p - Nitrobenzyl Esterase Mutant, Encoding Gene and Application

Abstract

The present invention discloses a p-nitrobenzyl esterase mutant and its encoding gene, a genetically engineered bacterium containing the p-nitrobenzyl esterase mutant, and its application in the chiral resolution of dl-menthyl esters by enzymatic catalysis to prepare l-menthol. The p-nitrobenzyl esterase mutant is obtained by combinatorial mutation of the amino acid residues at positions 109, 110, 193, 270, 273, 314 or 362 of the p-nitrobenzyl esterase with the amino acid sequence shown in SEQ ID NO.2. The beneficial effects of the present invention are mainly reflected in: ① The esterase mutant of the present invention does not need to add a cosolvent, and the enzymatic catalytic activity maintains a relatively high level; ② In the cosolvent-free system, the stereoselectivity of the mutant enzyme-catalyzed reaction is significantly improved compared with the wild type. These mutant enzymes are overexpressed in Escherichia coli and used for catalytic reactions, and dl-menthyl acetate at a concentration of 20-200 g / L is used as a substrate for hydrolysis resolution to prepare l-menthol. The results show that the ee p value of the product l-menthol > 99%, and the substrate conversion rate > 95%, showing good industrial application performance.

Description

(1) Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a p-nitrobenzyl esterase mutant, a coding gene thereof, and an application thereof in the chiral resolution of dl-menthyl ester by enzymatic catalysis to prepare l-menthol. (2) Background Art

[0002] L-menthol is a common substance used in the fields of essence and fragrance, cosmetics, food and medicine, etc. It has functions such as excitement, bactericidal analgesia, itching relief, aromatic coolness, etc., and has a unique mint aroma and cool effect. The market demand for l-menthol is large every year, and the global l-menthol market is expected to reach 1.4 billion US dollars in 2025.

[0003] At present, the main suppliers in the l-menthol industry include Agson Global, Symrise, Nantong Mint Factory, Takasago Perfumery, Tianyin Chemical Industry, Arora Aromatics, Anhui Fengle Perfumery, Swati Menthol&Allied Chem, NecLife, Bhagat Aromatics, KM Chemicals, Silverline Chemicals, Anhui Yinfeng Pharmaceutical, Jubang Perfumery, Xiangsheng Perfumery (Huai'an) Co., Ltd., BASF and other enterprises. The l-menthol on the market is mainly divided into natural l-menthol and synthetic l-menthol. With the rapid development of biotechnology, its green, environmentally friendly and high-efficiency low-cost production method has gradually attracted people's attention. Among them, the green synthesis technology of catalytic synthesis of l-menthol by bioenzymes has been continuously explored and improved. Bioenzymes have high catalytic efficiency, high selectivity, mild reaction conditions, low energy consumption and non-toxicity, which conform to the green development direction. Therefore, the biological preparation process based on high-performance bioenzymes has important application value.

[0004] There is currently a p-nitrobenzyl esterase derived from Bacillus subtilis, which has advantages such as fast reaction rate and large substrate feeding amount in the chiral resolution of dl-menthyl acetate to prepare l-menthol. However, its catalytic stereoselectivity is not high, and cosolvents such as n-butanol need to be added to improve the catalytic rate and catalytic stereoselectivity of the enzyme. Therefore, it is necessary to carry out directed modification on this esterase to improve its application performance. (3) Summary of the Invention

[0005] The object of the present invention is to provide a p-nitrobenzyl esterase mutant with excellent catalytic performance, a coding gene thereof, and an application thereof in the chiral resolution of dl-menthyl ester by enzymatic catalysis to prepare l-menthol.

[0006] The technical solution adopted by the present invention is:

[0007] A p-nitrobenzyl esterase mutant is obtained by combinatorial mutagenesis of two or more amino acid residues at positions 109, 110, 193, 270, 273, 314 or 362 of p-nitrobenzyl esterase with the amino acid sequence shown in SEQ ID NO.2.

[0008] In the present invention, a small and precise combinatorial mutant library, namely the three-codon saturation mutagenesis technique, is constructed at 10 amino acid residue sites at positions 109, 110, 190, 193, 270, 273, 314, 358, 362 and 362, and excellent mutants are obtained by combining multiple rounds of iterative mutagenesis and screening.

[0009] In the present invention, computer-aided analysis software is used to perform docking analysis on wild-type p-nitrobenzyl esterase with the substrates l-menthyl acetate and d-menthyl acetate respectively, and key amino acid residue sites are screened out. The results are as Figure 2 shown, that is, 10 amino acid residues around the active pocket. These amino acid residues have non-bonded interactions with the substrate and simultaneously affect the shape and size of the active pocket. By combinatorial mutagenesis and optimization of these residues, the spatial position and non-bonded interactions of the enzyme-substrate interaction can be optimized, so that the enzyme has stronger selectivity for specific substrates (such as l-menthyl acetate), thereby improving the stereoselectivity of the enzyme for the substrate.

[0010] The above-selected 10 amino acid residues are divided into three groups, namely group A (Y109, L110, F134), group B (A190, M193, M358) and group C (I270, L273, L362, F363), and are arranged as Figure 3The scheme shown constructs three small and refined libraries. At each site, three amino acids are used for substitution mutations, including valine, methionine, and tyrosine, that is, three-amino-acid codon saturation mutations are performed at each site. Through mixed degenerate primers, the PCR amplification method is applied to introduce randomly combined mutations, thereby amplifying the mixed mutant fragments. Then, the mixed fragments are ligated with the expression plasmid, and then, using the heat shock method, the recombinant mixed plasmid is transformed into the competent cell Escherichia coli BL21(DE3) to construct a three-codon saturation mutation library. A certain number of single colonies are picked from the library, transferred to liquid LB medium and induced for protein expression to obtain fermented cells containing different mutant enzymes respectively. Finally, the cells containing the mutant enzyme are reacted with the substrate, and the conversion rate of the substrate and the enantiomeric excess value (ee value) of the product are analyzed by gas chromatography to compare and analyze the enzyme activity and substrate stereoselectivity corresponding to each colony, so as to screen out excellent mutant enzymes in the library. By comparing the mutant strains of the three initially constructed libraries A, B, and C, the optimal mutant is screened, and the construction and screening of iterative mutation libraries of other groups in the next round are carried out. After successive iterations, the number of screened mutant enzymes reaches more than 95% coverage of the three-codon saturation mutation at 10 target sites, thus realizing the combinatorial optimization of the target sites and obtaining the optimal mutant.

[0011] Preferably, the amino acid sequence of the mutant is as shown in any one of SEQ ID NO.3-6. The esterase mutants include the quadruple mutant PNB-Y109M / L110Y / M193Y / F314Y (abbreviated as PNB-MYYY), the quintuple mutant PNB-Y109M / L110Y / M193Y / I270M / F314Y (abbreviated as PNB-MYYMY), the quintuple mutant PNB-Y109M / L110Y / M193Y / I270Y / F314Y (abbreviated as PNB-MYYYY), and the heptuple mutant PNB-Y109M / L110Y / M193Y / I270M / L273V / F314Y / L362V (abbreviated as PNB-MYYMVYV), and the corresponding amino acid sequences are as shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 respectively.

[0012] The amino acid sequence shown in SEQ ID NO.2 is that of wild-type p-nitrobenzyl esterase (PNB), which is derived from Bacillus subtilis CGMCC No.17904 (CN 110373366 A). This enzyme can rapidly perform chiral resolution on dl-menthyl ester to prepare l-menthol. The advantages of its catalytic reaction are its fast hydrolysis rate and high feed amount of the substrate dl-menthyl ester. However, during the catalytic process of this enzyme, an organic co-solvent is required and the stereoselectivity of the enzyme-catalyzed substrate is relatively low. The technical solution of the present invention is to optimize the substrate-binding pocket of wild-type p-nitrobenzyl esterase PNB, that is, to optimize and transform it by combinatorial mutagenesis at 10 sites, which improves the catalytic rate of the enzyme and significantly improves its stereoselectivity for the substrate. The ee value of l-menthol generated by the mutant screened by hydrolyzing dl-menthyl ester is >99%, showing very excellent stereoselectivity.

[0013] The present invention also relates to a gene encoding the p-nitrobenzyl esterase mutant.

[0014] The encoding genes of the esterase mutants PNB-MYYY, PNB-MYYMY, PNB-MYYYY, and PNB-MYYMVYV can be gene sequences synthesized after codon optimization according to their amino acid sequences, or can be obtained by PCR amplification from the genome of B. subtilis CGMCC No.17904 strain followed by mutagenesis and transformation. Preferably, the nucleotide sequence of the encoding gene is as shown in one of SEQ ID NO.7 to 10.

[0015] The present invention also relates to a recombinant vector and a genetically engineered bacterium containing the encoding gene.

[0016] The present invention also relates to the application of the p-nitrobenzyl esterase mutant in the chiral resolution of dl-menthyl ester by enzymatic catalysis to prepare l-menthol.

[0017] Specifically, the application is as follows: constructing a genetically engineered bacterium containing the encoding gene of the mutant, using the wet cells obtained by fermentation culture of the genetically engineered bacterium or the enzyme-containing cells obtained by disrupting the cells as a catalyst, and hydrolyzing dl-menthyl ester to obtain l-menthol.

[0018] Specifically, the genes of the esterase mutants PNB-MYYY, PNB-MYYMY, PNB-MYYYY, and PNB-MYYMVYV can be cloned into an expression plasmid and transformed into a host cell. After induced fermentation to prepare an esterase preparation, it is used to catalyze the selective hydrolysis of dl-menthyl ester to prepare l-menthol. The expression plasmid and host cell are preferably the pET28a plasmid and the Escherichia coli BL21(DE3) host cell, that is, the recombinant bacteria E. coli BL21(DE3)(pET28a-PNB-MYYY, pET28a-PNB-MYYMY, pET28a-PNB-MYYYY, pET28a-PNB-MYYMVYV) are constructed.

[0019] Preferably, the dl-menthyl ester is dl-menthyl acetate. The esterase mutant of the present invention catalyzes the hydrolysis of 1% to 20% of dl-menthyl acetate to prepare l-menthol in a solvent-free system, and the ee p value of the product l-menthol > 99%.

[0020] Preferably, in the hydrolysis reaction system, the cell density is 5 to 30 OD (preferably 20 OD), the dosage of dl-menthyl acetate is 1% to 20% (w / w) (preferably 15%), and the pH during the hydrolysis process is controlled at 6.5 to 8.5 (preferably pH 8.0), and the temperature is 25°C to 40°C (preferably 30°C).

[0021] Preferably, the hydrolysis is carried out under solvent-free conditions.

[0022] The beneficial effects of the present invention are mainly reflected in: ① The esterase mutant of the present invention does not need to add a co-solvent, and the enzyme catalytic activity maintains a relatively high level; ② In a solvent-free system, the stereoselectivity of the mutant enzyme catalysis is significantly improved compared with the wild type, and almost no d-menthol is produced in the catalytic reaction of the mutant enzyme. After the mutant enzyme is overexpressed by Escherichia coli and used for the catalytic reaction, and using 10 to 200 g / L of dl-menthyl acetate as the substrate, hydrolysis resolution is carried out to prepare l-menthol. The results show that the substrate conversion rate of l-menthyl acetate > 95%, and the ee p value of the product l-menthol > 99%, showing good industrial application performance. (IV) Description of the Drawings

[0023] Figure 1 It is a schematic structural diagram of wild-type p-nitrobenzyl esterase.

[0024] Figure 2 It is a schematic diagram of 10 amino acid residue sites around the substrate-binding pocket of wild-type esterase PNB.

[0025] Figure 3 It is a schematic diagram for constructing a small and precise library for triple-codon saturation mutagenesis in groups.

[0026] Figure 4 It is the GC chromatogram of the standard samples of dl-menthyl acetate and dl-menthol.

[0027] Figure 5 It is the GC chromatogram of the catalytic reaction solution for the preparation of l-menthol by the hydrolysis and resolution of dl-menthyl acetate catalyzed by the esterase mutant PNB-MYYY.

[0028] Figure 6 It is the GC chromatogram of the catalytic reaction solution for the preparation of l-menthol by the hydrolysis and resolution of dl-menthyl acetate catalyzed by the esterase mutant PNB-MYYMY.

[0029] Figure 7 It is the GC chromatogram of the catalytic reaction solution for the preparation of l-menthol by the hydrolysis and resolution of dl-menthyl acetate catalyzed by the esterase mutant PNB-MYYYY.

[0030] Figure 8 It is the GC chromatogram of the catalytic reaction solution for the preparation of l-menthol by the hydrolysis and resolution of dl-menthyl acetate catalyzed by the esterase mutant PNB-MYYMVYV.

[0031] Figure 9 It is a schematic diagram of the primary screening results of the mutant library in Group A of the first-generation library.

[0032] Figure 10 It is a schematic diagram of the primary screening results of the mutant library in Group B of the first-generation library.

[0033] Figure 11 It is a schematic diagram of the primary screening results of the mutant library in Group C of the first-generation library.

[0034] Figure 12 It is a schematic diagram of the primary screening results of the B-A group mutant library in the second-generation library.

[0035] Figure 13 It is a schematic diagram of the primary screening results of the B-C group mutant library in the second-generation library.

[0036] Figure 14 It is a schematic diagram of the primary screening results of the third-generation mutant library B-A-C (V) Specific implementation manners

[0037] To deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments. These embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0038] LB medium: yeast powder 5.0 g / L, peptone 10.0 g / L, NaCl 10.0 g / L, and the solvent is distilled water.

[0039] Fermentation medium: yeast powder 12.0 g / L, peptone 15.0 g / L, Na2HPO4·12H2O 8.9 g / L, KH2PO4 3.4 g / L, NH4Cl 2.67 g / L, Na2SO4 0.71 g / L, MgSO4·7H2O 0.49 g / L, kanamycin 50 μg / L, pH 7.0, and the solvent is distilled water.

[0040] pH 8.0 phosphate buffer solution (200 mmol / L): Na2HPO4·12H2O 67.8 g / L, NaH2PO4·2H2O 0.82 g / L, and the solvent is distilled water.

[0041] Example 1: Induced expression of esterase PNB gene

[0042] Induced expression of esterase PNB. The recombinant strain E. coli IEF-PNB containing the recombinant plasmid pET28a-pnbA (refer to CN113201516A) was streaked and cultured overnight from the preserved glycerol tube. Then, a single colony was inoculated into an LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with 200 rpm. It was inoculated into the fermentation medium according to an inoculation amount of 0.5% (v / v), cultured at 37°C for 2 h; IPTG with a final concentration of 1.0 mmol / L was added, the culture temperature was adjusted to 24°C, and fermentation was continued for 8 h to obtain a bacterial agent overexpressing esterase PNB.

[0043] Example 2: Design of esterase PNB mutation sites and construction of a small and precise mutant library

[0044] To improve the substrate stereoselectivity of esterase PNB in a cosolvent-free system, a computer-aided design method was used to select the key mutation sites of PNB. According to the reported crystal structure of esterase from Bacillus subtilis (PDB ID: 1QE3), homologous modeling was performed on esterase PNB from strain B. subtilis CGMCC No. 17904. Using protein three-dimensional structure analysis software and molecular docking software, simulation analysis was carried out. Considering the docking results of the esterase and the substrate, the characteristics of the enzyme's substrate-binding pocket, the structural characteristics of the enzyme's recognition of substrate stereoselectivity, and the catalytic mechanism of the enzyme, a total of 10 amino acid residue sites were finally determined, namely the 109th, 110th, 190th, 193rd, 270th, 273rd, 314th, 358th, 362nd, and 363rd sites of the amino acid sequence SEQ ID NO: 2. The specific positions are as Figure 2 shown (different colors represent different groups, green: Group A; blue: Group B; yellow: Group C), and they were divided into Group A (Y109, L110, F134), Group B (A190, M193, M358), and Group C (I270, L273, L362, F363).

[0045] Based on the gene sequence of wild-type esterase PNB shown in SEQ ID NO.1, three-codon saturation mutation primers for groups A, B, and C were designed respectively, as shown in Table 1. The three amino acids corresponding to the three codons are valine, methionine, and tyrosine. Using the vector pET28a-pnbA as a template, first perform fragment PCR amplification of the fragment containing the mutation site, randomly introducing mutations; then use the PCR product as one of the primers to further amplify the entire plasmid. After detection by 0.8% agarose gel electrophoresis, an amplification band with the correct size was obtained, indicating that the mutant fragment had been fused into the expression plasmid. After digesting the PCR-fused product with the restriction enzyme DpnI, perform a ligation reaction on the PCR product using the one-step cloning method, and then transform it into Escherichia coli BL21(DE3) cells, and spread it on an LB plate containing 50 μg / mL kanamycin to obtain a mutant library.

[0046] Table 1: Primers for three-codon saturation mutation

[0047]

[0048]

[0049] Example 3: Screening of three small and refined mutant libraries in groups A, B, and C

[0050] Transfer the transformants on the plate obtained in Example 2 to an LB liquid medium containing 50 μg / ml kanamycin, and culture them in a shaker until the mid-logarithmic growth phase. Then transfer them to the fermentation medium at an inoculation amount of 1% (v / v), culture at 37 °C for 2 h; add IPTG with a final concentration of 0.5 mmol / L, control the fermentation temperature at 24 °C, and continue fermentation for 8 h to obtain the inoculum after induced expression.

[0051] Take 2 mL of the cultured inoculum containing the esterase mutant, centrifuge at 10000×g for 5 min to collect the cells, and resuspend the cells in 1 mL of 200 mmol / L phosphate buffer at pH 8.0; add 20 μL of dl-menthyl acetate, and perform metal bath oscillation catalysis at 30 °C and 1200 rpm for 2 h. The catalytic reaction solution is used for GC analysis.

[0052] The screening results of the enzyme activity and substrate stereoselectivity of the mutants in library libA, library libB, and library libC are respectively as Figure 9 , Figure 10 , Figure 11 shown.

[0053] By comparing the substrate stereoselectivity with the catalytic rate of the enzyme, excellent mutant strains were selected for sequencing analysis. As a result, mutant strains PNB-Y109V / L110V, PNB-Y109M / L110M, and PNB-M193Y were obtained as the first-generation preferred mutants.

[0054] Example 4: Activity analysis of the first-generation preferred mutants in the catalytic hydrolysis of dl-menthyl acetate to prepare l-menthol

[0055] According to the method described in Example 1, bacterial agents containing esterase mutants PNB-Y109V / L110V, PNB-Y109M / L110M, and PNB-M193Y were prepared respectively. Each bacterial agent was 12 mL, and the cells were collected by centrifugation at 10,000×g for 5 min. The cells were resuspended in 6 mL of 200 mmol / L phosphate buffer at pH 8.0. The resuspended solution was divided into 6 tubes, and 20 μL of dl-menthyl acetate was added to each tube. The mixture was catalytically oscillated in a metal bath at 30 °C and 1200 rpm for 6 h, and the catalytic solution was used for GC analysis.

[0056] The analysis results of the enzyme activity and stereoselectivity of the first-generation preferred mutants are shown in Table 2. Among them, mutant strain PNB-M193Y had the best stereoselectivity for the substrate dl-menthyl acetate and was identified as the first-generation optimal mutant. It was used as the template esterase for the iterative combinatorial mutation of the second-generation library.

[0057] Table 2: Re-screening results of the first-generation preferred mutants

[0058]

[0059]

[0060] GC analysis method for sampling the catalytic solution:

[0061] 1) Pretreatment of the sample: 500 μL of the catalytic reaction solution was added to an equal proportion of ethyl acetate for mixing extraction; centrifuged at 12,000×g for 1 min, and the supernatant organic phase was taken for chromatographic analysis.

[0062] 2) Gas chromatography detection conditions. Chromatographic column: Agilent CYCLODEX-B 60 m×0.250 mm. Temperature programming: 100 °C, held for 3 min; heated to 145 °C at 2.5 °C / min, held for 1 min; heated to 160 °C at 1 °C / min, held for 1 min. Detector: FID detector. The peak time of the product l-menthol was about 33 min, and the peak time of the substrate menthyl ester was about 35 min. The GC chromatograms of dl-menthyl acetate and dl-menthol standards are as Figure 4 shown. From left to right, they are d-menthol, l-menthol, l-menthyl acetate, and d-menthyl acetate.

[0063] Example 5: Design of the Second-Generation Mutation Library and Construction and Screening of Mutants

[0064] To further improve the substrate stereoselectivity of the esterase mutant PNB-M193Y, using it as a template, the second-generation iterative mutation was continued. PNB-M193Y was screened into the first-generation library B, so the iterative libraries of group A and group C were constructed using it as a template respectively. The library construction method was as in Example 3, and the second-generation iterative libraries lib B-A and lib B-C were obtained.

[0065] The transformants on the plates of libraries lib B-A and lib B-C were transferred to LB liquid medium containing 50 μg / mL kanamycin respectively, and cultured in a shaker until the mid-logarithmic growth phase. Then, they were transferred to the fermentation medium according to an inoculation amount of 1% (v / v), cultured at 37 °C for 2 h; IPTG with a final concentration of 0.5 mmol / L was added, the fermentation temperature was controlled at 24 °C, and fermentation was continued for 8 h to obtain the inoculum after induced expression. Take 2 mL of the cultured inoculum containing the esterase mutant, centrifuge at 10000×g for 5 min to collect the cells, and resuspend the cells in 1 mL of 200 mmol / L phosphate buffer at pH 8.0; add 20 μL of dl-menthyl acetate, and perform metal bath oscillation catalysis at 30 °C and 1200 rpm for 2 h, and the catalytic solution was used for GC analysis.

[0066] The screening results of the enzyme activity and substrate stereoselectivity of the mutants in library libB-A and library libB-C are respectively as Figure 12 、 Figure 13 shown.

[0067] By comparing the substrate stereoselectivity and the catalytic rate of the enzyme of each colony, excellent mutant strains were selected for sequencing analysis, and the mutant strains PNB-M193Y / F314Y, PNB-M193Y / F314V, PNB-L110Y / M193Y, PNB-Y109V / L110V / M193Y, PNB-Y109M / L110Y / M193Y and PNB-Y109M / L110Y / M193Y / F314Y were obtained as the second-generation preferred mutants.

[0068] Example 6: Activity Analysis of the Second-Generation Preferred Mutant in Catalyzing the Hydrolysis of dl-Menthyl Acetate to Prepare l-Menthol

[0069] Bacterial agents of esterase mutants PNB-M193Y / F314Y, PNB-M193Y / F314V, PNB-L110Y / M193Y, PNB-Y109V / L110V / M193Y, PNB-Y109M / L110Y / M193Y, and PNB-Y109M / L110Y / M193Y / F314Y were prepared according to the method described in Example 1. Take 12 mL of each bacterial agent, centrifuge at 10,000×g for 5 min to collect cells, and resuspend the cells in 6 mL of 200 mmol / L phosphate buffer at pH 8.0; divide the resuspended solution into 6 tubes, add 20 μL of dl-menthyl acetate to each tube, and shake and catalyze in a metal bath at 30 °C and 1200 rpm for 6 h. The catalytic solution was used for GC analysis.

[0070] The results of the enzyme activity and stereoselectivity of the second-generation preferred mutants are shown in Table 3. Among them, the mutant strain PNB-Y109M / L110Y / M193Y / F314Y had the best stereoselectivity for the substrate dl-menthyl acetate, was identified as the second-generation optimal mutant, and was used as the template esterase for the iterative combinatorial mutagenesis of the third-generation library.

[0071] Table 3: Rescreening results of the second-generation preferred mutants

[0072]

[0073] Example 7: Design of the third-generation mutant library and construction and screening of mutants

[0074] To further improve the hydrolysis activity of the esterase mutant PNB-Y109M / L110Y / M193Y / F314Y, it was used as a template to continue the iterative mutagenesis of the third generation. PNB-Y109M / L110Y / M193Y / F314Y was screened into the second-generation library B-A, so the C-group iterative library was constructed using this as a template. The library construction method was as described in Example 3, and the third-generation iterative library lib B-A-C was obtained.

[0075] Transfer the transformants on the plate of library lib B-A-C to LB liquid medium containing 50 μg / mL kanamycin, and culture them in a shaker until the mid-logarithmic growth phase. Then transfer them to the fermentation medium at an inoculation amount of 1% (v / v), culture at 37 °C for 2 h; add IPTG with a final concentration of 0.5 mmol / L, control the fermentation temperature at 24 °C, and continue fermentation for 8 h to obtain the inoculum after induced expression. Take 2 mL of the cultured inoculum containing the esterase mutant, centrifuge at 10000×g for 5 min to collect the cells, and resuspend the cells in 1 mL of 200 mmol / L phosphate buffer at pH 8.0; add 20 μL of dl-menthyl acetate, and perform metal bath oscillation catalysis at 30 °C and 1200 rpm for 6 h, and the catalytic solution is used for GC analysis.

[0076] The screening results of the enzyme activity and substrate stereoselectivity of the mutants in library lib B-A-C are as Figure 14 shown.

[0077] By comparing the substrate selectivity and the catalytic rate of the enzyme, excellent mutant strains were selected for sequencing analysis, and the mutant strains PNB-Y109M / L110Y / M193Y / I270M / F314Y, mutant strain PNB-Y109M / L110Y / M193Y / I270Y / F314Y, mutant strain PNB-Y109M / L110Y / M193Y / I270M / L273M / F314Y, and mutant strain PNB-Y109M / L110Y / M193Y / I270M / L273V / F314Y / L362V were obtained as the third-generation preferred mutants.

[0078] Example 8: Activity analysis of the third-generation preferred mutants for catalyzing the hydrolysis of dl-menthyl acetate to prepare l-menthol

[0079] According to the method described in Example 1 respectively, the esterase mutants

[0080] PNB-Y109M / L110Y / M193Y / I270M / F314Y, PNB-Y109M / L110Y / M193Y / I270Y / F314Y, PNB-Y109M / L110Y / M193Y / I270M / L273M / F314Y, and

[0081] Bacterial agents of PNB-Y109M / L110Y / M193Y / I270M / L273V / F314Y / L362V. Take 12 mL of each bacterial agent respectively, centrifuge to collect cells at 10,000×g for 5 min, and resuspend the cells in 6 mL of 200 mmol / L phosphate buffer at pH 8.0; divide the resuspended solution into 6 tubes, add 20 μL of dl-menthyl acetate to each tube, and catalytically oscillate in a metal bath at 30 °C and 1200 rpm for 10 h. The catalytic solution is used for GC analysis.

[0082] The results of the enzyme activity and stereoselectivity of the third-generation preferred mutants are shown in Table 4. Among them, the conversion efficiencies of the mutant strains PNB-Y109M / L110Y / M193Y / I270Y / F314Y, PNB-Y109M / L110Y / M193Y / I270M / F314Y, and PNB-Y109M / L110Y / M193Y / I270M / L273V / F314Y / L362V mutants are relatively high, and the enantioselectivity for the substrate has been above 99%.

[0083] Table 4: Rescreening results of the third-generation preferred mutants

[0084]

[0085]

[0086] Example 9: Application of PNB-MYYY mutant strain in the preparation of l-menthol by hydrolysis of dl-menthyl acetate

[0087] Induced expression of esterase mutant strain. After streaking and culturing the strain E. coli BL21(DE3)(pET28a-PNB-MYYY) overnight from the preserved glycerol tube, pick a single colony and inoculate it into LB liquid medium containing 50 μg / mL kanamycin, and culture it overnight at 37 °C and 200 rpm. Inoculate the seed liquid into the fermentation medium containing kanamycin sulfate (50 μg / mL) at an inoculation amount of 2%, culture it in a shaker at 37 °C and 200 rpm for 4 - 6 h, and then inoculate it into a 2.5 L fermenter at an inoculation amount of 5%, and perform fermentation culture at 37 °C. When OD 600 reaches about 10, add lactose for induced expression of the target protein. The induction temperature is 24 °C, and the induction time is 12 - 16 h. When the rotation speed of the fermenter decreases and the pH rises, terminate the fermentation and centrifuge to collect cells.

[0088] After centrifuging to collect the bacterial cells, add phosphate buffer at pH 8.0 to prepare a cell density of OD 600160 mL of the whole-cell catalysis system with a concentration of 20 was poured into a round-bottom flask and incubated in a constant-temperature water bath at 30 °C for 10 min. Then, 40 mL of dl-menthyl acetate was added. Immediately, magnetic stirring was started to mix evenly, and the start of the reaction was recorded and timed. During the catalytic reaction, the reaction pH was adjusted to 8.0 with 2 mol / L NaOH solution. The reaction solution was used for gas chromatography analysis. As Figure 5 shown, from left to right are d-menthol, l-menthol, l-menthyl acetate, and d-menthyl acetate. The analysis results of the catalytic reaction solution at different reaction times are shown in Table 5, and the ee of the product l-menthyl acetate p remained above 99%.

[0089] Table 5: Catalytic results of mutant strain PNB-MYYY at different reaction times

[0090]

[0091] Example 10: Application of PNB-MYYMY mutant strain in the hydrolysis of dl-menthyl acetate to prepare l-menthol

[0092] Induced expression of the esterase mutant strain. After the strain E. coli BL21(DE3)(pET28a-PNB-MYYMY) was streaked and cultured overnight from the preserved glycerol tube, a single colony was inoculated into an LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37 °C and 200 rpm. The seed solution was inoculated into a fermentation medium containing kanamycin sulfate (50 μg / mL) at an inoculation amount of 2%, cultured in a shaker at 37 °C and 200 rpm for 4 - 6 h, and then inoculated into a 2.5 L fermenter at an inoculation amount of 5%. Fermentation culture was carried out at 37 °C. When the OD 600 reached about 10, lactose was added for the induced expression of the target protein. The induction temperature was 24 °C, and the induction time was 12 - 16 h. When the rotation speed of the fermenter decreased and the pH increased, the fermentation was terminated, and the cells were collected by centrifugation.

[0093] After centrifuging to collect the cells, 160 mL of a whole-cell catalysis system with an OD of 20 was prepared by adding a phosphate buffer solution with a pH of 8.0, poured into a round-bottom flask, and incubated in a constant-temperature water bath at 30 °C for 10 min; 40 mL of dl-menthyl acetate was added. Immediately, magnetic stirring was started to mix evenly, and the start of the reaction was recorded and timed. During the catalytic reaction, the reaction pH was adjusted to 8.0 with 2 mol / L NaOH solution. The reaction solution was used for gas chromatography analysis. As Figure 6 shown, from left to right are d-menthol, l-menthol, l-menthyl acetate, and d-menthyl acetate. The catalytic reaction results at different reaction times are shown in Table 6, and the ee of the product l-menthyl acetate p remained above 99%.

[0094] Table 6: Catalytic results of mutant strain PNB - MYYMY at different reaction durations

[0095]

[0096] Example 11: Application of PNB - MYYYY mutant strain in the hydrolysis of dl - menthyl acetate to prepare l - menthol

[0097] After streak - culturing the strain E.coli BL21(DE3)(pET28a - PNB - MYYYY) overnight from the preserved glycerol tube, a single colony was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with 200 rpm. The seed culture was inoculated into the fermentation medium containing kanamycin sulfate (50 μg / mL) at an inoculation amount of 2%, cultured in a shaker at 37°C with 200 rpm for 4 - 6 h, and then inoculated into a 2.5 L fermenter at an inoculation amount of 5% and fermented at 37°C. When the OD 600 reached about 10, lactose was added for the induced expression of the target protein. The induction temperature was 24°C and the induction time was 12 - 16 h. When the rotation speed of the fermenter decreased and the pH increased, the fermentation was terminated and the cells were collected by centrifugation.

[0098] After collecting the cells by centrifugation, 160 mL of a whole - cell catalytic system with an OD of 20 was prepared by adding phosphate buffer at pH 8.0 and poured into a round - bottom flask. It was first incubated in a constant - temperature water bath at 30°C for 10 min, and then 40 mL of dl - menthyl acetate was added. Immediately, magnetic stirring was started to mix evenly, and the start of the reaction was recorded and timed. During the catalytic reaction, the reaction pH was adjusted to 8.0 with 2 mol / L NaOH solution. The reaction solution was used for gas chromatography analysis. As Figure 7 shown, from left to right are d - menthol, l - menthol, l - menthyl acetate, and d - menthyl acetate. The catalytic reaction results at different reaction times are shown in Table 7, and the ee of the product l - menthyl acetate p was maintained above 99%.

[0099] Table 7: Catalytic results of mutant strain PNB - MYYYY at different reaction durations

[0100]

[0101] Example 12: Application of PNB - MYVMYYV mutant strain in the hydrolysis of dl - menthyl acetate to prepare l - menthol

[0102] After streaking the strain E. coli BL21(DE3)(pET28a-PNB-MYYMVYV) from the preserved glycerol tube overnight, a single colony was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with 200 rpm. The seed culture was inoculated into the fermentation medium containing kanamycin sulfate (50 μg / mL) at an inoculation amount of 2%, cultured in a shaker at 37°C with 200 rpm for 4 - 6 h, and then inoculated into a 2.5 L fermenter at an inoculation amount of 5%. Fermentation was carried out at 37°C. When OD 600 reached about 10, lactose was added for the induction expression of the target protein. The induction temperature was 24°C and the induction time was 12 - 16 h. When the rotation speed of the fermenter decreased and the pH increased, fermentation was terminated and the cells were collected by centrifugation.

[0103] After collecting the cells by centrifugation, 160 mL of a whole-cell catalytic system with an OD of 20 was prepared by adding phosphate buffer at pH 8.0, and then poured into a round-bottom flask. It was incubated in a constant temperature water bath at 30°C for 10 min, and then 40 mL of dl-menthyl acetate was added. Immediately, magnetic stirring was started to mix evenly, and the start of the reaction was recorded and timed. During the catalytic reaction, the reaction pH was adjusted to 8.0 with 2 mol / L NaOH solution. The reaction solution was used for gas chromatography analysis. As Figure 8 shown, from left to right are d-menthol, l-menthol, l-menthyl acetate, and d-menthyl acetate. The catalytic reaction results at different reaction times are shown in Table 8, and the ee p of the product l-menthyl acetate was maintained above 99%.

[0104] Table 8: Catalytic results of the mutant strain PNB-MYYMVYV at different reaction durations

[0105]

Claims

1. A p-nitrobenzyl esterase mutant, characterized in that The mutant amino acid sequence is as shown in any one of SEQ ID NO.3 to 6.

2. The encoding gene of the p-nitrobenzyl esterase mutant according to claim 1.

3. A recombinant vector containing the encoding gene according to claim 2.

4. A genetically engineered bacterium containing the encoding gene according to claim 2.

5. Use of the p-nitrobenzyl esterase mutant according to claim 1 in enzymatic catalysis dl - Preparation of chiral resolution of menthyl acetate l - Application in menthol 6. The application according to claim 5, characterized in that The application is as follows: constructing a genetically engineered bacterium containing the encoding gene of the mutant, using the enzyme-containing cells obtained by disrupting the cells of the genetically engineered bacterium obtained by fermentation culture as a catalyst, and hydrolyzing dl menthyl acetate to obtain l menthol.

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