
130 135 140
Asn Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His
145 150 155 160
Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly
165 170 175
Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly
180 185 190
Ala Leu Ile Cys Trp Glu Asn Lys Met Pro Leu Leu Arg Thr Ala Leu
195 200 205
Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Arg
210 215 220
Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys
225 230 235 240
Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro
245 250 255
Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp
260 265 270
Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu
275 280 285
Val Leu Ala Gly Pro Asn Tyr Glu Gly Glu Ala Leu Ile Thr Ala Asp
290 295 300
Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val
305 310 315 320
Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln
325 330 335
Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala
340 345 350
Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr Leu Glu His His His His
355 360 365
His His
370
<210> 5
<211> 370
<212> PRT
<213> Unknown (Unknown)
<400> 5
Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala
1 5 10 15
Glu Val Glu Met Asn Gly Gly Ala Thr Ser Gly Ala Ala Thr Val Arg
20 25 30
Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr
35 40 45
Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser
50 55 60
Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Tyr Pro Arg Gly
65 70 75 80
Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp
85 90 95
Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val
100 105 110
Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys
115 120 125
Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr
130 135 140
Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His
145 150 155 160
Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly
165 170 175
Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly
180 185 190
Ala Leu Ile Cys Trp Glu Asn Arg Met Pro Leu Leu Arg Thr Ala Leu
195 200 205
Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Trp
210 215 220
Gln Val Trp Gln Pro Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys
225 230 235 240
Phe Val Leu Ser Ala Asn Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro
245 250 255
Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp
260 265 270
Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu
275 280 285
Val Leu Ala Gly Pro Asn Tyr Glu Gly Glu Ala Leu Ile Thr Ala Asp
290 295 300
Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val
305 310 315 320
Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln
325 330 335
Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala
340 345 350
Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr Leu Glu His His His His
355 360 365
His His
370
<210> 6
<211> 370
<212> PRT
<213> Unknown (Unknown)
<400> 6
Met Ala Met Val Pro Ser Gly Ser Gly Gly Gly Pro Pro Val Ile Ala
1 5 10 15
Glu Val Glu Met Asn Gly Gly Ala Thr Ser Gly Ala Ala Thr Val Arg
20 25 30
Ala Thr Val Val Gln Ala Ser Thr Val Phe Tyr Asp Thr Pro Ala Thr
35 40 45
Leu Asp Lys Ala Glu Arg Leu Ile Glu Glu Ala Ala Gly Tyr Gly Ser
50 55 60
Gln Leu Val Val Phe Pro Glu Ala Phe Val Gly Gly Tyr Pro Arg Gly
65 70 75 80
Ser Thr Phe Gly Phe Gly Ala Asn Ile Ser Ile Gly Asn Pro Lys Asp
85 90 95
Lys Gly Lys Glu Glu Phe Arg Lys Tyr His Ala Ala Ala Ile Glu Val
100 105 110
Pro Gly Pro Glu Val Thr Arg Leu Ala Ala Met Ala Gly Lys Tyr Lys
115 120 125
Val Phe Leu Val Met Gly Val Ile Glu Arg Glu Gly Tyr Thr Leu Tyr
130 135 140
Cys Ser Val Leu Phe Phe Asp Pro Leu Gly Arg Tyr Leu Gly Lys His
145 150 155 160
Arg Lys Leu Met Pro Thr Ala Leu Glu Arg Ile Ile Trp Gly Phe Gly
165 170 175
Asp Gly Ser Thr Ile Pro Val Tyr Asp Thr Pro Leu Gly Lys Ile Gly
180 185 190
Ala Leu Ile Cys Trp Glu Asn Arg Met Pro Leu Leu Arg Thr Ala Leu
195 200 205
Tyr Gly Lys Gly Ile Glu Ile Tyr Cys Ala Pro Thr Ala Asp Ser Trp
210 215 220
Gln Val Trp Gln Ala Ser Met Thr His Ile Ala Leu Glu Gly Gly Cys
225 230 235 240
Phe Val Leu Ser Ala Val Gln Phe Cys Arg Arg Lys Asp Tyr Pro Pro
245 250 255
Pro Pro Glu Tyr Val Phe Thr Gly Leu Gly Glu Glu Pro Ser Pro Asp
260 265 270
Thr Val Val Cys Pro Gly Gly Ser Val Ile Ile Ser Pro Ser Gly Glu
275 280 285
Val Leu Ala Gly Pro Asn Tyr Glu Gly Glu Ala Leu Ile Thr Ala Asp
290 295 300
Leu Asp Leu Gly Glu Ile Val Arg Ala Lys Phe Asp Phe Asp Val Val
305 310 315 320
Gly His Tyr Ala Arg Pro Glu Val Leu Ser Leu Val Val Asn Asp Gln
325 330 335
Pro His Leu Pro Val Ser Phe Thr Ser Ala Ala Glu Lys Thr Thr Ala
340 345 350
Ala Lys Ser Asp Ser Thr Ala Lys Pro Tyr Leu Glu His His His His
355 360 365
His His
370