Carboxylesterase mutants and their use in the production of (r)-3-cyclohexene-1-carboxylic acid

Abstract

The application discloses a carboxyesterase mutant and application thereof in production of optically pure (R)-3-cyclohexene-1-carboxylic acid, wherein the carboxyesterase EstA1 mutant is obtained by single-point mutation or combined mutation of positions 70, 128, 133, 227, 248, 249, 297, 301, 326 or 329 of the amino acid sequence shown in SEQ ID No. 1. The application provides the carboxyesterase EstA1 mutant with high catalytic activity and high stereoselectivity to racemic (R,S)-3-cyclohexene-1-carboxylic acid methyl ester, and the obtained mutant has the characteristics of mild reaction condition, high enantiomer selectivity, short reaction time, small amount of catalyst, simple reaction system and environmental friendliness in the process of enantioselective resolution of (R)-3-cyclohexene-1-carboxylic acid.

Description

(I)TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a carboxylesterase mutant and application thereof in selective resolution of (R,S)-3-cyclohexen-1-methyl carbonate to produce (R)-3-cyclohexen-1-methyl carbonate. (II)BACKGROUND

[0002] (R)-3-cyclohexen-1-methyl carbonate is an important pharmaceutical intermediate, which is applied to the synthesis of edoxaban, oseltamivir phosphate and the like. The optical pure (R)-3-cyclohexen-1-methyl carbonate is usually prepared by using chiral resolution reagents for multiple crystallization, and the process has many steps and is complicated, and the total yield is low. The biological catalysis method has high catalytic efficiency, good selectivity and mild reaction conditions, and has become an important chiral synthesis technology in the pharmaceutical industry.

[0003] Carboxylesterase (CE) belongs to the esterase family, and can efficiently hydrolyze carboxylic acid esters, amides, thioesters and other compounds with diverse structures. In addition to catalyzing the hydrolysis reaction, carboxylesterase can also catalyze esterification and transesterification reactions. Carboxylesterase exists in almost all organisms and has a very wide source, and has become one of the most commonly used industrial enzymes in organic synthesis and industrial processes. However, the natural wild type (WT) carboxylesterase often has poor stereoselectivity and low catalytic activity, which hinders its industrial application. Therefore, it is of great research significance and industrial value to strengthen the catalytic performance of carboxylesterase by enzyme engineering technology and develop high-activity and high-selectivity carboxylesterase for catalytic preparation of optical pure (R)-3-cyclohexen-1-methyl carbonate. (III)SUMMARY

[0004] The application aims to provide a carboxylesterase EstA1 mutant, a coding gene, a recombinant vector carrying the coding gene, a recombinant genetically engineered bacterium obtained by transformation of the recombinant vector, and application in preparation of (R)-3-cyclohexen-1-methyl carbonate. The carboxylesterase EstA1 mutant provided by the application has excellent catalytic activity and enantiomer selectivity, and a green synthesis process of (R)-3-cyclohexen-1-methyl carbonate with mild reaction conditions, low production cost and environmental friendliness is constructed.

[0005] The technical scheme adopted by the application is:

[0006] The present application provides a carboxylesterase EstA1 mutant capable of selectively resolving (R,S)-3-cyclohexene-1-methyl carboxylate to prepare a single configuration (R)-3-cyclohexene-1-methyl carboxylate, wherein the carboxylesterase EstA1 mutant is obtained by single-point mutation or combined mutation of positions 70, 128, 133, 227, 248, 249, 297, 301, 326 or 329 of the amino acid sequence shown in SEQ ID No. 1.

[0007] Further, preferably, the carboxylesterase EstA1 mutant is one in which the amino acid sequence shown in SEQ ID No. 1 is mutated to one of the following: (1) valine at position 128 is mutated to glycine or tyrosine, V128G, V128Y; (2) alanine at position 70 is mutated to tyrosine, A70Y; (3) glycine at position 133 is mutated to leucine, G133L; (4) alanine at position 227 is mutated to arginine, A227R; (5) phenylalanine at position 248 is mutated to cysteine, glycine, F248C, F248G; (6) proline at position 326 is mutated to glutamic acid, P326E; (7) leucine at position 329 is mutated to cysteine, L329C; (8) phenylalanine at position 248 is mutated to cysteine, and valine at position 128 is mutated to glycine, F248C-V128G; (9) phenylalanine at position 248 is mutated to glycine, and alanine at position 70 is mutated to tyrosine, F248G-A70Y; (10) phenylalanine at position 248 is mutated to cysteine, and alanine at position 249 is mutated to cysteine, F248C-A249C; (11) phenylalanine at position 248 is mutated to cysteine, and alanine at position 301 is mutated to tyrosine, F248C-A301Y; (12) phenylalanine at position 248 is mutated to cysteine, alanine at position 249 is mutated to cysteine, and valine at position 128 is mutated to glycine, F248C-A249C-V128G; (13) phenylalanine at position 248 is mutated to cysteine, alanine at position 249 is mutated to cysteine, and alanine at position 227 is mutated to arginine, F248C-A249C-A227R; (14) phenylalanine at position 248 is mutated to cysteine, alanine at position 249 is mutated to cysteine, and leucine at position 329 is mutated to cysteine, F248C-A249C-L329C; (15) phenylalanine at position 248 is mutated to cysteine, alanine at position 249 is mutated to cysteine, valine at position 128 is mutated to glycine, and alanine at position 301 is mutated to tyrosine, F248C-A249C-V128G-A301Y; (16) phenylalanine at position 248 is mutated to cysteine, alanine at position 249 is mutated to cysteine, valine at position 128 is mutated to glycine, and glycine at position 133 is mutated to leucine, F248C-A249C-V128G-G133L.

[0008] Due to the specificity of the amino acid sequence, any peptide protein fragment or mutant thereof, such as its conservative mutant, bioactive fragment or derivative, containing the amino acid sequence described in the present application, as long as the peptide protein fragment or peptide protein mutant has more than 90% homology with the aforementioned amino acid sequence, belongs to the protection scope of the present application. Specifically, the changes include deletion, insertion or substitution of amino acids in the amino acid sequence; wherein for the conservative changes of the mutant, the substituted amino acid has similar structure or chemical properties to the original amino acid, such as substitution of leucine for isoleucine, and the mutant can also have non-conservative changes, such as substitution of tryptophan for glycine.

[0009] The present application provides a coding gene of the carboxyesterase EstA1 mutant.

[0010] Due to the specificity of the nucleotide sequence, any mutant of the polynucleotide shown in the present application, as long as it has more than 90% homology with the aforementioned polynucleotide, belongs to the protection scope of the present application. The mutant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide changes. The mutant of the polynucleotide can be a living or non-living variant, including substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution form of a polynucleotide, which can be a substitution, deletion or insertion of a polynucleotide, but will not substantially change the function of the encoded peptide protein.

[0011] The present application also provides a recombinant vector containing the coding gene of the carboxyesterase mutant EstA1 and an engineered bacterium constructed by the recombinant vector, wherein the base vector of the recombinant vector is pET28a(+) and the host bacterium of the engineered bacterium is E. coli BL21(DE3). The obtained recombinant gene engineered bacterium is induced and cultured, the culture solution is separated to obtain bacterium cell containing the recombinant esterase mutant, and the bacterium cell is separated to obtain the crude enzyme solution of the esterase mutant. The mutant esterase is compared with the wild esterase in catalytic activity, and a mutant with excellent catalytic performance is screened.

[0012] The present application relates to the application of the carboxyesterase EstA1 mutant in the preparation of (R)-3-cyclohexene-1-carboxylic acid from (R,S)-3-cyclohexene-1-carboxylic acid methyl ester, and specifically the application is as follows: the wet bacterium cell obtained by fermentation culture of the engineered bacterium containing the coding gene of the carboxyesterase EstA1 mutant or the crude enzyme solution extracted after ultrasonic crushing of the wet bacterium cell or the pure enzyme solution after purification is used as a catalyst, (R,S)-3-cyclohexene-1-carboxylic acid methyl ester is used as a substrate, a buffer solution with pH 6-10 is used as a reaction medium to form a reaction system, the reaction is carried out under the conditions of 20-60℃ (preferably 50℃) and 800 rpm, and after the reaction is completed, the reaction solution is extracted with ethyl acetate to obtain (R)-3-cyclohexene-1-carboxylic acid.

[0013] Preferably, the amount of catalyst in the reaction system is 0.1-5 g / L (preferably 1 g / L) based on the weight of the wet bacteria, the amount of crude enzyme solution is 1-10 mg / mL (preferably 2 mg / mL) based on the protein content, the amount of pure enzyme solution is 1-5 mg / mL (preferably 1 mg / mL) based on the protein content, and the initial concentration of the substrate is 100-700 g / L (preferably 700 g / L).

[0014] Preferably, the buffer is a phosphate buffer (PB) with a pH of 8.0 and a concentration of 100 mM.

[0015] The wet bacteria obtained by fermenting the engineered bacteria containing the gene encoding the carboxylic esterase EstA1 mutant are prepared as follows: the engineered bacteria containing the gene encoding the carboxylic esterase EstA1 mutant are inoculated into LB liquid medium containing kanamycin with a final concentration of 50 μg / mL, and cultured at 37°C and 200 rpm for 6 h; then the bacteria are inoculated into fresh LB liquid medium containing kanamycin with a final concentration of 50 μg / mL at a volume concentration of 2%, and cultured at 37°C and 200 rpm until the OD 600 After reaching 0.6-0.8, IPTG with a final concentration of 0.1 mM is added, and the bacteria are cultured at 16°C and 200 rpm for 10 h, then centrifuged at 8000 rpm and 4°C for 4 min, and the supernatant is discarded to obtain the wet bacteria.

[0016] The crude enzyme solution is prepared as follows: the wet bacteria obtained by fermenting the engineered bacteria containing the gene encoding the carboxylic esterase EstA1 mutant are resuspended in a phosphate buffer (PB) with a pH of 8.0 and a concentration of 100 mM, and an ultrasonic disrupter is used for crushing, with a power of 250 W, crushing for 2 s and resting for 3 s, for 15 min, to obtain the crude enzyme solution; and the volume of the buffer is 9 mL / g based on the weight of the wet bacteria.

[0017] The pure enzyme solution is prepared as follows: centrifuging the crude enzyme solution at 10000 rpm and 4°C for 10 min, passing the supernatant through a 0.22 μm microporous filter membrane, purifying the filtrate by nickel-NTA affinity chromatography, after loading, washing the column with 20 mM, pH 7.4 PB buffer containing 20 mM imidazole and 500 mM NaCl for 10 column volumes to elute the impurities, then eluting the target protein with 20 mM, pH 7.4 PB buffer containing 200 mM imidazole and 500 mM NaCl for 0.5 column volumes; finally, eluting the target protein with 20 mM, pH 7.4 PB buffer containing 500 mM imidazole and 500 mM NaCl until the eluate has the same absorbance value at 595 nm as the blank control sample, which is considered to be fully eluted from the chromatography column, collecting the eluate containing the target protein, centrifuging the eluate at 4°C and 3500 rpm for 15 min, and repeating the centrifugation three times with pH 7.4, 20 mM PB buffer, and collecting the pure enzyme solution.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The present application provides a carboxyesterase EstA1 mutant with high catalytic activity and high stereoselectivity for racemic (R,S)-3-cyclohexene-1-methyl carboxylate, and the obtained mutant is used for chiral resolution to produce (R)-3-cyclohexene-1-methyl carboxylate, which has the characteristics of mild reaction conditions, high enantiomer selectivity, short reaction time, less catalyst consumption, simplified reaction steps, and environmental friendliness. 1 g / L of the mutant EstA1-F248C-A249C-V128G-G133L can catalyze 700 g / L of (R,S)-3-cyclohexene-1-methyl carboxylate, and the conversion rate reaches 49.97% within 4 h, the enantioselectivity is strict, and the ee value of the product is >99%, which is significantly better than the catalytic performance of the carboxyesterases reported so far. (V) DESCRIPTION OF DRAWINGS

[0020] Figure 1 , SDS-PAGE gel electrophoresis results of the separation and purification process of E. coli BL21 (DE3) / EstA1, lane 1 is a protein standard, lane 2 is the precipitate of cell lysate, lane 3 is the supernatant of cell lysate, lane 4 is the flow-through of nickel-NTA affinity chromatography, and lane 5 is the pure enzyme solution.

[0021] Figure 2 , gas chromatogram of the reaction solution obtained by the mutant EstA1-F248C-A249C-V128G-G133L wet bacteria body catalysis in Example 6. (V) SPECIFIC EMBODIMENTS

[0022] The application will be further described in conjunction with specific examples, but the scope of protection of the application is not limited thereto: the terms used in the application generally have the meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

[0023] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0024] Example 1, cloning of wild-type carboxyesterase EstA1

[0025] Using ancestral sequence reconstruction (ASR), the nucleotide sequence of cyclohexene carboxylate esterase from Acinetobacter sp. WCHAc010052 (GenBank accession number: AXY61874.1) was selected as a probe, and a carboxyesterase ancestral enzyme (amino acid sequence as shown in SEQ ID No. 2) was screened by FireProt-ASR online tool (https: / / loschmidt.chemi.muni.cz / fireprotasr / ), and its coding gene (nucleotide sequence as shown in SEQ ID No. 1) was obtained by codon optimization, and the full-length sequence was artificially synthesized by a gene synthesis company, and was transferred into the vector pET28a to construct the recombinant plasmid pET28a-EstA1 and the recombinant genetically engineered bacteria E. coli BL21 (DE3) / EstA1.

[0026] Example 2, random mutation of wild-type carboxyesterase EstA1

[0027] Using the recombinant plasmid pET28a-EstA1 in Example 1 as a template, random base mutations were introduced into the carboxyesterase EstA1 gene sequence by error-prone PCR technology, wherein the primers used are as follows:

[0028] Upstream primer:

[0029] 5'-CAGCAAATGGGTCGCGGATCCATGCCTAGTGCCGCAATGG-3'

[0030] Downstream primer:

[0031] 5'-GTGGTGGTGGTGGTGCTCGAGTTATGCTGCAGCCGGTGC-3'

[0032] The system of the PCR reaction is as follows: 0.5 ng of template, 1.5 μL of upstream and downstream primers (10 μM) each, 8.75 μL of MnCl2 (1 mM), 25 μL of 2x Taq mix, and adding sterile distilled water to a total volume of 50 μL.

[0033] The procedure of error-prone PCR amplification is as follows: (1) denaturation at 95 °C for 3 min, (2) denaturation at 95 °C for 30 s, (3) annealing at 55 °C for 30 s, (4) extension at 72 °C for 1 min, steps (2)-(4) for 30 cycles, and finally extension at 72 °C for 10 min, and preservation at 16 °C.

[0034] After purification of the PCR amplification product, the PCR amplification product and the vector plasmid pET-28a were double-digested with restriction endonucleases BamH I and Xho I at 37 °C for 12 h, the digested products were recovered, and T4 DNA ligase was used for ligation at 16 °C overnight, then the E. coli BL21 (DE3) competent cells were transformed, and uniformly coated on LB agar medium plates containing 50 μg / mL kanamycin, and cultured at 37 °C overnight to construct the random mutant library. Single clones were selected and cultured in a deep well plate, each well of the primary plate contained 300 μL of LB medium (containing 50 μg / mL kanamycin), and cultured at 37 °C, 220 rpm overnight, 50 μL of the primary seed liquid was transferred to each well of the secondary plate containing 600 μL of LB medium (containing 50 μg / mL kanamycin), and cultured at 37 °C, 220 rpm for 3 h, when the OD 600 was about 1.0, 0.2 mM IPTG was added for induction, and the culture was further incubated at 16 °C for 24 h, then centrifuged at 3500 rpm for 10 min, the supernatant was discarded, 200 μL of lysis solution (containing 750 mg / L lysozyme and 12 mg / L DNase) was added to each well, the cells were fully suspended by shaking, and incubated at 37 °C for 1 h, then centrifuged at 3500 rpm for 10 min again, and the supernatant was collected to obtain the crude enzyme solution of the mutant library.

[0035] Example 3, screening of carboxyesterase EstA1 mutants

[0036] 50 μL of the supernatant in each well of the mutant plate constructed in Example 2 was taken, 1 mL of Tris-HCl buffer (100 mM, pH 8.0) containing 20 mM (R,S)-3-cyclohexene-1-carboxylic acid methyl ester was added, and the reaction was carried out at 30 °C, 800 rpm for 1 h, then an equal amount of ethyl acetate was used to extract the product, and the upper ethyl acetate phase was directly subjected to gas chromatography (GC) detection of the characteristic peak area of the substrate and the product (R)-3-cyclohexene-1-carboxylic acid, the concentration of the substrate and the product was calculated according to the standard curve of the concentration and the peak area of the standard sample, and then the substrate conversion rate, the enantiomeric excess value ee p (%) of the target product, and the enantiomeric excess value ee s (%) of the substrate were calculated.

[0037] The calculation formula of the enantiomeric excess value (%) is as follows:

[0038]

[0039] S, P represent substrate and product, respectively; S s , S R represents the concentration (g / L) of substrate (S)-3-cyclohexen-1-carboxylic acid methyl ester and (R)-3-cyclohexen-1-carboxylic acid methyl ester; P s , P R represents the concentration (g / L) of product (S)-3-cyclohexen-1-carboxylic acid and (R)-3-cyclohexen-1-carboxylic acid, respectively.

[0040] Gas chromatography (GC) analysis: analysis was performed using a gas chromatograph 7890B with a B-DM gas chromatography column, split ratio 25:1, column flow 2 mL / min, tail gas 25 mL / min; the injection port and detector were set at 280℃, initial temperature 50℃ for 5 min; then increased at 2℃ / min to 100℃, and then at 5℃ / min to 150℃ for 10 min.

[0041] Enzyme activity definition: the amount of enzyme required to produce 1 μmol 3-cyclohexen-1-carboxylic acid per minute is defined as 1 U. The specific activity of wild-type EstA1 is defined as 100%, and the specific activity of the dominant mutant is compared with that of the wild type to obtain the relative activity of the mutant.

[0042] The catalytic performance of the constructed dominant mutants A70Y, V128G, G133L, A227R, F248C, A249C, L329C, F248C-V128G, F248G-A70Y, F248C-A249C-V128G, F248C-A249C-A227R, F248C-A249C-L329C, F248C-A249C-V128G-A301Y, F248C-A249C-V128G-G133L is shown in Table 1, and the optimal four mutants are EstA1-F248C-A249C-V128G-G133L, with a relative activity of 237.53% of the wild type (EstA1-WT). When the substrate conversion rate is 49.97%, the ee p value of the product (R)-3-cyclohexen-1-carboxylic acid is >99%.

[0043] Table 1. Screening results of mutants

[0044]

[0045]

[0046] Example 4. Preparation of carboxyesterase mutant wet cells

[0047] The wild-type carboxyesterase EstA1 of Example 1, and the recombinant genetically engineered bacterial strain of the advantage mutant constructed in Example 3 were streaked on LB agar medium plates containing 50 μg / mL kanamycin and incubated at 37°C for 12 h; single colonies grown on the medium were picked and inoculated into 10 mL of LB test tube medium containing 50 μg / mL kanamycin, and incubated at 200 rpm and 37°C for 6 h. 1 mL was inoculated into 50 mL of TB medium containing 50 μg / mL kanamycin at a final concentration, and incubated at 37°C and 200 rpm for 2 h; then 0.1 mM of inducer IPTG was added at a final concentration, and induced at 16°C and 200 rpm for 12 h; after the induction was completed, the wet bacterial cells were obtained by centrifugation at 4°C and 8000 rpm.

[0048] The composition of the TB medium is: peptone 12 g / L, yeast powder 24 g / L, KH2PO4 2.3 g / L, K2HPO4 12.54 g / L, glycerol 5 g / L, and water as the solvent.

[0049] Example 5, Isolation and purification of carboxyesterase mutants

[0050] The wet bacterial cells obtained by the method of Example 4 were resuspended in 4.5 mL of pH 8.0, 100 mM PB buffer at a ratio of 0.5 g per 4.5 mL, and broken using an ultrasonic disrupter at a power of 250 W, 2 s of breaking and 3 s of interval, for 15 min, to obtain a cell lysate. The cell lysate was centrifuged at 10000 rpm and 4°C for 10 min, and the supernatant (crude enzyme solution) was collected. The supernatant was microfiltered through a 0.22 μm microporous filter membrane, and the permeate obtained was stored at 4°C. Nickel-NTA affinity chromatography was used for separation and purification of the target protein, and the following reagents were prepared using 20 mM, pH 7.4 PB buffer containing 500 mM NaCl unless otherwise specified, and the operation steps were as follows:

[0051] a. Open the upper and lower covers of the gravity column, add an extension hose to the sample outlet to speed up the flow rate, and let the 20% ethanol in it flow out as much as possible, and then use 20 mM, pH 7.4 PB buffer containing 20 mM imidazole and 500 mM NaCl to rinse 5 column volumes (50 mL), and then load the sample after rinsing.

[0052] b. Elute the unbound impurities with 20 mM, pH 7.4 PB buffer containing 20 mM imidazole and 500 mM NaCl for 10 column volumes (100 mL), and then elute the target enzyme protein with 20 mM, pH 7.4 PB buffer containing 200 mM imidazole and 500 mM NaCl for 0.5 column volumes (5 mL); after elution, use 20 mM, pH 7.4 PB buffer containing 500 mM imidazole and 500 mM NaCl to elute the target enzyme protein until the absorbance value at 595 nm of the eluate is the same as that of the blank control sample, which is considered to have washed the column clean, and collect the eluate containing the target protein.

[0053] c. Try to make the imidazole flow as much as possible, and then wash with 2 column volumes (20 mL) of 20% (v / v) aqueous ethanol solution, and finally fill with 20% (v / v) aqueous ethanol solution, cover, and store the nickel-NTA affinity chromatography at 4°C.

[0054] d. Use an ultrafiltration tube to replace and concentrate the 5 mL eluate collected in step b containing the target protein. Centrifuge at 4°C and 3500 rpm for 15 min, discard the waste liquid; make up with 20 mM, pH 7.4 PB buffer and mix well with a pipette, continue to centrifuge at 4°C and 3500 rpm for 15 min, and repeat the replacement for 3 times. Finally, concentrate to 1 mL to obtain the pure enzyme solution, add 50% glycerol, and store in a -80°C ultra-low temperature refrigerator.

[0055] During the preparation of the pure enzyme solution, the supernatant of the cell lysate, the precipitate part, the flow-through collected after loading, and the pure enzyme solution were subjected to SDS-PAGE gel electrophoresis, respectively. EstA1-WT is shown in Figure 1 .

[0056] Example 6, Optimum pH determination of EstA1-WT and mutant F248C-A249C-V128G-G133L

[0057] Replace the buffer with 100 mM PB (pH 6.0, 7.0, 8.0), Tris-HCl (pH 8.0, 9.0), and Gly-NaOH (pH 9.0, 10.0), and use the pure enzyme solution of EstA1-WT and mutant F248C-A249C-V128G-G133L prepared in Example 5 to determine the enzyme activity of EstA1-WT and mutant F248C-A249C-V128G-G133L at each pH according to the method of Example 3. The relative enzyme activity at other pH is calculated based on the enzyme activity at the optimum pH as 100%, as shown in Table 2. EstA1-WT and the mutant both exhibit the best activity in PB buffer at pH 8.0.

[0058] Table 2 Effect of pH on the activity of EstAl-WT and mutant enzymes

[0059]

[0060]

[0061] a The relative activity of EstAl-WT was calculated based on the highest specific activity of EstAl-WT (pH 8.0 PB). b The relative activity of EstAl-F248C-A249C-V128G-G133L was calculated based on the highest specific activity of EstAl-F248C-A249C-V128G-G133L (pH 8.0 PB).

[0062] Example 7 Determination of the optimal reaction temperature of EstAl-WT and mutant F248C-A249C-V128G-G133L

[0063] The pure enzyme solutions of EstAl-WT and mutant F248C-A249C-V128G-G133L were prepared according to the method of Example 5, and the enzyme activity at different temperatures (20°C, 30°C, 40°C, 50°C, 60°C) was determined in the optimal buffer system (pH 8.0, 100 mM PB buffer) obtained in Example 6 using the method of Example 3. As shown in Table 3, both the wild type and the mutant exhibited the best activity at 50°C.

[0064] Table 3 Effect of temperature on the activity of EstAl-WT and mutant enzymes

[0065]

[0066] a The relative activity of EstAl-WT was calculated based on the highest specific activity of EstAl-WT (50°C).

[0067] b The relative activity of EstAl-F248C-A249C-V128G-G133L was calculated based on the highest specific activity of EstAl-F248C-A249C-V128G-G133L (50°C).

[0068] Example 8 Determination of the substrate tolerance of EstAl-WT and mutant F248C-A249C-V128G-G133L

[0069] The EstAl-WT and mutant F248C-A249C-V128G-G133L wet cell bodies prepared by the method of Example 4 were used as catalysts, and the concentration was 0.5-1 g / L, (R,S)-3-cyclohexen-1-methyl carboxylate was added to a final concentration of 100 g / L, 200 g / L, 300 g / L, 500 g / L, 700 g / L, and PB buffer (100 mM, pH 8.0) was used as the reaction medium to form a 1 mL reaction system, and the reaction was carried out at 50°C, 800 rpm for 1 h. The conversion rate and selectivity were determined by the method of Example 3, and the results are shown in Table 4.

[0070] The concentration of the substrate (R,S)-3-cyclohexen-1-methyl carboxylate was 100 g / L, and equal amounts of EstAl-WT and F248C-A249C-V128G-G133L were added. When the substrate conversion rate was close to 50%, the ee of the product after 6 h of reaction of EstAl-WT was 81%, while the substrate conversion rate of F248C-A249C-V128G-G133L was 49.9% after 2 h of reaction, and the ee of the product was >99%. Therefore, the mutant F248C-A249C-V128G-G133L has better substrate conversion ability. p p >99%, and the required reaction time was significantly shortened. Therefore, the mutant F248C-A249C-V128G-G133L has better substrate conversion ability.

[0071] In addition, as the substrate concentration increases, EstAl-WT requires more time to reach a conversion rate close to 50% when catalyzing the same concentration of substrate, while F248C-A249C-V128G-G133L requires significantly less enzyme and reaction time to reach a conversion rate close to 50% when catalyzing the same concentration of substrate, showing potential for industrial application.

[0072] Table 4 Reaction of EstAl-WT and mutant enzymes under different substrate concentrations

[0073]

[0074] Example 9, Enzymatic preparation of (R)-3-cyclohexen-1-methyl carboxylate by mutant F248C-A249C-V128G-G133L

[0075] ​A reaction mixture containing 100 mL PB buffer (pH 8.0, 200 mM), 1 g / L mutant F248C-A249C-V128G-G133L wet cell paste prepared according to the method of Example 4, 700 g / L (R,S)-3-cyclohexen-1 -methylate (70 g) was placed in a three-necked flask and subjected to magnetic stirring at 50 °C and 800 rpm. The reaction was titrated with 1.0 M Na2CO3 to maintain the pH of the reaction solution at 8.0. Samples were taken at intervals and analyzed immediately for conversion and selectivity according to the method of Example 3. After 4 h, the conversion of the substrate was nearly 50%, and the results of gas chromatographic analysis of the reaction solution are shown in Table 1. Figure 2 The reaction solution was extracted with an equal volume of ethyl acetate three times, the organic layers were combined, dried over anhydrous Na2SO4, filtered, and rotary evaporated to dryness to give 33.6 g of an oily product with a special odor, i.e., (R)-3-cyclohexen-1 -methylate, in a yield of 45% and an ee value of 99%. p Table 1

Claims

1. A mutant of the carboxyesterase EstAl that selectively resolves the preparation of a single configuration (R)-3-cyclohexene-1-carboxylic acid methyl ester, characterized in that, R -3-cyclohexene-1-carboxylic acid methyl ester, characterized in that, The carboxylesterase EstA1 mutant is the amino acid sequence shown in SEQ ID No. 2 is mutated into one of the following: (1) the valine at position 128 is mutated into glycine or tyrosine, V128G, V128Y; (2) the glycine at position 133 is mutated into leucine, G133L; (3) the alanine at position 227 is mutated into arginine, A227R; (4) the phenylalanine at position 248 is mutated into cysteine, glycine, F248C, F248G; (5) the proline at position 326 is mutated into glutamic acid, P326E; (6) the leucine at position 329 is mutated into cysteine, L329C; (7) the phenylalanine at position 248 is mutated into cysteine, and the valine at position 128 is mutated into glycine, F248C-V128G; (8) the phenylalanine at position 248 is mutated into glycine, and the alanine at position 70 is mutated into tyrosine, F248G-A70Y; (9) the phenylalanine at position 248 is mutated into cysteine, and the alanine at position 249 is mutated into cysteine, F248C-A249C; (10) the phenylalanine at position 248 is mutated into cysteine, and the alanine at position 301 is mutated into tyrosine, F248C-A301Y; (11) the phenylalanine at position 248 is mutated into cysteine, and the alanine at position 249 is mutated into cysteine, and the valine at position 128 is mutated into glycine, F248C-A249C-V128G; (12) the phenylalanine at position 248 is mutated into cysteine, and the alanine at position 249 is mutated into cysteine, and the alanine at position 227 is mutated into arginine, F248C-A249C-A227R; (13) the phenylalanine at position 248 is mutated into cysteine, and the alanine at position 249 is mutated into cysteine, and the leucine at position 329 is mutated into cysteine, F248C-A249C-L329C; (14) the phenylalanine at position 248 is mutated into cysteine, and the alanine at position 249 is mutated into cysteine, and the valine at position 128 is mutated into glycine, and the alanine at position 301 is mutated into tyrosine, F248C-A249C-V128G-A301Y; (15) the phenylalanine at position 248 is mutated into cysteine, and the alanine at position 249 is mutated into cysteine, and the valine at position 128 is mutated into glycine, and the glycine at position 133 is mutated into leucine, F248C-A249C-V128G-G133L.

2. An engineered bacterium containing the carboxylesterase mutant EstA1 coding gene of claim 1.

3. Use of a mutant of the carboxyesterase EstAl according to claim 1 for the resolution of (S)-3-cyclohexene-1-carboxylic acid methyl ester to produce optically pure (S)-3-cyclohexene-1-carboxylic acid. R , S R (S)-3-cyclohexene-1-carboxylic acid.​ 4. Use according to claim 3, wherein the compound is ###0002### The application is: using wet bacteria obtained by fermentation culture of engineered bacteria containing carboxylesterase EstA1 mutant encoding gene or crude enzyme liquid extracted after ultrasonic crushing of wet bacteria or purified pure enzyme liquid as catalyst, R , S )-3-cyclohexene-1-carboxylic acid methyl ester was used as substrate and the reaction mixture was stirred at pH 6. 10% buffer solution was used as the reaction medium to form the reaction system. The reaction was carried out at 60 °C and 800 rpm. After the reaction was complete, the reaction solution was extracted with ethyl acetate to obtain ( R )-3-cyclohexene-1-carboxylic acid.

5. The use according to claim 4, wherein the compound is ###0002### The catalyst is used in an amount of 0.1 5 g / L, and the crude enzyme solution is added in an amount of 1 10 mg / mL; and the pure enzyme solution is added in an amount of 1 5 mg / mL; the initial concentration of the substrate is 100 700 g / L.

6. The use according to claim 4, wherein the compound is ###0002### The buffer is a phosphate buffer of pH 8.0, 100 mM.

7. The use according to claim 4, wherein the compound is ###0002### The wet bacteria are prepared as follows: the engineering bacteria containing the gene encoding the carboxylesterase EstA1 mutant are inoculated into LB liquid medium containing kanamycin with a final concentration of 50 μg / mL, and cultured at 37 ℃ and 200 rpm for 6 h; then the bacteria are inoculated into fresh LB liquid medium containing kanamycin with a final concentration of 50 μg / mL at a volume concentration of 2%, and cultured at 37 ℃ and 200 rpm until the OD 600 0.6 After 0.8, IPTG with a final concentration of 0.1 mM is added, and the bacteria are cultured at 16 ℃ and 200 rpm for 10 h, then centrifuged at 8000 rpm and 4 ℃ for 4 min, the supernatant is discarded, and the wet bacteria are obtained.

8. The use according to claim 4, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The preparation method of the crude enzyme solution: the wet bacteria obtained by fermentation of the engineered bacteria containing the mutant gene of carboxylesterase EstA1 are resuspended in 100 mM PB buffer solution with pH 8.0, and are broken by using an ultrasonic disrupter with a power of 250 W, a breaking time of 2 s and a gap of 3 s, and a duration of 15 min, to obtain a cell lysate, i.e. a crude enzyme solution; the volume of the buffer solution is 9 mL / g based on the weight of the wet bacteria.

9. The use according to claim 4, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The pure enzyme solution is prepared as follows: the cell lysate is centrifuged at 10000 rpm and 4 DEG C for 10 min, the supernatant is micro-filtered through a 0.22 mu m micro-pore filter membrane, the permeate is loaded into a nickel-NTA affinity chromatography, the impure proteins are eluted by using 20 mM PB buffer solution containing 20 mM imidazole and 500 mM NaCl with a volume of 10 column volumes, and then the target proteins are eluted by using 20 mM PB buffer solution containing 200 mM imidazole and 500 mM NaCl with a volume of 0.5 column volumes; finally, the target proteins are eluted by using 20 mM PB buffer solution containing 500 mM imidazole and 500 mM NaCl with pH 7.4, and the eluent containing the target proteins is collected, centrifuged at 3500 rpm and 4 DEG C for 15 min by using an ultrafiltration tube, and then the precipitate is dissolved in 20 mM PB buffer solution with pH 7.4, and the operation of centrifugation is repeated for 3 times, and then the concentrated solution is obtained, to obtain a pure enzyme solution.

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