Nitrilase mutants and their use in the synthesis of gamma-aminobutyric acid derivatives

Abstract

The application discloses a nitrilase mutant and application thereof in synthesis of gamma-aminobutyric acid derivatives, and relates to a nitrilase mutant with improved catalytic activity and stereoselectivity, which is used for efficiently catalyzing hydrolysis of 2-substituted succinonitrile to synthesize 3-substituted-3-cyanopropionic acid, and further synthesizing gamma-aminobutyric acid derivatives through hydrogenation, thereby laying a foundation for industrialized production of gamma-aminobutyric acid drugs synthesized by the nitrilase method.

Description

(I) Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically relates to nitrile hydrolase mutants, encoding genes, engineered bacteria, and their application in the hydrolysis of 2-substituted succinic acid to synthesize γ-aminobutyric acid derivatives. (II) Background Technology

[0002] Gamma-aminobutyric acid (GABA) drugs are a class of non-protein amino acids. As a major inhibitory neurotransmitter in the central nervous system, GABA maintains the homeostasis of the nervous system by regulating neuronal excitability. Its physiological activities include sedation, antidepressant effects, sleep promotion, blood pressure reduction, bone growth promotion, and metabolic improvement, making it an important therapeutic agent for nervous system diseases. For example, brivacertan is a novel high-affinity ligand for synaptic vesicle protein 2A (SV2A), which inhibits epileptic seizures by regulating the release of neurotransmitters. Pregabalin, by binding to the α2-δ subunit of calcium ion channels in the nervous system, inhibits the influx of Ca ions into the presynaptic membrane of neurons, reducing the release of excitatory neurotransmitters, thereby achieving anti-epileptic, analgesic, and anti-anxiety therapeutic effects.

[0003] In recent years, due to the rapid growth in market demand for GABA drugs, their synthesis methods have become a research hotspot in the field of pharmaceutical engineering. Typical chemical synthesis methods for GABA derivatives include: using aldehydes and diethyl malonate as raw materials, condensation and addition are used to prepare ethyl 3-substituted-3-cyano-2-(ethyl ester)propionate, followed by hydrolysis, decarboxylation, hydrogenation, and resolution to synthesize optically pure GABA derivatives (J.Org.Chem.2015,80:5704-5712); using unsaturated pyrazolamides as raw materials, asymmetric addition and cleavage catalyzed by Gd(III) complexes are used to obtain chiral γ-nitro ester compounds, followed by cyclization, hydrogenation, and hydrolysis to synthesize the target GABA derivative (Chem.Rev.2021,122:3637-3710). However, existing chemical synthesis methods for GABA drugs suffer from drawbacks such as difficulty in reusing ineffective enantiomers, expensive catalysts, and poor atom economy, making industrial application difficult. Therefore, developing green and efficient methods for synthesizing GABA-based drugs is of great significance.

[0004] Nitrile hydrolases catalyze the hydrolysis of nitrile compounds to synthesize carboxylic acids. Their mediated biocatalytic processes offer advantages such as high catalytic efficiency, stereoselectivity, strict regioselectivity, and environmental friendliness, making them important tool enzymes for the industrial synthesis of carboxylic acids. Utilizing the regio- and stereoselective hydrolysis of 2-substituted butadionitrile to synthesize 3-substituted-3-cyanopropionic acid via nitrile hydrolases, and then preparing GABA-like compounds through one-step hydrogenation, offers significant advantages such as simple steps and high atom economy, making it an ideal route for their industrial preparation.

[0005] This invention utilizes protein engineering techniques to modify nitrile hydrolases, enhancing their activity and stereoselectivity in catalyzing the hydrolysis of 2-substituted succinic acid regions, thus laying the foundation for the efficient synthesis of GABA-based drugs. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a nitrile hydrolase mutant, its encoding gene, an engineered bacterium, and its application in the catalytic synthesis of GABA derivatives. By constructing a nitrile hydrolase mutant with improved catalytic activity and stereoselectivity, it efficiently catalyzes the hydrolysis of 2-substituted succinic acid to synthesize 3-substituted-3-cyanopropionic acid, and further synthesizes GABA derivatives through hydrogenation, laying the foundation for the industrial production of GABA-like drugs by nitrile hydrolase method.

[0007] The technical solution adopted in this invention is:

[0008] In a first aspect, the present invention provides a nitrile hydrolase mutant with high catalytic activity and high stereoselectivity, wherein the nitrile hydrolase mutant is obtained by single or multiple mutations of the amino acid at position 126 or 184 of the amino acid shown in SEQ ID NO.2.

[0009] Preferably, the nitrile hydrolase mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.2 to one of the following: (1) arginine at position 126 is mutated to serine (Pg NITmut / R126S, amino acid sequence as shown in SEQ ID NO.4, nucleotide sequence as shown in SEQ ID NO.3); (2) arginine at position 126 is mutated to serine and serine at position 184 is mutated to valine (Pg NITmut / R126S / S184V, amino acid sequence as shown in SEQ ID NO.6, nucleotide sequence as shown in SEQ ID NO.5).

[0010] Mutations that retain nitrile hydrolase activity by conserved substitutions, additions or deletions of one or more amino acids, amino-terminal truncation, or carboxyl-terminal truncation of other amino acid sites in the nitrile hydrolase mutants shown in SEQ ID NO.4 or SEQ ID NO.6 are also included within the scope of this invention.

[0011] In a second aspect, the present invention provides a coding gene for the nitrile hydrolase mutant, the nucleotide sequence of which is shown in SEQ ID NO.3 or SEQ ID NO.5.

[0012] Thirdly, the present invention provides a recombinant expression vector containing the coding gene of the nitrile hydrolase mutant; the original vector of the recombinant expression vector is pET-28b(+).

[0013] Fourthly, the present invention provides a recombinant genetically engineered bacterium comprising the recombinant expression vector; the host cell of the recombinant genetically engineered bacterium can be any conventional host cell in the art, preferably Escherichia coli BL21. The culture of the engineered bacterium in the present invention can be carried out using any culture medium capable of supporting its growth and inducing the production of the specific nitrile hydrolase of the present invention, preferably LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, water as solvent, pH 7.0. There are no specific limitations on the culture method and conditions, as long as it is ensured that the engineered bacterium can grow normally and effectively produce nitrile hydrolase.

[0014] The method for preparing the nitrile hydrolase mutant includes the following steps:

[0015] (1) Design site-directed mutagenesis primers, use recombinant plasmids carrying gene fragments with nucleotide sequences as shown in SEQ ID NO.1 as templates, perform overlap extension PCR to obtain the mutant product with R mutated to S at position 126 in the amino acid sequence of parent nitrile hydrolase.

[0016] (2) Using the mutant product carrying the Pg NITmut / R126S gene obtained in step (1) as a template, overlap extension PCR was performed to obtain the mutant product with the S at position 184 mutated to V.

[0017] (3) The mutant products obtained in steps (1) and (2) are transformed into host bacteria, and strains expressing nitrile hydrolase mutants are screened. The strains with correct sequencing results are the target strains. The nitrile hydrolase mutants PgNITmut / R126S and Pg NITmut / R126S / S184V are obtained by induction expression.

[0018] Fifthly, the present invention provides an application of the nitrile hydrolase mutant in the catalytic synthesis of γ-aminobutyric acid derivatives from 2-substituted succinic acid.

[0019] Preferably, the method of application is as follows: using wet cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the coding gene of the nitrile hydrolase mutant or pure enzyme extracted by breaking wet cells as a catalyst, using 2-substituted butadionitrile (Ⅰ) as a substrate, and using a buffer solution of pH 6-8 as a reaction medium to form a transformation system, the transformation reaction is carried out at 20-40℃ and 100-200 rpm to obtain a reaction solution containing 3-substituted-3-cyanopropionic acid (Ⅱ), and then subjected to hydrogenation reaction to obtain γ-aminobutyric acid derivative;

[0020]

[0021] In formula (I), R includes phenyl, m-chlorophenyl, p-fluorophenyl, p-chlorophenyl, p-methylphenyl, o-chlorophenyl, isobutyl, and propyl. In formula (II), R is the same as R in formula (I).

[0022] Preferably, the substrate concentration in the conversion system is 100-200 g / L (preferably 100 g / L); the catalyst concentration, calculated as wet cells, is 5-20 g / L (preferably 10 g / L).

[0023] Preferably, the reaction medium is a 50mM phosphate buffer (PB) at pH 7.4; the conversion reaction is carried out at 30°C and 150 rpm.

[0024] Preferably, the 2-substituted succinic anion includes 2-phenylsuccinic anion, 2-m-chlorophenylsuccinic anion, 2-p-fluorophenylsuccinic anion, 2-p-chlorophenylsuccinic anion, 2-p-methylphenylsuccinic anion, 2-o-chlorophenylsuccinic anion, 2-isobutylsuccinic anion, and 3-cyanohexanoic anion.

[0025] Preferably, the wet bacterial cells are prepared according to the following method:

[0026] (1) Plate culture: Recombinant Escherichia coli containing the gene encoding nitrile hydrolase mutant was streaked onto LB solid medium containing 50 μg / mL kanamycin and activated overnight at 37°C to obtain single colonies;

[0027] (2) Seed culture: Single colonies were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 180 rpm for 10-12 h to obtain seed culture;

[0028] (3) Fermentation culture: The seed culture was inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 2% (v / v), and cultured at 37°C until the cell concentration reached OD. 600 Once the concentration reaches 0.5–0.7, add isopropyl-β-D-thiopyranogalactopyranoside (IPTG) to the culture medium to a final concentration of 0.1 mM, induce culture at 28°C for 12 h, centrifuge at 4°C and 8000 rpm for 10 min, and collect the wet cells.

[0029] The nitrile hydrolase mutant of the present invention has flexible application forms. It can be used directly in the form of whole cells of engineered bacteria, in the form of unpurified crude enzyme, or in the form of partially or completely purified pure enzyme. In addition, using immobilization techniques known in the art, the nitrile hydrolase mutant of the present invention can also be prepared into immobilized enzymes or immobilized cell-based biocatalysts, further broadening its application scope.

[0030] Preferably, the hydrogenation reaction involves hydrogenating 3-substituted-3-cyanopropionic acid (II) with Raney nickel at a reaction pressure of 2 MPa, a reaction temperature of room temperature, a stirring speed of 600 rpm, and a reaction time of 4 h; the mass ratio of Raney nickel to 3-substituted-3-cyanopropionic acid is 0.5:1.

[0031] Compared with the prior art, the beneficial effects of this application are mainly reflected in:

[0032] The nitrile hydrolase mutant screened in this invention exhibits improved activity and stereoselectivity in catalyzing the synthesis of 3-substituted-3-cyanopropionic acid from 2-substituted succinic acid. This one-step hydrogenation process prepares GABA derivatives, laying the foundation for the efficient synthesis of GABA drugs.

[0033] The superior nitrile hydrolase mutant Pg NITmut / R126S / S184V of this invention exhibits significantly improved activity and stereoselectivity in catalyzing the synthesis of 3-substituted-3-cyanopropionic acid from 2-substituted succinic acid, specifically reflected in:

[0034] Using 2-phenylbutadionitrile as a substrate, the specific activity of Pg NITmut was 42 U / mg, the product ee value was 95.9%, and the E value was 86. The specific activity of Pg NITmut / R126S / S184V was 23 U / mg, the product ee value was 98.4%, and the E value was 167.

[0035] Using 2-m-chlorophenylbutadionitrile as a substrate, the specific activity of Pg NITmut was 95 U / mg, the product ee value was 56.5%, and the E value was 7. The specific activity of Pg NITmut / R126S / S184V was 89 U / mg, the product ee value was 63.3%, and the E value was 18.

[0036] Using 2-p-fluorophenylbutadionitrile as a substrate, the specific activity of Pg NITmut was 61 U / mg, the product ee value was 87.7%, and the E value was 22. The specific activity of Pg NITmut / R126S / S184V was 22 U / mg, the product ee value was 92.3%, and the E value was 47.

[0037] Using 2-p-chlorophenylbutadionitrile as a substrate, the specific activity of Pg NITmut was 74 U / mg, the product ee value was 82.6%, and the E value was 36; the specific activity of Pg NITmut / R126S / S184V was 76 U / mg, the product ee value was 93.2%, and the E value was 47.

[0038] Using 2-p-tolylsuccinate as a substrate, the specific activity of Pg NITmut was 25 U / mg, the product ee value was 90.3%, and the E value was 52; the specific activity of Pg NITmut / R126S / S184V was 66 U / mg, the product ee value was 96.8%, and the E value was 134.

[0039] Using 2-o-chlorophenylbutadionitrile as a substrate, the specific activity of Pg NITmut was 64 U / mg, the product ee value was 95.2%, and the E value was 102; the specific activity of Pg NITmut / R126S / S184V was 66 U / mg, the product ee value was 98.4%, and the E value was 189.

[0040] Using isobutylsuccinate as a substrate, the specific activity of Pg NITmut was 66 U / mg, the product ee value was 84.1%, and the E value was 52; the specific activity of Pg NITmut / R126S / S184V was 93 U / mg, the product ee value was 94.9%, and the E value was 124.

[0041] Using 3-cyanohexonitrile as a substrate, the specific activity of PgNITmut was 35 U / mg, the product ee value was 99.2%, and the E value was 569; the specific activity of PgNITmut / R126S / S184V was 46 U / mg, the product ee value was 99.4%, and the E value was 674. (IV) Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0043] The parental nitrile hydrolase used in this embodiment of the invention is a nitrile hydrolase mutant Pg NITmut / F135L / R199W / T59D (hereinafter referred to as Pg NITmut) derived from Paraburkholderia graminis. Its amino acid sequence is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.1. The recombinant engineered parental nitrile hydrolase strain E.coli BL21(DE3) / pET-28b(+)-Pg NITmut has been disclosed in patent application CN202310202126.6.

[0044] LB liquid medium composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, water as solvent, pH 7.0.

[0045] LB solid medium is LB liquid medium with 20 g / L agar added.

[0046] Example 1: Construction and screening of nitrile hydrolase mutants

[0047] 1. Screening for mutation sites

[0048] The nitrile hydrolase mutant Pg Nit / F135L / R199W / T59D (hereinafter referred to as Pg NITmut) derived from Paraburkholderia graminis was used as the parent, and its amino acid sequence is shown in SEQ ID NO.2 and its nucleotide sequence is shown in SEQ ID NO.1.

[0049] Homology modeling and molecular docking were performed on Pg NITmut. Based on the possible substrate or product channels of nitrile hydrolase, combined with DCCM analysis, key amino acids on the periphery of the catalytic pocket were selected, and site 126 was selected for modification. Furthermore, based on the tetramer analysis of the "A interface" structure of nitrile hydrolase, key amino acids with hydrogen bond interactions on the key loop were located, and site 184 was selected for modification.

[0050] SEQ ID NO.2

[0051] MGKVVKAAAVQFSPVLYSREATVAKVVQKIHELGLKGVQFATFPETVVPYYPYFAAVQDGIELLSGSEHLRLLEQAVTVPSAATDAIGKAAREAGMVVSIGVNERDGGTLYNTQLLFDADGTLIQRRRKITPTHLERMIWGQGDGSGLRAVDSAVGRIGQLACFEHNNPL ARYAMIADGEQIHSAMYPGSAFGEGFAQWMEINIRQHALESGAFVVNATAWLDADQQAQIMKDTGCGIGPISGGCFTTIVSPDGMLMAEPLRSGEGEVIVDLDFAQIDRRKMLMDAAGHYNRPELLSLMIDRTPTAHVHERAPHSLPVSDKADDDVRTQAAAVAGSRLEI.

[0052] 2. Design of site-directed mutagenesis primers

[0053] Based on the mutation sites screened in step 1, corresponding primers were designed, as shown in Table 1.

[0054] Table 1. Primers for site-directed saturation mutagenesis

[0055]

[0056] Note: N=A / G / C / T, K=G / T, M=A / C

[0057] 3. Construction of mutant Pg NITmut / R126S

[0058] Using the recombinant plasmid pET-28b(+)-Pg NITmut containing the target gene fragment as a template, the template was amplified using the primers in Table 1 according to the overlapping extension PCR method, and site-directed mutations were performed at positions 126 and 184 in the parent amino acid sequence.

[0059] The PCR amplification system consisted of (50 μL): dNTPs (1 μL, 2 mM), buffer (25 μL), ultrapure water (20 μL), Pg NITmut template plasmid DNA (1 μL, 10 ng / μL), primers (2 μL of F and R, 10 pmol / μL) and Phanta Max Super-Fdelity DNA polymerase (1 μL, 5 U / μL).

[0060] PCR reaction parameters: (1) 95℃ pre-denaturation for 30s; (2) 95℃ denaturation for 30s; (3) 61℃ annealing for 30s; (4) 72℃ extension for 4min, repeat steps (2)-(4) 30 times; (5) 72℃ extension for 5min, store at 16℃.

[0061] After the PCR product showed a positive result by 0.9% agarose gel electrophoresis, 20 μL of the PCR reaction solution was taken, and 1 μL of the restriction enzyme Dpn I was added for digestion at 37℃ for 2 h to remove the template plasmid DNA. The DNA was then inactivated at 65℃ for 10 min. The cells were heat-shocked into E. coli BL21(DE3) competent cells, and after recovery, they were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37℃.

[0062] 4. Cell Culture and High-Throughput Screening: First, using sterilized toothpicks, single-clone colonies grown on the plates in step 3 were picked and transferred to a pre-prepared 96-well plate containing 600 μL of sterile LB liquid medium (final concentration 50 μg / mL kanamycin). Single-clone colonies of the control group (recombinant engineered parenteral nitrile hydrolase) were added to the first three and last three wells of the 96-well plate, respectively. The 96-well plate was then tightly sealed. The culture was carried out at 37°C and 180 rpm in a constant-temperature shaker for 20 to 24 hours to obtain the seed culture. Next, 200 μL of the seed culture was removed from the cultured 96-well plate and transferred to a new 96-well plate pre-filled with 600 μL of sterile LB liquid medium (final concentration 50 μg / mL kanamycin and 0.1 mM IPTG). Induction culture was then carried out at 28°C and 180 rpm for 12 hours. Subsequently, the cells were centrifuged at 4000 rpm at room temperature for 10 minutes, the supernatant was discarded, and the expressed bacterial cells were collected for subsequent high-throughput screening.

[0063] Next, the bacterial cells in the wells were resuspended in 200 μL of phosphate buffer (10 mM, pH 8), and racemic 3-cyanohexonitrile (final concentration 20 mM) was added as a model substrate. The reaction was carried out in a 30°C incubator at 200 rpm for 30 min. After the reaction, 30 μL of 2 M HCl was added to each well to terminate the reaction, followed by centrifugation at 4000 rpm at room temperature for 10 min to obtain the supernatant. 30 μL of the supernatant was transferred to a 96-well microplate, and 150 μL of fluorescent reagent (prepared by dissolving 0.1 g of o-phthalaldehyde in 20 mL of anhydrous ethanol, adding 50 μL of mercaptoethanol, and then diluting 4 times with phosphate buffer (10 mM, pH 8)) was added to each well. The plate was incubated in a 37°C incubator for 30 min to allow for sufficient color development. After incubation, the fluorescence intensity in the 96-well microplate was measured using a microplate reader (excitation wavelength 412 nm, emission wavelength 467 nm). The difference in reaction between the bacterial culture and the two mono-configurations of racemic 3-cyanohexonitrile was calculated based on the fluorescence intensity values. Strains with larger differences compared to the control group were preliminarily identified as strains with significantly enhanced catalytic stereoselectivity.

[0064] Superior mutants with improved activity and stereoselectivity were obtained through screening. Subsequently, 4-5 clones were selected and cultured in LB liquid medium at 37°C for 8 hours. After sequencing, the bacterial culture was collected to obtain the recombinant engineered strain E. coli BL21(DE3) / pET-28b(+)-Pg NITmut / R126S, which is shown in SEQ ID NO.4 and SEQ ID NO.3.

[0065] 5. Construction of mutant Pg NITmut / R126S / S184V

[0066] Using the constructed recombinant plasmid pET-28b(+)-Pg NITmut / R126S as a template, PCR was performed using primers S184V-Forward and S184V-Reverse in Table 1. The recombinant engineered bacteria E. coli BL21(DE3)pET28b(+)-Pg NITmut / R126S / S184V was obtained using the method described in section 4 above. Its amino acid sequence is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.5.

[0067] SEQ ID NO.6

[0068] MGKVVKAAAVQFSPVLYSREATVAKVVQKIHELGLKGVQFATFPETVVPYYPYFAAVQDGIELLSGSEHLRLLEQAVTVPSAATDAIGKAAREAGMVVSIGVNERDGGTLYNTQLLFDADGTLIQSRRKITPTHLERMIWGQGDGSGLRAVDSAVGRIGQLACFEHNNPL ARYAMIADGEQIHVAMYPGSAFGEGFAQWMEINIRQHALESGAFVVNATAWLDADQQAQIMKDTGCGIGPISGGCFTTIVSPDGMLMAEPLRSGEGEVIVDLDFAQIDRRKMLMDAAGHYNRPELLSLMIDRTPTAHVHERAPHSLPVSDKADDDVRTQAAAVAGSRLEI.

[0069] Example 2: Induced expression of recombinant Escherichia coli containing nitrile hydrolase mutant

[0070] 1. Wet bacteria

[0071] Recombinant *E. coli* containing the parental nitrile hydrolase *E. coli* BL21(DE3) / pET-28b(+)-Pg NITmut, as well as the mutant *E. coli* BL21(DE3) / pET-28b(+)-Pg NITmut / R126S and *E. coli* BL21(DE3) / pET-28b(+)-Pg NITmut / R126S / S184V obtained in Example 1, were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 6-8 h. Then, at a 2% (v / v) inoculation rate, the bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm until the bacterial concentration reached OD0.05. 600 =0.6, add IPTG to a final concentration of 0.1mM, induce culture at 28℃ for 12h, centrifuge at 4℃ and 8000rpm for 10min, collect wet cells, wash the wet cells with 0.85% physiological saline, and store at -20℃ for later use.

[0072] 2. Pure enzymes

[0073] Pure enzymes were prepared according to the methods and conditions described in the literature (ACS Catalysis, 2023, 13: 9078-9089).

[0074] Example 3: Determination of the activity and stereoselectivity of recombinant Escherichia coli containing nitrile hydrolase mutants in hydrolyzing 2-substituted succinic acid compounds.

[0075] 1 mg of purified enzyme was placed in an EP tube, and the volume was brought up to 900 μL with PB buffer (50 mM, pH 7.4). The EP tube was incubated in a metal bath at 30 °C for 5 min. 100 μL of substrate stock solution (1 mol / L substrate concentration, N,N dimethylformamide solvent) was added to the EP tube, and the reaction was carried out at 30 °C for 6 h. 200 μL of the reaction solution was mixed with 50 μL of 6 M hydrochloric acid to terminate the reaction, and the mixture was extracted with ethyl acetate. 200 μL of the upper organic phase was dried over anhydrous sodium sulfate, and 7.5 μL of methanol and 3.75 μL of diazomethane were added. The product content was determined by gas chromatography, and the substrate conversion rate and enantiomeric excess (ee) were calculated.

[0076] The gas chromatograph was a TRACE 1300 (Thermo Fisher Scientific), and the capillary columns were BGB 175 (BGB Analytik Switzerland) and BGB 174 (BGB Analytik AG). Chromatographic conditions were as follows: injection volume 2.0 μL, injector and detector temperatures both 250 °C, column temperature 120 °C held for 20 min, then ramped to 200 °C at a rate of 5 °C / min and held for 20 min. The carrier gas was high-purity helium, maintained at a constant pressure of 180 kPa, with a split ratio of 20:1.

[0077] The conversion rate and enantiomeric excess (ee) were calculated using the method of Rakels et al. (Enzyme & Microbial. Technology, 1993, 15(15): 1051-1056).

[0078] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of product under reaction conditions at 30°C. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of product.

[0079] Specific activity is defined as the enzyme activity per milligram of enzyme protein.

[0080] Example 4: Application of nitrile hydrolase mutant in the synthesis of 2-phenyl-γ-aminobutyric acid

[0081] 1. Enzyme catalysis

[0082] Using the wet bacterial cells of nitrile hydrolases Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, 25 g of 2-phenylbutadionitrile substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer. The amount of catalyst added was 10 g / L based on the weight of the wet bacterial cells. The reaction was carried out at 30 °C and 150 rpm. The reaction solution was sampled, and the specific enzyme activity, product ee value, and product concentration were detected using the method in Example 3.

[0083] The results are shown in Table 2. The specific activity of Pg NITmut in the synthesis of (R)-3-phenyl-3-cyanopropionic acid from 2-phenylbutadionitrile was 42 U / mg, the conversion rate was 37.5% after 10 h of reaction, the product ee value was 95.9%, and the E value was 86. The specific activity of Pg NITmut / R126S / S184V was 23 U / mg, the conversion rate was 30.3% after 10 h of reaction, the product ee value was 98.4%, and the E value was 167.

[0084] 2. Hydrogenation reaction

[0085] The product of the mutant catalytic reaction in step 1 was extracted using the method described in Example 2 of patent application CN112941122A and prepared into an aqueous solution with a final concentration of 100 g / L. 10 mL of this aqueous solution was taken, and 0.5 g of Raney nickel was added for hydrogenation. The reaction was carried out at a pressure of 2 MPa, a temperature of room temperature, and a stirring speed of 600 rpm for 4 h. The results, as described in Example 3, showed that the conversion rate of (R)-3-phenyl-3-cyanopropionic acid was 100%, and the yield of (R)-2-phenyl-γ-aminobutyric acid was 73.4%.

[0086] Example 5: Application of nitrile hydrolase mutant in the synthesis of 2-m-chlorophenyl-γ-aminobutyric acid

[0087] 1. Enzyme catalysis

[0088] Using the wet bacterial cells containing nitrile hydrolases Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, 25 g of 2-m-chlorophenylbutadionitrile substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer. The amount of catalyst added was 10 g / L based on the weight of the wet cells. The reaction was carried out at 30 °C and 150 rpm. Samples of the reaction solution were taken, and the specific enzyme activity, product ee value, and product concentration were detected using the method in Example 3. The results are shown in Table 2. The specific activity of Pg NITmut in the synthesis of (R)-3-m-chlorophenyl-3-cyanopropionic acid from 2-m-chlorophenylbutadionitrile was 95 U / mg, the conversion rate was 47.6% after 4 h of reaction, the product ee value was 56.5%, and the E value was 7. The specific activity of Pg NITmut / R126S / S184V was 89 U / mg, the conversion rate was 49.9% after 6 h of reaction, the product ee value was 63.3%, and the E value was 18.

[0089] 2. Hydrogenation reaction

[0090] The product (R)-3-m-chlorophenyl-3-cyanopropionic acid from the catalytic reaction of the mutant in step 1 was extracted using the method of Example 2 in patent application CN112941122A and prepared into an aqueous solution with a final concentration of 100 g / L. 10 mL of the above aqueous solution was taken, and 0.5 g of Raney nickel was added for hydrogenation. The reaction was carried out at a pressure of 2 MPa, a temperature of room temperature, and a stirring speed of 600 rpm for 4 h. The results, as described in Example 3, showed that the conversion rate of (R)-3-m-chlorophenyl-3-cyanopropionic acid was 100%, and the yield of (R)-2-m-chlorophenyl-γ-aminobutyric acid was 86.7%.

[0091] Example 6: Application of nitrile hydrolase mutant in the synthesis of 2-p-fluorophenyl-γ-aminobutyric acid

[0092] 1. Enzyme catalysis

[0093] Using the wet bacterial cells containing nitrile hydrolases Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, 25 g of 2-p-fluorophenylbutadionitrile substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer. The amount of catalyst added was 10 g / L based on the weight of the wet bacterial cells. The reaction was carried out at 30 °C and 150 rpm. Samples of the reaction solution were taken, and the specific enzyme activity, product ee value, and product concentration were detected using the method in Example 3. The results are shown in Table 2. The specific activity of Pg NITmut catalyzing the synthesis of (R)-3-p-fluorophenyl-3-cyanopropionic acid from 2-p-fluorophenylbutadionitrile was 61 U / mg, the conversion rate was 40.4% after 6 h of reaction, the product ee value was 87.7%, and the E value was 22. The specific activity of Pg NITmut / R126S / S184V was 22 U / mg, the conversion rate was 35.9% after 8 h of reaction, the product ee value was 92.3%, and the E value was 47.

[0094] 2. Hydrogenation reaction

[0095] The mutant catalytic reaction product (R)-3-p-fluorophenyl-3-cyanopropionic acid was extracted using the method in Example 2 of patent application CN112941122A and prepared into an aqueous solution with a final concentration of 100 g / L. 10 mL of this aqueous solution was taken, and 0.5 g of Raney nickel was added for hydrogenation. The reaction was carried out at a pressure of 2 MPa, a temperature of room temperature, and a stirring speed of 600 rpm for 4 h. The results, as described in Example 3, showed that the conversion rate of (R)-3-p-fluorophenyl-3-cyanopropionic acid was 100%, and the yield of (R)-2-p-fluorophenyl-γ-aminobutyric acid was 97.5%.

[0096] Example 7: Application of nitrile hydrolase mutant in the synthesis of 2-p-chlorophenyl-γ-aminobutyric acid

[0097] 1. Enzyme catalysis

[0098] Using the wet bacterial cells containing nitrile hydrolases Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, 25 g of 2-p-chlorophenylbutadionitrile substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer. The amount of catalyst added was 10 g / L based on the weight of the wet bacterial cells. The reaction was carried out at 30 °C and 150 rpm. Samples of the reaction solution were taken, and the specific enzyme activity, product ee value, and product concentration were detected using the method in Example 3. The results are shown in Table 2. The specific activity of Pg NITmut catalyzing the synthesis of (R)-3-p-chlorophenyl-3-cyanopropionic acid from 2-p-chlorophenylbutadionitrile was 74 U / mg, the conversion rate was 45.4% after 6 h of reaction, the product ee value was 82.6%, and the E value was 36. The specific activity of Pg NITmut / R126S / S184V was 76 U / mg, the conversion rate was 35.9% after 6 h of reaction, the product ee value was 93.2%, and the E value was 47.

[0099] 2. Hydrogenation reaction

[0100] The mutant catalytic reaction product (R)-3-p-chlorophenyl-3-cyanopropionic acid was extracted using the method in Example 2 of patent application CN112941122A and prepared into an aqueous solution with a final concentration of 100 g / L. 10 mL of this aqueous solution was added to 0.5 g of Raney nickel for hydrogenation. The reaction was carried out at a pressure of 2 MPa, a temperature of room temperature, and a stirring speed of 600 rpm for 4 h. The results, as described in Example 3, showed that the conversion rate of (R)-3-p-chlorophenyl-3-cyanopropionic acid was 100%, and the yield of (R)-2-p-chlorophenyl-γ-aminobutyric acid was 98.5%.

[0101] Example 8: Application of nitrile hydrolase mutant in the synthesis of 2-p-tolyl-γ-aminobutyric acid

[0102] 1. Enzyme catalysis

[0103] Using the wet bacterial cells containing nitrile hydrolases Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, 25 g of 2-p-tolylbutanilide substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer. The amount of catalyst added was 10 g / L based on the weight of the wet bacterial cells. The reaction was carried out at 30 °C and 150 rpm. Samples of the reaction solution were taken, and the specific enzyme activity, product ee value, and product concentration were determined using the method in Example 3. The results are shown in Table 2. The specific activity of Pg NITmut in the synthesis of (R)-3-p-tolyl-3-cyanopropionic acid from 2-p-tolyl succinate was 25 U / mg, the conversion rate was 45.1% after 10 h of reaction, the product ee value was 90.3%, and the E value was 52. The specific activity of Pg NITmut / R126S / S184V was 66 U / mg, the conversion rate was 44.2% after 6 h of reaction, the product ee value was 96.8%, and the E value was 134.

[0104] 2. Hydrogenation reaction

[0105] The mutant catalytic reaction product (R)-3-p-tolyl-3-cyanopropionic acid was extracted using the method in Example 2 of patent application CN112941122A and prepared into an aqueous solution with a final concentration of 100 g / L. 10 mL of this aqueous solution was taken, and 0.5 g of Raney nickel was added for hydrogenation. The reaction was carried out at a pressure of 2 MPa, a temperature of room temperature, and a stirring speed of 600 rpm for 4 h. The results, as described in Example 3, showed that the conversion rate of (R)-3-p-tolyl-3-cyanopropionic acid was 100%, and the yield of (R)-2-p-tolyl-γ-aminobutyric acid was 96.2%.

[0106] Example 9: Application of nitrile hydrolase mutant in the synthesis of 2-o-chlorophenyl-γ-aminobutyric acid

[0107] 1. Enzyme catalysis

[0108] Using the wet bacterial cells containing nitrile hydrolases Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, 25 g of 2-o-chlorophenylbutadionitrile substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer. The amount of catalyst added was 10 g / L based on the weight of the wet bacterial cells. The reaction was carried out at 30 °C and 150 rpm. Samples of the reaction solution were taken, and the specific enzyme activity, product ee value, and product concentration were detected using the method in Example 3. The results are shown in Table 2. The specific activity of Pg NITmut catalyzing the synthesis of (R)-3-o-chlorophenyl-3-cyanopropionic acid from 2-o-chlorophenylbutadionitrile was 64 U / mg, the conversion rate was 45.6% after 6 h of reaction, the product ee value was 95.2%, and the E value was 102. The specific activity of Pg NITmut / R126S / S184V was 66 U / mg, the conversion rate was 45.3% after 6 h of reaction, the product ee value was 98.4%, and the E value was 189.

[0109] 2. Hydrogenation reaction

[0110] The mutant catalytic reaction product (R)-3-o-chlorophenyl-3-cyanopropionic acid was extracted using the method in Example 2 of patent application CN112941122A and prepared into an aqueous solution with a final concentration of 100 g / L. 10 mL of this aqueous solution was added to 0.5 g of Raney nickel for hydrogenation. The reaction was carried out at a pressure of 2 MPa, a temperature of room temperature, and a stirring speed of 600 rpm for 4 h. The results, as described in Example 3, showed that the conversion rate of (R)-3-o-chlorophenyl-3-cyanopropionic acid was 100%, and the yield of (R)-2-o-chlorophenyl-γ-aminobutyric acid was 96.9%.

[0111] Example 10: Application of nitrile hydrolase mutant in the synthesis of 3-aminomethyl-5-methylhexanoic acid

[0112] 1. Enzyme catalysis

[0113] Using the wet bacterial cells containing nitrile hydrolases Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, 25 g of isobutylsuccinate substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer. The amount of catalyst added was 10 g / L based on the weight of the wet bacterial cells. The reaction was carried out at 30 °C and 150 rpm. Samples of the reaction solution were taken, and the specific enzyme activity, product ee value, and product concentration were detected using the method in Example 3. The results are shown in Table 2. The specific activity of Pg NITmut catalyzing the synthesis of (R)-3-cyano-5-methylhexanoic acid from isobutylsuccinate was 66 U / mg, the conversion rate was 49.8% after 12 h of reaction, the product ee value was 84.1%, and the E value was 52. The specific activity of Pg NITmut / R126S / S184V was 93 U / mg, the conversion rate was 47.3% after 10 h of reaction, the product ee value was 94.9%, and the E value was 124.

[0114] 2. Hydrogenation reaction

[0115] The extraction method (ZL202110204296.9), previously constructed using the method in Example 2 of patent application CN112941122A, was used to extract the mutant catalytic reaction product (R)-3-cyano-5-methylhexanoic acid, which was then prepared into an aqueous solution with a final concentration of 100 g / L. 10 mL of this aqueous solution was taken, and 0.5 g of Raney nickel was added for hydrogenation. The reaction was carried out at a pressure of 2 MPa, a temperature of room temperature, and a stirring speed of 600 rpm for 4 h. The results, as described in Example 3, showed that the conversion rate of (R)-3-cyano-5-methylhexanoic acid was 100%, and the yield of (R)-3-aminomethyl-5-methylhexanoic acid was 97.8%.

[0116] Example 11: Application of nitrile hydrolase mutant in the synthesis of 3-aminomethylhexanoic acid

[0117] 1. Enzyme catalysis

[0118] Using wet bacterial cells containing nitrile hydrolases Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, 25 g of 3-cyanohexonitrile substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer. The catalyst addition was 10 g / L based on the weight of the wet bacterial cells. The reaction was carried out at 30 °C and 150 rpm. Samples of the reaction solution were taken, and the specific enzyme activity, product ee value, and product concentration were determined using the method in Example 3. The results are shown in Table 2. The specific activity of Pg NITmut in catalyzing the synthesis of (R)-3-cyanohexonitrile from 3-cyanohexonitrile was 35 U / mg, with a conversion rate of 45.2% after 16 h, a product ee value of 99.2%, and an E value of 569. The specific activity of PgNITmut / R126S / S184V was 46 U / mg, with a conversion rate of 46.3% after 16 h, a product ee value of 99.4%, and an E value of 674.

[0119] 2. Hydrogenation reaction

[0120] The mutant catalytic reaction product (R)-3-cyanohexanoic acid was extracted using the method described in Example 2 of patent application CN112941122A and prepared into an aqueous solution with a final concentration of 100 g / L. 10 mL of this aqueous solution was taken, and 0.5 g of Raney nickel was added for hydrogenation. The reaction was carried out at a pressure of 2 MPa, a temperature of room temperature, and a stirring speed of 600 rpm for 4 h. The results, as described in Example 3, showed that the conversion rate of (R)-3-cyanohexanoic acid was 100%, and the yield of (R)-3-aminomethylhexanoic acid was 98.5%.

[0121] Table 2 Results of stereoselective hydrolysis of 2-substituted succinitrile by Pg NITmut and its mutants

[0122]

[0123] This invention is not limited to the specific textual description above. Various changes can be made to this invention within the scope outlined in the claims, and all such changes are within the scope of this invention.

Claims

1. A nitrile hydrolase mutant, characterized in that, The nitrile hydrolase mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.2 to one of the following: (1) arginine at position 126 is mutated to serine; (2) arginine at position 126 is mutated to serine and serine at position 184 is mutated to valine.

2. A recombinant genetically engineered bacterium comprising the encoding gene of the nitrile hydrolase mutant of claim 1.

3. The application of the nitrile hydrolase mutant of claim 1 in the catalytic synthesis of γ-aminobutyric acid derivatives from 2-substituted succinic acid, characterized in that, The application method is as follows: using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the coding gene of the nitrile hydrolase mutant or pure enzyme extracted by breaking wet bacterial cells as a catalyst, using 2-substituted butadionitrile in Formula I as a substrate, and using a buffer solution with pH 6-8 as a reaction medium to form a transformation system, the transformation reaction is carried out at 20-40℃ and 100-200 rpm to obtain a reaction solution containing 3-substituted-3-cyanopropionic acid in Formula II, and then subjected to a hydrogenation reaction to obtain a γ-aminobutyric acid derivative; Ⅰ Ⅱ In Formula I, R is phenyl, m-chlorophenyl, p-fluorophenyl, p-chlorophenyl, p-methylphenyl, o-chlorophenyl, isobutyl, or propyl; in Formula II, R is the same as R in Formula I.

4. The application as described in claim 3, characterized in that, The substrate concentration in the conversion system is 10-200 g / L; the catalyst concentration, calculated as wet bacterial cells, is 5-20 g / L.

5. The application as described in claim 3, characterized in that, The reaction medium is a 50mM phosphate buffer solution with a pH of 7.

4.

6. The application as described in claim 3, characterized in that, The wet bacterial cells were prepared according to the following method: (1) Plate culture: Recombinant Escherichia coli containing the gene encoding nitrile hydrolase mutant was streaked onto LB solid medium containing 50 μg / mL kanamycin and activated overnight at 37°C to obtain single colonies; (2) Seed culture: Single colonies were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 180 rpm for 10-12 h to obtain seed culture; (3) Fermentation culture: The seed culture was inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 2%, and cultured at 37°C until the cell concentration reached OD. 600 Once the concentration reaches 0.5–0.7, add 0.1 mM isopropyl-β-D-thiopyranoside to the culture medium and induce culture at 28°C for 12 h. Centrifuge at 4°C and 8000 rpm for 10 min and collect the wet cells.

7. The application as described in claim 3, characterized in that, The hydrogenation reaction involves hydrogenating 3-substituted-3-cyanopropionic acid from Formula II with Raney nickel at a pressure of 2 MPa, a temperature of room temperature, a stirring speed of 600 rpm, and a reaction time of 4 h. The mass ratio of Raney nickel to 3-substituted-3-cyanopropionic acid is 0.5:1.

Images