2-Valeroylbenzonitrile reductase mutant and its application in the preparation of optically pure butylphthalide

Abstract

The present invention relates to a 2-pentanoylbenzonitrile reductase mutant and its use in preparing optically pure n-butylphthalide. Specifically, it relates to a 2-pentanoylbenzonitrile reductase mutant with improved activity, stability, and enantioselectivity, a gene encoding the 2-pentanoylbenzonitrile reductase mutant, a recombinant expression vector containing the gene sequence, and a recombinant expression transformant, as well as the use of the 2-pentanoylbenzonitrile reductase mutant as a catalyst for catalyzing the asymmetric reduction of 2-pentanoylbenzonitrile compounds, particularly catalyzing the asymmetric reduction of 2-pentanoylbenzonitrile to prepare optically pure n-butylphthalide (n-butylphthalide). Compared with the prior art, the present invention has the advantages of high enzymatic reaction substrate concentration, mild reaction conditions, environmental friendliness, high yield, and high product optical purity, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a 2-pentanoylbenzonitrile reductase mutant with significantly improved catalytic performance, a gene encoding the 2-pentanoylbenzonitrile reductase mutant, a recombinant expression vector containing the gene sequence, and a recombinant expression transformant, as well as the use of the 2-pentanoylbenzonitrile reductase mutant or a recombinant 2-pentanoylbenzonitrile reductase mutant catalyst in catalyzing 2-pentanoylbenzonitrile compounds to prepare optically pure butylphthalide. Background Art

[0002] n-Butylphthalide (dl-3-n-butylphthalide, NBP), also known as butylphthalide, is a Class I new drug independently developed in my country for the treatment of cerebral infarction. Studies have shown that NBP can block multiple pathological pathways of brain damage caused by cerebral infarction and has a strong brain-protective effect. NBP can significantly reduce the infarct size of localized cerebral infarction in experimental animals, improve brain energy metabolism, promote microcirculation and blood flow in the ischemic brain region, reduce cerebral edema, and inhibit neuronal apoptosis. Continuous or sequential treatment with NBP has been shown to improve neurological function impairment and is included in the "Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke in China 2014" (Grade II recommendation, Level B evidence). Therefore, the synthesis of high-optical-purity n-butylphthalide has significant economic value.

[0003] dl-NBP is a racemic compound that can be separated into d- and l-isomers due to the chiral carbon atom on the furan ring. Like many other optical isomers, significant differences exist in the pharmacological activities of d-NBP and l-NBP. l-NBP has been reported to be more effective as an antiplatelet agent than both d-NBP and dl-NBP, outperforming dl-NBP at twice the dose in reducing cytochrome c release and inhibiting DNA fragmentation after ischemic injury. Furthermore, l-NBP is superior to both d-NBP and dl-NBP in reducing cerebral infarct volume and alleviating cognitive deficits. These differences may be due to the stereoselectivity of the drug molecules, ultimately manifesting in their pharmacological, toxicological, and pharmacokinetic properties. With the increasing emphasis on chiral separation and analysis of single enantiomers, many existing drugs have been converted from racemic mixtures to one of their isomers. In this context, l-NBP exhibits superior in vivo pharmacological activity and is likely to have a broader market in the future.

[0004] Li Shaobai et al. refluxed phthalic anhydride and n-valeric anhydride in the presence of anhydrous sodium acetate at 300°C to produce 3-butenylphthalide. Subsequently, they used Pd / C reduction to obtain butylphthalide in yields of 25% and 95%, respectively. This method involves high temperatures, making it unsuitable for industrial production and resulting in relatively low yields. Yang Hua et al. used a Grignard reagent and anhydrous CdCl₂ to prepare a cadmium reagent, which reacted with phthalic anhydride in ether and then was reduced with NaBH₄ to obtain butylphthalide in a yield of 42.7% and a purity of 99.5%. This method offered a low yield, and the cadmium reagent was highly toxic. Chen Xiaokang et al. prepared a Grignard reagent from methyl o-bromobenzoate, which was then refluxed with n-valeraldehyde and subjected to intramolecular cyclization. This yielded butylphthalide in a one-pot process with an 83.4% yield. This reaction offered a high yield, but the product required purification by vacuum distillation. The preparation of the Grignard reagent was crucial to this method, ensuring absolute anhydrous conditions during the operation. Activation of magnesium powder was also crucial. Kuethe et al. reacted methyl o-iodobenzoate with isopropylmagnesium chloride-lithium chloride at -50-70°C, followed by the addition of n-valeraldehyde for reaction at room temperature, to produce butylphthalide in an 82% yield. This reaction required a low-temperature reaction in the first step, and the product required column chromatography purification. Suchand et al. used a Pd-catalyzed, environmentally friendly carbonylation reaction in the presence of palladium acetate and silver oxide, using TBHP aqueous solution as the oxidant and solvent, to couple ethyl o-iodobenzoate with valeraldehyde to produce ethyl o-valerylbenzoate (65%). This was then reduced with NaBH4 in the presence of CeCl3, followed by intramolecular ring closure to yield butylphthalide in a 78% yield.

[0005] In summary, there are currently multiple reaction routes for the synthesis of butylphthalide, each with its own advantages and disadvantages. Most routes utilize compounds containing a benzene ring as starting materials, constructing ester rings or introducing butyl groups. Furthermore, most employ metal reagents such as Grignard reagents and alkyl zinc reagents. While all of these pathways can produce NBP, these reactions suffer from low substrate conversion rates and low ee values ​​for the resulting pharmaceutical products, limiting their practical applications.

[0006] The most direct precursor of NBP is the aromatic ketone before its cyclization. If the chiral hydroxy acid is obtained by asymmetric reduction of the prochiral keto acid and finally cyclized to the lactone, it is the most atom-economical path. The inventor's Chinese patent CN109797140A discloses a carbonyl reductase mutant, encoding gene, recombinant vector and expression transformant and its application in the preparation of (R)-alkyl lactone. The carbonyl reductase SmCR is molecularly modified to obtain a (R)-4-carbonyldecanoic acid (ester) reductase mutant with improved reducing activity, stability and enantioselectivity. The mutant is more stable than SmCR at 30°C, with a half-life of up to 124h and a K of 4-carbonyldecanoic acid methyl ester. m The value is 0.79mM, k cat 179 minutes -1, and the reduction activity of other 4- and 5-carbonyl compounds was improved to varying degrees. In the preparation of chiral lactones, substrate loadings of up to 1500 mM and catalyst loadings of 75 g / L were achieved, resulting in short reaction times and high space-time yields. However, the reactivity for 2-pentanoylbenzonitrile was extremely low, with an ee(S) of only 1.1%.

[0007] In summary, in the research on the synthesis of optically pure NBP compounds, known (R)-4-oxodecanoate reductase mutants still suffer from low catalytic activity, low substrate loading, and low product yield towards 2-pentanoylbenzonitrile. Therefore, enzyme catalysts with better catalytic performance are needed for the asymmetric reduction of 2-pentanoylbenzonitrile to meet the industrial requirements of high reaction efficiency, high substrate concentration, simple operation, and high yield. Summary of the Invention

[0008] The purpose of the present invention is to overcome the defects of the above-mentioned prior art and provide a 2-pentanoylbenzonitrile reductase mutant and its application in the preparation of optically pure butylphthalide.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] One of the technical solutions of the present invention: The present invention provides a carbonyl reductase mutant with significantly improved catalytic performance and enantioselectivity.

[0011] The present inventors screened a mutant with improved catalytic performance, stability and enantioselectivity using carbonyl reductase SmCR from Serratia marcescens through random mutagenesis and semi-rational modification. The amino acid sequence of the mutant is shown in SEQ ID No. 2 in the sequence listing.

[0012] In the present invention, the carbonyl reductase SmCR gene is used as the parent, and a combination of mutation strategies such as error-prone PCR, iterative saturation mutagenesis, and site-directed saturation mutagenesis is used for further directed evolution. Combined with high-throughput primary screening using a microplate reader and secondary screening using an ultraviolet spectrophotometer, 2-pentanoylbenzonitrile reductase with significantly improved catalytic performance and thermal stability is identified.

[0013] The 2-pentanoylbenzonitrile reductase mutant (hereinafter referred to as the SmCR mutant) is a derivative protein having a new amino acid sequence formed by replacing one or more amino acid residues at positions 145 N, 92 D, 183 Y, 127 K, and 90 S of the amino acid sequence shown in SEQ ID No. 2 with other amino acid residues. The derivative protein has higher catalytic performance and thermal stability than the protein composed of the amino acid sequence shown in SEQ ID No. 2.

[0014] The amino acid sequence of the SmCR mutant is selected from any one of the following sequences:

[0015] (1) replacing asparagine at position 145 of the amino acid sequence shown in SEQ ID No. 2 with arginine;

[0016] (2) replacing aspartic acid at position 92 of the amino acid sequence shown in SEQ ID No. 2 with tyrosine;

[0017] (3) replacing tyrosine at position 183 of the amino acid sequence shown in SEQ ID No. 2 with leucine;

[0018] (4) replacing the threonine at position 90 of the amino acid sequence shown in SEQ ID No. 2 with methionine;

[0019] (5) replacing lysine at position 127 of the amino acid sequence shown in SEQ ID No. 2 with arginine;

[0020] (6) the asparagine at position 145 of the amino acid sequence shown in SEQ ID No. 2 is replaced by arginine, and the aspartic acid at position 92 is replaced by tyrosine;

[0021] (7) in the amino acid sequence shown in SEQ ID No. 2, the asparagine at position 145 is replaced by arginine, the aspartic acid at position 92 is replaced by tyrosine, and the tyrosine at position 183 is replaced by leucine;

[0022] (8) in the amino acid sequence shown in SEQ ID No. 2, asparagine at position 145 is replaced by arginine, aspartic acid at position 92 is replaced by tyrosine, tyrosine at position 183 is replaced by leucine, and threonine at position 90 is replaced by methionine;

[0023] (9) in the amino acid sequence shown in SEQ ID No. 2, asparagine at position 145 is replaced by arginine, aspartic acid at position 92 is replaced by tyrosine, tyrosine at position 183 is replaced by leucine, and lysine at position 127 is replaced by arginine;

[0024] (10) In the amino acid sequence shown in SEQ ID No. 2, asparagine at position 145 is replaced by arginine, aspartic acid at position 92 is replaced by tyrosine, tyrosine at position 183 is replaced by leucine, threonine at position 90 is replaced by methionine, and lysine at position 127 is replaced by arginine.

[0025] The second technical solution of the present invention: The present invention provides the nucleic acid encoding the SmCR mutant.

[0026] The nucleic acid encodes any one of the SmCR mutants described in one of the technical solutions.

[0027] The nucleotide sequence encoding the SmCR mutant is a nucleic acid sequence encoding the SmCR mutant as described in one of the technical solutions.

[0028] The method for obtaining the nucleic acid encoding the SmCR mutant of the present invention is conventional in the art: cloning the gene sequence of the SmCR mutant as described in one of the technical solutions by genetic engineering technology or obtaining it by artificial full sequence synthesis.

[0029] Technical solution three of the present invention: The present invention provides a recombinant expression vector.

[0030] The recombinant expression vector comprises the nucleic acid as described in the second technical solution.

[0031] The recombinant expression vector can be constructed by connecting the SmCR mutant nucleic acid of the present invention to various commercially available empty vectors using conventional methods in the art. The commercially available empty vector can be any conventional plasmid vector in the art, as long as the recombinant expression vector can replicate normally in the corresponding expression host and express the corresponding reductase. The preferred plasmid in this scheme is pET-28a(+).

[0032] Preferably, the recombinant expression vector of the present invention can be prepared by the following method: the DNA fragment of the SmCR mutant obtained by PCR amplification is double-digested with restriction endonucleases EcoR I and Hind III, and the empty plasmid pET-28a(+) is also double-digested with restriction endonucleases EcoR I and Hind III, the DNA fragment of the SmCR mutant and the empty plasmid after the above enzyme digestion are recovered, and they are ligated using T4 DNA ligase to construct a recombinant expression vector containing the nucleic acid sequence of the SmCR mutant.

[0033] Technical solution 4 of the present invention: The present invention provides a recombinant expression transformant.

[0034] The recombinant expression transformant of the present invention can be prepared by transforming the recombinant expression vector into a host microorganism using conventional methods in the art. The host microorganism can be any of a variety of conventional host microorganisms in the art, as long as the recombinant expression vector can stably replicate on its own and effectively express after induction with an inducer. The host microorganism is preferably Escherichia coli BL21 (DE3).

[0035] Technical solution 5 of the present invention: The present invention provides a recombinant SmCR mutant catalyst, wherein the SmCR mutant catalyst is any one of the following forms:

[0036] (1) culturing the recombinant expression transformant as described in the fourth technical solution, and isolating the transformant cells containing the SmCR mutant;

[0037] (2) culturing the recombinant expression transformant as described in the fourth technical solution, and isolating the crude enzyme solution containing the SmCR mutant;

[0038] (3) Drying the crude enzyme liquid of the SmCR mutant to obtain crude enzyme powder.

[0039] To obtain the SmCR mutant catalyst, the culture method and conditions for the recombinant expression transformant are conventional methods and conditions in the art. The culture medium is any culture medium in the art that can grow the transformant and produce 2-pentanoylbenzonitrile reductase.

[0040] The activity determination method of the SmCR mutant described in the present invention is as follows: a reaction system is added to a 1 mL cuvette: 970 μL PBS (pH 7.0) + 10 μL pure enzyme + 10 μL NADPH (0.2 mM) + 10 μL substrate (2 mM), the reaction time is 1 min, and the activity of the mutant is measured in a constant temperature spectrophotometer by observing the decrease in absorbance.

[0041] Enzyme activity (U) = EW × V × 103 / (6220 × l)

[0042] Where EW is the change in absorbance at 340 nm over 1 minute; V is the volume of the reaction solution in mL; 6220 is the molar extinction coefficient of NADPH in L / (mol·cm); and l is the optical path length in cm. One unit (U) of enzyme activity is defined as the amount of enzyme required to catalyze the oxidation of 1 μmol of NADPH per minute under the above conditions.

[0043] Technical solution six of the present invention: The present invention provides an application of a SmCR mutant or a SmCR mutant catalyst in the asymmetric reduction of a 2-pentanoylbenzonitrile compound.

[0044] The specific synthesis method of optically pure NBP is as follows:

[0045]

[0046] In some embodiments of the present invention, NADPH or NADP required in the reaction +The dosage of 2-pentanoylbenzonitrile is 0.2-0.5 mmol / L. During the reaction, glucose can be used as a cosubstrate, and the NADPH coenzyme cycle in the reaction system is achieved through catalysis by BmGDH dehydrogenase. The dosage of the glucose dehydrogenase can be 50-200 U / mmol of the 2-pentanoylbenzonitrile compound, the dosage of the glucose can be 1.5 times the molar amount of the 2-pentanoylbenzonitrile compound, and the dosage of the coenzyme NADPH or NADP+ is 0.1-0.5 mmol / L. The concentration of the 2-pentanoylbenzonitrile compound can be 2-20 mmol / L, and the dosage of the SmCR mutant or SmCR mutant catalyst can be 5-20 U / mmol of the 2-pentanoylbenzonitrile compound.

[0047] The phosphate buffer required in the asymmetric reduction reaction is a conventional phosphate buffer in the art, preferably a PBS buffer, with a concentration of 100 mmol / L.

[0048] Preferably, the reaction is carried out under shaking or stirring conditions; the temperature is 20-40°C, more preferably 30°C; the reaction time is based on the time when the substrate is completely reacted or the reaction terminates on its own, and more preferably the reaction time is less than 24h.

[0049] The present invention provides a 2-pentanoylbenzonitrile reductase mutant with enhanced catalytic performance, which efficiently catalyzes the asymmetric reduction of the carbonyl group of 2-pentanoylbenzonitrile to produce optically pure apigenin (n-butylphthalide). This product is commercially available (National Pharmaceutical Approval No. H20050299) for the treatment of mild to moderate acute ischemic stroke.

[0050] Compared with the prior art, the invention has the following innovations and beneficial effects:

[0051] (1) The present invention couples the SmCR mutant with glucose dehydrogenase to achieve in situ regeneration of the coenzyme, greatly reducing the amount of coenzyme used; and can achieve a conversion rate of 98% within 3 hours, and the optical purity of the product can reach 93.5% (S).

[0052] (2) Compared with the (R)-4-carbonyldecanoate reductase mutant, the 2-pentanoylbenzonitrile reductase mutant obtained in the present invention has the advantages of high catalytic activity, tolerance to high concentration substrates, high optical purity of the reaction product, and high final separation yield when targeting 2-pentanoylbenzonitrile compounds, and therefore has better industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the liquid phase diagram of n-butylphthalide product and standard product. DETAILED DESCRIPTION

[0054] The various reaction or detection conditions described in the present invention may be combined or modified according to common knowledge in the art and may be verified by experiments. The technical solutions and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, the scope of protection of the present invention is not limited to these embodiments, and any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0055] Sources of materials in the following examples:

[0056] The parent recombinant plasmid pET28a-SmCR contains the nucleic acid sequence shown in SEQ ID No. 1 in the sequence listing, was constructed by the inventors themselves, and is also disclosed in patent CN109797140A.

[0057] The plasmid vector pET28a was purchased from Novagen.

[0058] E. coli BL21 (DE3) competent cells, 2× Taq PCR MasterMix, and agarose gel DNA recovery kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0059] Restriction endonucleases EcoRI and HindIII were commercially available from New England Biolabs (NEB).

[0060] Unless otherwise stated, the specific experiments in the following examples were performed according to conventional methods and conditions in the art, or in accordance with the commercial instructions of the kits.

[0061] Example 1 Semi-rational construction of SmCR mutants

[0062] Homology modeling of the carbonyl reductase SmCR was performed, and the resulting mutant models were then used for molecular docking with 2-pentanoylbenzonitrile. An appropriate docking pose was selected based on the catalytic mechanism and binding energy of SmCR. Enzyme activity was further enhanced by site-directed saturation mutagenesis and combinatorial mutagenesis of amino acids near the substrate pocket. In the three-dimensional structure of SmCR, as shown in the amino acid sequence of SEQ ID No. 2, the amino acid residues surrounding the substrate 2-pentanoylbenzonitrile binding site include: asparagine 145, aspartic acid 92, tyrosine 183, threonine 90, lysine 127, asparagine 110, serine 138, valine 139, threonine 142, methionine 143, glycine 147, tyrosine 151, lysine 155, and arginine 167. The amino acid residues at these sites were subjected to saturation mutation using site-directed saturation mutagenesis technology. The primers used are shown in Table 1.

[0063] Table 1 Primers for mutation sites

[0064]

[0065]

[0066] PCR amplification was performed using PrimeStar HS premix using pET28a-SmCR as a template. The PCR system was as follows: 10 μL of 2× PrimeStar HS premix, 1 μL of upstream and downstream primers, 40 ng of pET28a-SmCR plasmid, 1 μL of DMSO, and sterile distilled water to make up to 20 μL. PCR reaction procedure: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 94°C for 30 s; (3) annealing at 55°C for 30 s; (4) extension at 72°C for 6.3 min; steps (2) to (4) were repeated for a total of 30 cycles; and finally, extension was performed at 72°C for 10 min. Add 1 μL of Dpn I enzyme to 20 μL of PCR product and incubate at 37°C for 3 hours to digest the template. The digestion product is transformed into E. coli BL21 (DE3) competent cells and evenly spread on LB agar plates containing 50 μg / mL kanamycin. Incubate in a 37°C incubator for approximately 12 hours. The resulting monoclonal colonies are picked and cultured in 96-well deep-well plates. The cultured cells are broken and the expressed proteins are screened for high-throughput activity in 96-well plates using NADPH as a coenzyme. Mutants with high activity are purified and characterized, and the corresponding genes are sequenced.

[0067] Through the high-throughput screening using an ELISA as described in Example 1, it was found that mutants such as those in which asparagine at position 145 was replaced with arginine (N145R) had improved activity and selectivity for 2-pentanoylbenzonitrile.

[0068] Method for determining the enzyme activity of the SmCR mutant: Preheat 1 mL of a reaction system (100 mmol / L sodium phosphate buffer, pH 6.0) containing 2 mmol / L 2-pentanoylbenzonitrile and 0.1 mmol / L NADPH to 40°C, then add an appropriate amount of the SmCR mutant enzyme solution. Incubate the reaction at 40°C and measure the absorbance change at 340 nm on a spectrophotometer. Record the absorbance change within 1 min and calculate the enzyme activity.

[0069] Through screening, mutants with significantly improved activity towards 2-pentanoylbenzonitrile were obtained. The sequences of these mutants and their activities towards 2-pentanoylbenzonitrile are listed in Table 2. Table 2 provides a list of carbonyl reductase SmCR mutants with specific sequences having relevant activities disclosed in the present invention. In Table 2, the mutant proteins have improved specific activity and enantioselectivity compared to the protein consisting of the amino acid sequence shown in SEQ ID No. 2.

[0070] Table 2 Enzyme activity and enantioselectivity of mutants

[0071]

[0072] K1: replacing the asparagine at position 145 of the amino acid sequence shown in SEQ ID No. 2 with arginine;

[0073] K2: The asparagine at position 145 of the amino acid sequence shown in SEQ ID No. 2 is replaced by arginine, and the aspartic acid at position 92 is replaced by tyrosine;

[0074] K3: The asparagine at position 145 of the amino acid sequence shown in SEQ ID No. 2 is replaced by arginine, the aspartic acid at position 92 is replaced by tyrosine, and the tyrosine at position 183 is replaced by leucine;

[0075] K4: in the amino acid sequence shown in SEQ ID No. 2, the asparagine at position 145 is replaced by arginine, the aspartic acid at position 92 is replaced by tyrosine, the tyrosine at position 183 is replaced by leucine, and the threonine at position 90 is replaced by methionine;

[0076] K5: in the amino acid sequence shown in SEQ ID No. 2, asparagine at position 145 is replaced by arginine, aspartic acid at position 92 is replaced by tyrosine, tyrosine at position 183 is replaced by leucine, and lysine at position 127 is replaced by arginine;

[0077] K6: In the amino acid sequence shown in SEQ ID No. 2, asparagine at position 145 is replaced by arginine, aspartic acid at position 92 is replaced by tyrosine, tyrosine at position 183 is replaced by leucine, threonine at position 90 is replaced by methionine, and lysine at position 127 is replaced by arginine.

[0078] Example 2 Recombinant E. coli BL21 (DE3) / pET28a-SmCR K6 expression and enzymatic preparation.

[0079] Preferably, the recombinant E. coli BL21 (DE3) / pET28a-SmCR of mutant K6 obtained in Example 1 is used. K6The cells were inoculated into LB medium containing 50 μg / mL kanamycin and cultured on a shaker at 37°C for 12 hours. Afterwards, a 1% (v / v) inoculum was transferred to 100 mL of LB medium (containing 50 μg / mL kanamycin) in a 500 mL Erlenmeyer flask and cultured on a shaker at 37°C at 180 rpm. When the OD600 of the culture reached 0.6, IPTG was added at a final concentration of 0.2 mmol / L as an inducer and induced at 16°C for 24 hours. The culture was centrifuged at 8000 × g for 10 minutes, and the cells were harvested and washed twice with saline to obtain resting cells. The cells from 100 mL of culture were suspended in 15 mL of 100 mM sodium phosphate buffer (pH 7.0) and disrupted by sonication in an ice-water bath as follows: 350 W power, 4 seconds on, 6 seconds off, 10 minutes of disruption. The cells were then centrifuged at 12000 × g for 40 minutes at 4°C, and the crude enzyme supernatant was collected. In addition, the harvested crude enzyme solution can be freeze-dried to obtain freeze-dried enzyme powder.

[0080] Example 3 Recombinant SmCR K6 Catalytic synthesis of n-butylphthalide

[0081] In a 100 mL jacketed reactor, 40 g / L of the mutant recombinant expression transformant (E. coli BL21 (DE3) / pET28a-SmCR) as described in Example 2 was preferably added to 50 mL of sodium phosphate buffer (100 mM, pH 6.0) containing 50 mM 2-pentanoylbenzonitrile substrate and 75 mM glucose. K6 ) wet cells, and add 2g / L freeze-dried enzyme powder of glucose dehydrogenase, and finally add 0.2mM NADP + Used for the recycling of coenzymes. The reaction was carried out in a 30°C water bath under stirring with a stirring paddle, and 1 mol / L sodium carbonate solution was added by an automatic potentiometric titrator to control the pH at 6.0. After 12 hours of reaction, concentrated sulfuric acid was added to terminate the reaction and the pH of the reaction solution was adjusted to 2.0. It was extracted twice with 1.5 times the volume of ethyl acetate, the extracts were combined, and then the solvent ethyl acetate was removed by rotary evaporation. The intermediate product was dissolved in 5mL [bmim] HSO4 and heated with stirring at 60°C in a water bath for 6 hours to complete the cyanocarboxylation and lactone cyclization reactions. The [bmim] HSO4 was then precipitated with an ice-water mixture, and the organic layer was separated; and dried with anhydrous sodium sulfate overnight. Finally, the crude product was purified by silica gel chromatography column, and the target product was eluted with a mobile phase of ethyl acetate and petroleum ether (1:10) to obtain 270mg of product with a purity of 99%. Liquid chromatography determined that the substrate conversion rate was 98% and the product ee value was 93.5% (S). Figure 1 shown.

[0082] It can be seen that the recombinant mutant enzyme preparation obtained by the method of the present invention can efficiently catalyze the 2-pentanoylbenzonitrile compound, and then combine the chemical method to carboxylate the cyano acid / ester compound and heat it to cyclize it to form n-butylphthalide. This optically pure compound can be used to prepare drugs for treating mild to moderate acute ischemic stroke and Alzheimer's disease (senile dementia), and has very great medicinal and application value.

[0083] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A 2-pentanoylbenzonitrile reductase mutant, characterized in that: The amino acid sequence of the 2-pentanoylbenzonitrile reductase mutant is selected from any one of the following sequences: (1) The asparagine at position 145 of the amino acid sequence shown in SEQ ID No. 2 is replaced by arginine, and the aspartic acid at position 92 is replaced by tyrosine; (2) In the amino acid sequence shown in SEQ ID No. 2, asparagine at position 145 is replaced by arginine, aspartic acid at position 92 is replaced by tyrosine, and tyrosine at position 183 is replaced by leucine; (3) In the amino acid sequence shown in SEQ ID No. 2, asparagine at position 145 is replaced by arginine, aspartic acid at position 92 is replaced by tyrosine, tyrosine at position 183 is replaced by leucine, and threonine at position 90 is replaced by methionine; (4) In the amino acid sequence shown in SEQ ID No. 2, asparagine at position 145 is replaced by arginine, aspartic acid at position 92 is replaced by tyrosine, tyrosine at position 183 is replaced by leucine, and lysine at position 127 is replaced by arginine; (5) In the amino acid sequence shown in SEQ ID No. 2, asparagine at position 145 is replaced by arginine, aspartic acid at position 92 is replaced by tyrosine, tyrosine at position 183 is replaced by leucine, threonine at position 90 is replaced by methionine, and lysine at position 127 is replaced by arginine.

2. An isolated nucleic acid, characterized in that The nucleic acid encodes the 2-pentanoylbenzonitrile reductase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 2.

4. A recombinant expression transformant, characterized in that: The recombinant expression transformant comprises the recombinant expression vector according to claim 3.

5. A recombinant 2-pentanoylbenzonitrile reductase mutant catalyst, characterized in that: The recombinant 2-pentanoylbenzonitrile reductase mutant catalyst is any one of the following forms: (1) culturing the recombinant expression transformant according to claim 4, and isolating transformant cells containing the 2-pentanoylbenzonitrile reductase mutant; (2) culturing the recombinant expression transformant according to claim 4, and isolating a crude enzyme solution containing the 2-pentanoylbenzonitrile reductase mutant; (3) Drying the crude enzyme liquid of the 2-pentanoylbenzonitrile reductase mutant to obtain crude enzyme powder.

6. Use of the 2-pentanoylbenzonitrile reductase mutant according to claim 1 or the recombinant 2-pentanoylbenzonitrile reductase mutant catalyst according to claim 5 in the asymmetric reduction of 2-pentanoylbenzonitrile compounds.

7. The use according to claim 6, characterized in that The 2-pentanoylbenzonitrile reductase mutant or recombinant 2-pentanoylbenzonitrile reductase mutant catalyst is used in the process of asymmetric reduction of 2-pentanoylbenzonitrile compound in the presence of glucose dehydrogenase, glucose; coenzyme NADPH or NADP + If present.

8. The use according to claim 7, characterized in that The amount of glucose dehydrogenase used is 50 to 200 U / mmol of 2-valerylbenzonitrile compound, the amount of glucose used is 1.5 times the molar amount of 2-valerylbenzonitrile compound, the coenzyme NADPH or NADP + The dosage is 0.1~0.5 mmol / L.

9. The use according to claim 6, characterized in that The concentration of the 2-pentanoylbenzonitrile compound is 2 to 20 mmol / L, and the dosage of the 2-pentanoylbenzonitrile reductase mutant or the recombinant 2-pentanoylbenzonitrile reductase mutant catalyst is 5 to 20 U / mmol of the 2-pentanoylbenzonitrile compound.

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