Nitrilase mutant and application thereof in synthesis of gamma-aminobutyric acid derivative

Abstract

The invention discloses a nitrilase mutant and application thereof in synthesis of a gamma-aminobutyric acid derivative, the nitrilase mutant with improved catalytic activity and stereoselectivity is constructed to efficiently catalyze hydrolysis of 2-substituted succinonitrile to synthesize 3-substituted-3-cyanopropionic acid, and the gamma-aminobutyric acid derivative is further synthesized through hydrogenation. And a foundation is laid for industrial production of gamma-aminobutyric acid drugs synthesized by a nitrilase method.

Description

(1) Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to a nitrilase mutant, a coding gene, an engineered bacterium, and their application in hydrolyzing 2-substituted succinonitrile to synthesize γ-aminobutyric acid derivatives. (2) Background Art

[0002] γ-Aminobutyric Acid (GABA) drugs are a class of non-protein amino acids. As the main inhibitory neurotransmitter in the central nervous system, GABA maintains the stable state of the nervous system by regulating the excitability of neurons. Its physiological activities include sedation, antidepressant, promoting sleep, lowering blood pressure, promoting bone growth, and improving metabolism, etc., and it is an important therapeutic drug for nervous system diseases. For example, brivaracetam is a new type of high-affinity ligand for synaptic vesicle protein 2A (SV2A), and it inhibits epileptic seizures by regulating the release of neurotransmitters. Pregabalin binds to the α2-δ subunit of calcium channels in the nervous system, inhibits the influx of Ca ions in the presynaptic membrane of neurons, reduces the release of excitatory neurotransmitters, and thus achieves the therapeutic effects of anti-epilepsy, analgesia, and anti-anxiety.

[0003] In recent years, due to the sharp increase in the 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 aldehyde and diethyl malonate as raw materials, through condensation and addition to prepare ethyl 3-substituted-3-cyano-2-(ethoxycarbonyl)propionate, and then through multiple steps such as hydrolysis, decarboxylation, hydrogenation, and resolution to synthesize optically pure GABA derivatives (J. Org. Chem. 2015, 80: 5704-5712); using unsaturated pyrazolamide as a raw material, through Gd(III) complex-catalyzed asymmetric addition and cleavage to obtain a chiral γ-nitroester compound, and then through cyclization, hydrogenation, and hydrolysis to synthesize the target GABA derivative (Chem. Rev. 2021, 122: 3637-3710). However, the existing chemical methods for synthesizing GABA drugs have defects such as difficult reuse of ineffective enantiomers, expensive catalysts, and poor atom economy, and it is difficult to achieve industrial application. Therefore, it is of great significance to develop green and efficient synthesis methods for GABA drugs.

[0004] Nitrilase can catalyze the hydrolysis of nitrile compounds to synthesize carboxylic acids. The biocatalytic process mediated by it has the advantages of high catalytic efficiency, strict stereo- and regioselectivity, and environmental friendliness, and it is an important tool enzyme for industrial synthesis of carboxylic acids. Using nitrilase to regioselectively and stereoselectively hydrolyze 2-substituted succinonitrile to synthesize 3-substituted-3-cyanopropionic acid and then preparing GABA compounds through one-step hydrogenation has significant advantages such as simple steps and high atom economy, and it is an ideal way for its industrial preparation.

[0005] The present invention uses protein engineering means to modify nitrilase to enhance its activity and stereoselectivity for the regioselective and stereoselective hydrolysis of 2-substituted succinonitrile, laying a foundation for the efficient synthesis of GABA drugs. (III) Summary of the Invention

[0006] The object of the present invention is to provide a nitrilase mutant, a coding gene, an engineered bacterium, and their application in the catalytic synthesis of GABA derivatives. By constructing a nitrilase mutant with improved catalytic activity and stereoselectivity, it can efficiently catalyze the hydrolysis of 2-substituted succinonitrile to synthesize 3-substituted-3-cyanopropionic acid, and further synthesize GABA derivatives by hydrogenation, laying a foundation for the industrial production of GABA drugs by the nitrilase method.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides a nitrilase mutant with high catalytic activity and high stereoselectivity, which is obtained by single mutation or multiple mutations of the 126th or 184th amino acid of the amino acid sequence shown in SEQ ID NO.2.

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

[0010] Mutant forms that retain nitrilase activity, such as conservative substitution, addition or deletion of one or several amino acids, amino-terminal truncation, or carboxyl-terminal truncation at other amino acid sites of the nitrilase mutant shown in SEQ ID NO.4 or SEQ ID NO.6, are also included within the scope of the present invention.

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

[0012] In the third aspect, the present invention provides a recombinant expression vector containing the coding gene for the nitrilase mutant; the original vector of the recombinant expression vector is pET-28b(+).

[0013] Fourthly, the present invention provides a recombinant genetically engineered bacterium containing the above-mentioned recombinant expression vector; the host cell of the recombinant genetically engineered bacterium can be various conventional host cells in the art, and the preferred host cell is Escherichia coli E. coli BL21. For the cultivation of the engineered bacterium in the present invention, any medium that can support its growth and induce the production of the specific nitrilase of the present invention can be used, and the preferred medium is LB medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent is water, and the pH is 7.0. As for the cultivation method and conditions, there are no specific limitations, as long as it is ensured that the engineered bacterium can grow normally and effectively produce nitrilase.

[0014] The preparation method of the nitrilase mutant includes the following steps:

[0015] (1) Design site-directed mutagenesis primers, and use the recombinant plasmid carrying the gene fragment with the nucleotide sequence shown in SEQ ID NO.1 as a template to perform overlap extension PCR to obtain a mutant product in which the 126th R in the amino acid sequence of the parental nitrilase is mutated to S;

[0016] (2) Use the mutant product carrying the Pg NITmut / R126S gene obtained in step (1) as a template to perform overlap extension PCR to obtain a mutant product in which the 184th S is mutated to V;

[0017] (3) Transform the mutant products obtained in steps (1) and (2) into the host bacterium respectively, screen to obtain nitrilase mutant expression strains, and the strains with correct sequencing results are the target strains. The target nitrilase mutants PgNITmut / R126S and Pg NITmut / R126S / S184V are obtained through induced expression.

[0018] Fifthly, the present invention provides an application of the nitrilase mutant in catalyzing the synthesis of γ-aminobutyric acid derivatives from 2-substituted succinonitrile.

[0019] Preferably, the method of the application is as follows: using the wet cells obtained by fermentation culture of the recombinant genetically engineered bacterium containing the nitrilase mutant coding gene or the pure enzyme extracted by crushing the wet cells as a catalyst, using 2-substituted succinonitrile (Ⅰ) as a substrate, using a buffer solution with a pH of 6-8 as a reaction medium to form a transformation system, and performing a transformation reaction at 20-40 °C and 100-200 rpm to obtain a reaction solution containing 3-substituted-3-cyanopropionic acid (Ⅱ), and then performing a hydrogenation reaction to obtain γ-aminobutyric acid derivatives;

[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 concentration of the substrate added to the conversion system is 100 - 200 g / L (preferably 100 g / L); the addition amount of the catalyst, calculated as wet bacterial cells, is 5 - 20 g / L (preferably 10 g / L).

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

[0024] Preferably, the 2-substituted succinonitrile includes 2-phenylsuccinonitrile, 2-m-chlorophenylsuccinonitrile, 2-p-fluorophenylsuccinonitrile, 2-p-chlorophenylsuccinonitrile, 2-p-methylphenylsuccinonitrile, 2-o-chlorophenylsuccinonitrile, 2-isobutylsuccinonitrile, and 3-cyanohexanenitrile.

[0025] Preferably, the wet bacterial cells are prepared as follows:

[0026] (1) Plate culture: The recombinant Escherichia coli containing the nitrilase mutant encoding gene is inoculated onto an LB solid medium containing 50 μg / mL kanamycin by streaking, and activated overnight at 37 °C to obtain single colonies;

[0027] (2) Seed culture: The single colonies are inoculated into an LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37 °C and 180 rpm for 10 - 12 h to obtain a seed solution;

[0028] (3) Fermentation culture: The seed solution is inoculated into a fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at an inoculation amount of 2% (v / v) by volume, cultured at 37 °C until the cell concentration OD 600 reaches 0.5 - 0.7, then isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM is added to the culture solution, induced to culture at 28 °C for 12 h, centrifuged at 4 °C and 8000 rpm for 10 min, and the wet bacterial cells are collected.

[0029] The nitrilase mutant of the present invention has a flexible application form. It can be directly used in the form of whole cells of the engineered bacteria, can also be applied in the form of unpurified crude enzyme, or can be in the form of partially purified or completely purified pure enzyme. In addition, by means of the immobilization techniques known in the art, the nitrilase mutant of the present invention can also be prepared into an immobilized enzyme or an immobilized cell form of biocatalyst, further broadening its application scope.

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

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

[0032] The activity and stereoselectivity of the nitrilase mutant screened in the present invention for catalyzing the synthesis of 3-substituted-3-cyanopropionic acid from 2-substituted succinonitrile are both improved, and the one-step hydrogenation is used to prepare GABA derivatives, laying a foundation for the efficient synthesis of GABA drugs.

[0033] The activity and stereoselectivity of the advantageous nitrilase mutant Pg NITmut / R126S / S184V of the present invention for catalyzing the synthesis of 3-substituted-3-cyanopropionic acid from 2-substituted succinonitrile are both significantly improved, specifically reflected in:

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

[0035] Using 2-(3-chlorophenyl)succinonitrile as the substrate, the specific activity of Pg NITmut is 95 U / mg, the ee value of the product is 56.5%, and E is 7; the specific activity of Pg NITmut / R126S / S184V is 89 U / mg, the ee value of the product is 63.3%, and E is 18;

[0036] Using 2-(4-fluorophenyl)succinonitrile as the substrate, the specific activity of Pg NITmut is 61 U / mg, the ee value of the product is 87.7%, and E is 22; the specific activity of Pg NITmut / R126S / S184V is 22 U / mg, the ee value of the product is 92.3%, and E is 47;

[0037] Using 2-(4-chlorophenyl)succinonitrile as the substrate, the specific activity of Pg NITmut is 74 U / mg, the ee value of the product is 82.6%, and E is 36; the specific activity of Pg NITmut / R126S / S184V is 76 U / mg, the ee value of the product is 93.2%, and E is 47;

[0038] Using 2-(4-methylphenyl)succinonitrile as the substrate, the specific activity of Pg NITmut is 25 U / mg, the ee value of the product is 90.3%, and E is 52; the specific activity of Pg NITmut / R126S / S184V is 66 U / mg, the ee value of the product is 96.8%, and E is 134;

[0039] Using 2-(2-chlorophenyl)succinonitrile as the substrate, the specific activity of Pg NITmut is 64 U / mg, the ee value of the product is 95.2%, and E is 102; the specific activity of Pg NITmut / R126S / S184V is 66 U / mg, the ee value of the product is 98.4%, and E is 189;

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

[0041] Using 3-cyanohexanenitrile as the substrate, the specific activity of Pg NITmut is 35 U / mg, the ee value of the product is 99.2%, and E is 569; the specific activity of Pg NITmut / R126S / S184V is 46 U / mg, the ee value of the product is 99.4%, and E is 674. (IV) Specific implementation manners

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

[0043] The parental nitrilase used in the embodiments of the present invention is a nitrilase 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 engineering bacterium E. coli BL21(DE3) / pET-28b(+)-Pg NITmut of the parental nitrilase has been disclosed in the patent application CN202310202126.6.

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

[0045] LB solid medium is obtained by adding 20 g / L agar to LB liquid medium.

[0046] Example 1: Construction and screening of nitrilase mutants

[0047] 1. Screening of mutation sites

[0048] Using the nitrilase mutant Pg Nit / F135L / R199W / T59D (hereinafter referred to as Pg NITmut) derived from Paraburkholderia graminis as the parent, 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 nitrilase and combined with DCCM analysis, key amino acids on the periphery of the catalytic pocket were selected, and site 126 was chosen for modification; further, based on the analysis of the "A interface" structure of the nitrilase tetramer, key amino acids with hydrogen bond interactions on the key Loop were located, and site 184 was chosen for modification.

[0050] SEQ ID NO.2

[0051] MGKVVKAAAVQFSPVLYSREATVAKVVQKIHELGLKGVQFATFPETVVPYYPYFAAVQDGIELLSGSEHLRLLEQAVTVPSAATDAIGKAAREAGMVVSIGVNERDGGTLYNTQLLFDADGTLIQRRRKITPTHLERMIWGQGDGSGLRAVDSAVGRIGQLACFEHNNPLARYAMIADGEQIHSAMYPGSAFGEGFAQWMEINIRQHALESGAFVVNATAWLDADQQAQIMKDTGCGIGPISGGCFTTIVSPDGMLMAEPLRSGEGEVIVDLDFAQIDRRKMLMDAAGHYNRPELLSLMIDRTPTAHVHERAPHSLPVSDKADDDVRTQAAAVAGSRLEI.

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

[0053] According to the mutant sites screened in step 1, the corresponding primers were designed as shown in Table 1.

[0054] Table 1 Site-directed saturation mutagenesis primers

[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 by the method of overlap extension PCR with the primers in Table 1, and site-directed mutations were introduced at positions 126 and 184 in the parental amino acid sequence.

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

[0060] PCR reaction parameters: (1) pre-denaturation at 95 °C for 30 s; (2) denaturation at 95 °C for 30 s; (3) annealing at 61 °C for 30 s; (4) extension at 72 °C for 4 min, steps (2)-(4) were cycled 30 times; (5) extension at 72 °C for 5 min, and stored at 16 °C.

[0061] After the PCR product was analyzed as positive by 0.9% agarose gel electrophoresis, 20 μL of the PCR reaction solution was taken, 1 μL of restriction enzyme Dpn I was added, and the template plasmid DNA was digested at 37 °C for 2 h, and inactivated at 65 °C for 10 min. Heat shock transformation was carried out into E. coli BL21(DE3) competent cells, and after recovery, it was spread on an LB plate containing 50 μg / mL kanamycin and cultured overnight at 37 °C.

[0062] 4. Bacterial culture and high-throughput screening: First, use a sterilized toothpick to pick the monoclonal colonies grown on the plate in step 3 and transfer them to a 96-deep well plate containing 600 μL of sterile LB liquid medium (final concentration 50 μg / mL kanamycin). The first three wells and the last three wells of the 96-deep well plate were respectively added with monoclonal colonies of the control group (parental nitrilase recombinant engineering bacteria), and then the 96-deep well plate was strictly sealed. Cultivate in a constant temperature shaker at 37 °C and 180 r / min for 20 to 24 hours to obtain the seed liquid. Then, take out 200 μL of the seed liquid from the cultured 96-deep well plate and transfer it to a new 96-deep well plate, which was pre-filled with 600 μL of sterile LB liquid medium (final concentration 50 μg / mL kanamycin and 0.1 mM IPTG), and induce culture at 28 °C and 180 r / min for 12 h. Subsequently, centrifuge at 4000 r / min at room temperature for 10 minutes, discard the supernatant, and collect the expressed bacterial cells for subsequent high-throughput screening.

[0063] Next, resuspend the bacterial cells in the wells with 200 μL of phosphate buffer (10 mM, pH 8), and then add racemic 3-cyanohexanenitrile (final concentration 20 mM) as the model substrate. React at a speed of 200 r / min in a constant temperature reaction oscillator at 30 °C for 30 min. After the reaction, add 30 μL of 2 M HCl to each well to terminate the reaction, and then centrifuge at a speed of 4000 r / min at room temperature for 10 min to obtain the supernatant. Take 30 μL of the supernatant and transfer it to a 96-well microreaction plate. Pre-add 150 μL of the fluorescence chromogenic solution to each well (the preparation method is: dissolve 0.1 g of o-phthalaldehyde in 20 mL of absolute ethanol, add 50 μL of mercaptoethanol, and then dilute 4 times with phosphate buffer (10 mM, pH 8) to make the fluorescence reaction solution), and incubate in a constant temperature incubator at 37 °C for 30 minutes to fully develop color. After the incubation, use an enzyme-linked immunosorbent assay (ELISA) reader (excitation wavelength 412 nm, emission wavelength 467 nm) to measure the fluorescence intensity in the 96-well microreaction plate. Calculate the difference in the reaction of the bacterial solution with two single-configuration racemic 3-cyanohexanenitriles according to the fluorescence intensity value. The strain with a larger difference compared to the control group is initially determined to be the strain with significantly enhanced catalytic stereoselectivity.

[0064] Screen out the dominant mutants with certain improvements in both activity and stereoselectivity. Subsequently, pick 4-5 clones into LB liquid medium, culture at 37 °C for 8 h, and then take the bacterial solution for sequencing to obtain the recombinant engineering bacterium E. coli BL21(DE3) / pET-28b(+)-Pg NITmut / R126S, whose amino acid sequence is shown in SEQ ID NO.4 and nucleotide sequence is shown in SEQ ID NO.3.

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

[0066] Using the constructed recombinant plasmid pET-28b(+)-Pg NITmut / R126S as a template, perform PCR with the primers S184V-Forward and S184V-Reverse in Table 1, and obtain the recombinant engineering bacterium E. coli BL21(DE3)pET28b(+)-Pg NITmut / R126S / S184V by the method in 4 above. Its amino acid sequence is shown in SEQ ID NO.6 and nucleotide sequence is shown in SEQ ID NO.5.

[0067] SEQ ID NO.6

[0068] MGKVVKAAAVQFSPVLYSREATVAKVVQKIHELGLKGVQFATFPETVVPYYPYFAAVQDGIELLSGSEHLRLLEQAVTVPSAATDAIGKAAREAGMVVSIGVNERDGGTLYNTQLLFDADGTLIQSRRKITPTHLERMIWGQGDGSGLRAVDSAVGRIGQLACFEHNNPLARYAMIADGEQIHVAMYPGSAFGEGFAQWMEINIRQHALESGAFVVNATAWLDADQQAQIMKDTGCGIGPISGGCFTTIVSPDGMLMAEPLRSGEGEVIVDLDFAQIDRRKMLMDAAGHYNRPELLSLMIDRTPTAHVHERAPHSLPVSDKADDDVRTQAAAVAGSRLEI。

[0069] Example 2: Induced Expression of Recombinant Escherichia coli Containing Nitrilase Mutant

[0070] 1. Wet Bacterial Cells

[0071] Inoculate the parental nitrilase E. coli BL21(DE3) / pET-28b(+)-Pg NITmut and the recombinant Escherichia coli E. coli BL21(DE3) / pET-28b(+)-Pg NITmut / R126S, E. coli BL21(DE3) / pET-28b(+)-Pg NITmut / R126S / S184V obtained in Example 1 into LB liquid medium containing 50 μg / mL kanamycin, culture at 37 °C and 180 rpm for 6 - 8 h, then inoculate into LB liquid medium containing 50 μg / mL kanamycin at an inoculum size of 2% (v / v), culture at 37 °C and 180 rpm until the cell concentration OD 600 = 0.6, add IPTG with a final concentration of 0.1 mM, induce culture at 28 °C for 12 h, then centrifuge at 4 °C and 8000 rpm for 10 min to collect wet bacterial cells. Wash the wet bacterial cells with 0.85% normal saline and store at -20 °C for later use.

[0072] 2. Pure Enzyme

[0073] Refer to the method and conditions in the literature (ACS Catalysis, 2023, 13: 9078 - 9089) to prepare pure enzyme.

[0074] Example 3: Method for Determining the Activity and Stereoselectivity of Recombinant Escherichia coli Containing Nitrilase Mutant in Hydrolyzing 2-Substituted Succinonitrile Compounds

[0075] Take 1 mg of pure enzyme and place it in an eppendorf tube. Use PB buffer (50 mM, pH 7.4) to make up the volume to 900 μL. Place the eppendorf tube in a metal bath at 30 °C for 5 min. Take 100 μL of the substrate stock solution (substrate concentration is 1 mol / L, solvent is N-N dimethylformamide) and add it to the eppendorf tube. React at 30 °C for 6 h. Take 200 μL of the reaction solution and mix it with 50 μL of 6 M hydrochloric acid to terminate the reaction and extract with ethyl acetate. After drying 200 μL of the upper organic phase with anhydrous sodium sulfate, add 7.5 μL of methanol and 3.75 μL of diazomethane, and determine the product content by gas chromatography, and calculate the substrate conversion rate and enantiomeric excess value (ee).

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

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

[0078] Definition of enzyme activity: Under the reaction conditions at 30 °C, the amount of enzyme required to catalyze the formation of 1 μmol of product is 1 enzyme activity unit U.

[0079] Definition of specific activity: The enzyme activity per milligram of enzyme protein.

[0080] Example 4: Application of nitrilase mutants in the synthesis of 2-phenyl-γ-aminobutyric acid

[0081] 1. Enzyme catalysis

[0082] Using the wet cell bodies of nitrilase Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, add 25 g of 2-phenylbutyronitrile substrate to 500 mL of PB (50 mM, pH 7.4) buffer. The catalyst addition amount is 10 g / L based on the weight of the wet cell bodies. React at 30 °C and 150 rpm. Take samples of the reaction solution and detect the specific enzyme activity, product ee value and product concentration by the method of Example 3.

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

[0084] 2. Hydrogenation reaction

[0085] After extracting the product of the mutant catalytic reaction in the above step 1 by the method of Example 2 of Patent Application CN112941122A, an aqueous solution with a final concentration of 100 g / L was prepared. Take 10 mL of the above aqueous solution, add 0.5 g of Raney nickel for hydrogenation reaction. The reaction pressure is 2 MPa, the reaction temperature is room temperature, and the stirring speed is 600 rpm. The reaction is carried out for 4 h. Detected by the method of Example 3, the results show that the conversion rate of (R)-3-phenyl-3-cyanopropionic acid is 100%, and the yield of (R)-2-phenyl-γ-aminobutyric acid is 73.4%.

[0086] Example 5: Application of nitrilase mutant in the synthesis of 2-(3-chlorophenyl)-γ-aminobutyric acid

[0087] 1. Enzymatic catalysis

[0088] Using the wet cell bodies containing nitrilase Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, add 25 g of 2-(3-chlorophenyl)butanedinitrile substrate to 500 mL of PB (50 mM, pH 7.4) buffer solution. The addition amount of the catalyst is 10 g / L based on the weight of the wet cells. The reaction is carried out at 30 °C and 150 rpm. Samples of the reaction solution are taken and the specific enzyme activity, the ee value of the product and the product concentration are detected by the method of Example 3. The determination results are shown in Table 2. The specific activity of Pg NITmut in catalyzing the synthesis of (R)-3-(3-chlorophenyl)-3-cyanopropionic acid from 2-(3-chlorophenyl)butanedinitrile is 95 U / mg. After reacting for 4 h, the conversion rate is 47.6%, the ee value of the product is 56.5%, and the E value is 7; the specific activity of Pg NITmut / R126S / S184V is 89 U / mg. After reacting for 6 h, the conversion rate is 49.9%, the ee value of the product is 63.3%, and the E value is 18.

[0089] 2. Hydrogenation reaction

[0090] The method of Example 2 of Patent Application CN112941122A was used to extract the catalytic reaction product (R)-3-(3-chlorophenyl)-3-cyanopropionic acid of the mutant in Step 1, and then it was formulated into an aqueous solution with a final concentration of 100 g / L. Take 10 mL of the above aqueous solution, add 0.5 g of Raney nickel for hydrogenation reaction. The reaction pressure was 2 MPa, the reaction temperature was room temperature, and the reaction was carried out for 4 h under the condition of a stirring speed of 600 rpm. Detection was carried out by the method of Example 3. The results showed that the conversion rate of (R)-3-(3-chlorophenyl)-3-cyanopropionic acid was 100%, and the yield of (R)-2-(3-chlorophenyl)-γ-aminobutyric acid was 86.7%.

[0091] Example 6: Application of nitrilase mutant in the synthesis of 2-(4-fluorophenyl)-γ-aminobutyric acid

[0092] 1. Enzymatic catalysis

[0093] Using the wet cell bodies containing nitrilase Pg NITmut, Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, 25 g of 2-(4-fluorophenyl)succinonitrile substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer solution. The addition amount of the catalyst was 10 g / L based on the weight of the wet cell bodies. 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 by the method of Example 3. The measurement results are shown in Table 2. The specific activity of Pg NITmut for catalyzing the synthesis of (R)-3-(4-fluorophenyl)-3-cyanopropionic acid from 2-(4-fluorophenyl)succinonitrile was 61 U / mg. After reacting for 6 h, the conversion rate was 40.4%, 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. After reacting for 8 h, the conversion rate was 35.9%, the product ee value was 92.3%, and the E value was 47.

[0094] 2. Hydrogenation reaction

[0095] The method of Example 2 of Patent Application CN112941122A was used to extract the catalytic reaction product (R)-3-(4-fluorophenyl)-3-cyanopropionic acid of the mutant, and then it was formulated into an aqueous solution with a final concentration of 100 g / L. Take 10 mL of the above aqueous solution, add 0.5 g of Raney nickel for hydrogenation reaction. The reaction pressure was 2 MPa, the reaction temperature was room temperature, and the reaction was carried out for 4 h under the condition of a stirring speed of 600 rpm. Detection was carried out by the method of Example 3. The results showed that the conversion rate of (R)-3-(4-fluorophenyl)-3-cyanopropionic acid was 100%, and the yield of (R)-2-(4-fluorophenyl)-γ-aminobutyric acid was 97.5%.

[0096] Example 7: Application of nitrilase mutant in the synthesis of 2-(4-chlorophenyl)-γ-aminobutyric acid

[0097] 1. Enzymatic catalysis

[0098] Using the wet cell bodies containing nitrilase Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2 as catalysts, 25 g of 2-(4-chlorophenyl)succinonitrile substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer. The addition amount of the catalyst was 10 g / L based on the weight of the wet cell bodies. The reaction was carried out at 30 °C and 150 rpm. Samples of the reaction solution were taken, and the specific enzyme activity, ee value of the product, and product concentration were detected by the method of Example 3. The measurement results are shown in Table 2. The specific activity of Pg NITmut in catalyzing the synthesis of (R)-3-(4-chlorophenyl)-3-cyanopropionic acid from 2-(4-chlorophenyl)succinonitrile was 74 U / mg. The conversion rate after 6 h of reaction was 45.4%, the ee value of the product was 82.6%, and the E value was 36. The specific activity of Pg NITmut / R126S / S184V was 76 U / mg. The conversion rate after 6 h of reaction was 35.9%, the ee value of the product was 93.2%, and the E value was 47.

[0099] 2. Hydrogenation reaction

[0100] After extracting the catalytic reaction product (R)-3-(4-chlorophenyl)-3-cyanopropionic acid by the method of Example 2 of Patent Application CN112941122A, an aqueous solution with a final concentration of 100 g / L was prepared. Take 10 mL of the above aqueous solution, add 0.5 g of Raney nickel for hydrogenation reaction. The reaction pressure was 2 MPa, the reaction temperature was room temperature, and the reaction was carried out for 4 h under the stirring speed of 600 rpm. Detection by the method of Example 3 showed that the conversion rate of (R)-3-(4-chlorophenyl)-3-cyanopropionic acid was 100%, and the yield of (R)-2-(4-chlorophenyl)-γ-aminobutyric acid was 98.5%.

[0101] Example 8: Application of nitrilase mutant in the synthesis of 2-(p-tolyl)-γ-aminobutyric acid

[0102] 1. Enzymatic catalysis

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

[0104] 2. Hydrogenation reaction

[0105] After extracting the catalytic reaction product (R)-3-p-tolyl-3-cyanopropionic acid by the method of Example 2 of Patent Application CN112941122A, an aqueous solution with a final concentration of 100 g / L was prepared. Take 10 mL of the above aqueous solution, add 0.5 g of Raney nickel for hydrogenation reaction. The reaction pressure was 2 MPa, the reaction temperature was room temperature, and the reaction was carried out for 4 h under the stirring speed of 600 rpm. Detection by the method of 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 nitrilase mutant in the synthesis of 2-o-chlorophenyl-γ-aminobutyric acid

[0107] 1. Enzymatic catalysis

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

[0109] 2. Hydrogenation reaction

[0110] After extracting the catalytic reaction product (R)-3-o-chlorophenyl-3-cyanopropionic acid by the method of Example 2 of Patent Application CN112941122A, an aqueous solution with a final concentration of 100 g / L was prepared. 10 mL of the above aqueous solution was taken, and 0.5 g of Raney nickel was added for hydrogenation reaction. The reaction pressure was 2 MPa, the reaction temperature was room temperature, and the reaction was carried out for 4 h under the stirring speed of 600 rpm. Detection by the method of 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 nitrilase mutants in the synthesis of 3-aminomethyl-5-methylhexanoic acid

[0112] 1. Enzymatic catalysis

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

[0114] 2. Hydrogenation reaction

[0115] The mutant-catalyzed reaction product (R)-3-cyano-5-methylhexanoic acid was extracted by the extraction method (ZL202110204296.9) constructed in advance by the method of Example 2 of Patent Application CN112941122A and formulated into an aqueous solution with a final concentration of 100 g / L. Take 10 mL of the above aqueous solution, add 0.5 g of Raney nickel for hydrogenation reaction. The reaction pressure was 2 MPa, the reaction temperature was room temperature, and the reaction was carried out for 4 h under a stirring speed of 600 rpm. Detection by the method of 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 nitrilase mutants in the synthesis of 3-aminomethylhexanoic acid

[0117] 1. Enzymatic catalysis

[0118] Using the wet cell catalysts containing nitrilase Pg NITmut and Pg NITmut / R126S / S184V prepared in Example 2, 25 g of 3-cyanohexanenitrile substrate was added to 500 mL of PB (50 mM, pH 7.4) buffer. The addition amount of the catalyst was 10 g / L based on the weight of the wet cell catalyst. The reaction was carried out at 30 °C and 150 rpm. Samples of the reaction solution were taken and the specific enzyme activity, the ee value of the product and the product concentration were detected by the method of Example 3. The measurement results are shown in Table 2. The specific activity of Pg NITmut for catalyzing the synthesis of (R)-3-cyanohexanoic acid from 3-cyanohexanenitrile was 35 U / mg. After reacting for 16 h, the conversion rate was 45.2%, the ee value of the product was 99.2%, and the E value was 569; the specific activity of PgNITmut / R126S / S184V was 46 U / mg. After reacting for 16 h, the conversion rate was 46.3%, the ee value of the product was 99.4%, and the E value was 674.

[0119] 2. Hydrogenation reaction

[0120] The mutant-catalyzed reaction product (R)-3-cyanohexanoic acid was extracted by the method of Example 2 of Patent Application CN112941122A and formulated into an aqueous solution with a final concentration of 100 g / L. Take 10 mL of the above aqueous solution, add 0.5 g of Raney nickel for hydrogenation reaction. The reaction pressure was 2 MPa, the reaction temperature was room temperature, and the reaction was carried out for 4 h under the condition of a stirring speed of 600 rpm. Detection by the method of 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 succinonitrile by Pg NITmut and its mutants

[0122]

[0123] The present invention is not limited by the above specific text descriptions. The present invention can make various changes within the scope summarized by the claims, and these changes are all within the scope of the present invention.

Claims

1. A nitrilase mutant, characterized in that The nitrilase mutant is obtained by subjecting the 126th or 184th amino acid shown in SEQ ID NO.2 to single mutation or multiple mutation.

2. The nitrilase mutant according to claim 1, wherein The nitrilase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO. 2 to one of the following: (1) the arginine at position 126 is mutated to serine; (2) the arginine at position 126 is mutated to serine and the serine at position 184 is mutated to valine.

3. A recombinant genetically engineered bacterium comprising a gene encoding the nitrilase mutant according to claim 1.

4. Use of the nitrilase mutant according to claim 1 in catalyzing the synthesis of γ-aminobutyric acid derivatives from 2-substituted succinonitrile.

5. The use according to claim 4, characterized in that The application method comprises: using wet bacteria obtained by fermentation culture of a recombinant genetically engineered bacterium containing the gene encoding the nitrilase mutant or pure enzyme extracted by crushing the wet bacteria as a catalyst, using 2-substituted succinonitrile (I) as a substrate, and using a buffer solution with a pH of 6-8 as a reaction medium to form a conversion system, carrying out a conversion reaction at 20-40° C. and 100-200 rpm to obtain a reaction solution containing 3-substituted-3-cyanopropionic acid (II), and then carrying out a hydrogenation reaction to obtain a γ-aminobutyric acid derivative; 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).

6. The use according to claim 5, characterized in that The substrate addition concentration in the conversion system is 10-200 g / L; the catalyst addition amount is 5-20 g / L based on wet bacteria.

7. The use according to claim 5, characterized in that The reaction medium is a 50 mM phosphate buffer at pH 7.

4.

8. The use according to claim 5, characterized in that The 2-substituted succinonitrile includes 2-phenylsuccinonitrile, 2-m-chlorophenylsuccinonitrile, 2-p-fluorophenylsuccinonitrile, 2-p-chlorophenylsuccinonitrile, 2-p-methylphenylsuccinonitrile, 2-o-chlorophenylsuccinonitrile, 2-isobutylsuccinonitrile, and 3-cyanohexanenitrile.

9. The use according to claim 5, characterized in that The wet cells are prepared as follows: (1) Plate culture: The recombinant Escherichia coli containing the gene encoding the nitrilase mutant was inoculated onto a LB solid medium containing 50 μg / mL kanamycin by streaking, and activated at 37°C overnight to obtain a single colony; (2) Seed culture: A single colony was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 10-12 h to obtain seed solution; (3) Fermentation culture: The seed solution 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 bacterial concentration OD 600 When it reaches 0.5-0.7, add isopropyl-β-D-thiogalactopyranoside with a final concentration of 0.1 mM to the culture medium, induce culture at 28°C for 12 h, centrifuge at 4°C and 8000 rpm for 10 min, and collect wet bacteria.

10. The use according to claim 5, characterized in that The hydrogenation reaction is to carry out a hydrogenation reaction of 3-substituted-3-cyanopropionic acid (II) and Raney nickel, the reaction pressure is 2MPa, the reaction temperature is room temperature, the stirring speed is 600rpm, and the reaction time is 4h; the mass ratio of the Raney nickel to the 3-substituted-3-cyanopropionic acid is 0.5:1.

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