High-enantioselectivity pinyl hydrate esterase mutant as well as construction method and application thereof

Abstract

The invention discloses a pinyl hydrate esterase mutant with high enantioselectivity as well as a construction method and application of the pinyl hydrate esterase mutant, and belongs to the technical field of enzyme engineering. The pinyl hydrate esterase mutant provided by the invention is obtained by carrying out single-point mutation or combined mutation on the 273 site leucine, the 314 site phenylalanine, the 362 site leucine and the 193 site methionine of a pinyl hydrate esterase amino acid sequence derived from bacillus subtilis through a high-throughput screening method based on fluorescence coupling. Wherein the mutant M4 shows the best catalytic performance for racemization-acetic acid pinyl hydrate, the enantiomer excess value of the mutant M4 is 99.35%, and the conversion rate of the (1S, 5R)-acetic acid pinyl hydrate is 83.90%. The pinyl hydrate esterase mutant provided by the invention is high in enzyme activity of catalyzing (1S, 5R)-acetic acid pinyl hydrate, high in eep value and high in conversion rate.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme engineering, in particular to a high enantioselective sobrerol esterase mutant, a construction method and application thereof. BACKGROUND

[0002] (1 S , 5 R )-sobrerol, also known as trans-sobrerol, has a molecular formula of C 10 H 18 O2, is a chiral monocyclic monoterpene compound with a wide source, and is widely used in organic synthesis and pharmaceutical industry. Biological studies have shown that (1 S , 5 R )-sobrerol has the effects of mucus dilution, promotion of mucus cilia clearance, antioxidant and increase of secretory IgA, and is often prepared into various dosage forms such as syrup and aerosol for treating mucus hypersecretion respiratory tract infection diseases such as bronchus and asthma. In addition, (1 S , 5 R )-sobrerol also shows the pharmacological effect of inducing tumor cell apoptosis.

[0003] Due to the low efficiency of extracting natural (1 S , 5 R )-sobrerol from plants (pine oil, citrus essential oil and other plant sources), the yield cannot meet the market demand, and industrial production relies more on chemical synthesis. At present, the synthesis methods of (1 S , 5 R )-sobrerol mainly include 8-step reaction synthesis of optically pure (1 S , 5 R )-sobrerol from 3, 5-dihydroxy-4-methyl benzoic acid methyl ester and oxidation synthesis of (1 S , 5 R )-sobrerol from α-pinene. Hydrolysis of (-)-α-pinene oxide at different temperatures can produce different forms of sobrerol. In these chemical synthesis methods, asymmetric synthesis or chemical resolution of (1 S , 5 R )-sobrerol requires a large amount of heavy metal catalysts and organic solvents, which is harmful to the environment and human health, so using biological catalysts is a more environmentally friendly alternative method. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a high enantioselective sobrerol esterase mutant, a construction method and application thereof to solve the above problems in the prior art. The present application is based on obtaining 1S , 5RA method for hydrating pinol, which proposes that racemic or low enantioselective trans-acetic acid hydrate pinol ester can be dynamically hydrolyzed and separated by an esterase catalyst to obtain a single configuration chiral intermediate 1S , 5R A finished product of hydrating pinol.

[0005] To achieve the above object, the technical scheme adopted by the present application is: the first aspect of the present application provides a hydrating pinol esterase mutant with high enantioselectivity, wherein the hydrating pinol esterase mutant is obtained by mutating the amino acid sequence of wild-type hydrating pinol esterase pnbA shown in SEQ ID NO. 1 in the following manner: the methionine (M) at position 193 is mutated to glycine (G), the leucine (L) at position 273 is mutated to phenylalanine (F), the phenylalanine (F) at position 314 is mutated to tyrosine (Y), and the leucine (L) at position 362 is mutated to arginine (R).

[0006] Preferably, the hydrating pinol esterase mutant is obtained by mutating the amino acid sequence of hydrating pinol esterase pnbA shown in SEQ ID NO. 1 in any of the following (1)-(4): (1) the phenylalanine (F) at position 314 is mutated to tyrosine (Y), and the obtained single-site mutant is denoted as F314Y; (2) the phenylalanine (F) at position 314 is mutated to tyrosine (Y) and the leucine (L) at position 362 is mutated to arginine (R), and the obtained combined mutant is denoted as F314Y / L362R; (3) the leucine (L) at position 273 is mutated to phenylalanine (F), the phenylalanine (F) at position 314 is mutated to tyrosine (Y), and the leucine (L) at position 362 is mutated to arginine (R), and the obtained combined mutant is denoted as L273F / F314Y / L362R; (4) the methionine (M) at position 193 is mutated to glycine (G), the leucine (L) at position 273 is mutated to phenylalanine (F), the phenylalanine (F) at position 314 is mutated to tyrosine (Y), and the leucine (L) at position 362 is mutated to arginine (R), and the obtained combined mutant is denoted as M193G / L273F / F314Y / L362R.

[0007] The amino acid sequences of the mutants F314Y, F314Y / L362R, L273F / F314Y / L362R and M193G / L273F / F314Y / L362R are SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

[0008] In a second aspect of the present application, a gene encoding the mutant pinostrobin acetylhydrolase is provided.

[0009] Preferably, the nucleotide sequence encoding the wild-type pinostrobin acetylhydrolase pnbA as shown in SEQ ID NO. 1 is a polynucleotide optimized based on the codon bias of E. coli, and the sequence is SEQ ID NO. 6, and the gene encoding the mutant pinostrobin acetylhydrolase is obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO. 5.

[0010] In a third aspect of the present application, a recombinant plasmid comprising the gene is provided.

[0011] Preferably, the plasmid vector of the recombinant plasmid is pET-28a(+).

[0012] In a fourth aspect of the present application, a host cell comprising the gene or the recombinant plasmid is provided.

[0013] Preferably, the host cell is a prokaryotic cell.

[0014] Preferably, the host cell is E. coli BL21 (DE3) carrying the recombinant plasmid. E. coli

[0015] In a fifth aspect of the present application, a method for constructing the mutant pinostrobin acetylhydrolase with high enantioselectivity is provided, and the method comprises the following steps: culturing the host cell, inducing expression of the mutant pinostrobin acetylhydrolase, collecting the host cell, crushing and centrifuging, and separating and purifying the mutant pinostrobin acetylhydrolase from the supernatant.

[0016] In a sixth aspect of the present application, the mutant pinostrobin acetylhydrolase, the gene, the recombinant plasmid, and the host cell are used for catalyzing the production of a pre-ester compound in a hydrate pinostrobin. 1S 5R Preferably, the pre-ester compound is racemic trans-pinostrobin formate or racemic trans-pinostrobin acetate.

[0017] Preferably, the pre-ester compound is racemic trans-pinostrobin formate or racemic trans-pinostrobin acetate.

[0018] In a seventh aspect of the present application, a green synthesis method for producing (1 S , 5 R )-pinostrobin is provided, and the method comprises the following steps: adding the mutant pinostrobin acetylhydrolase to a reaction system to obtain a reaction liquid; and extracting the product from the reaction liquid to obtain the target product (1 S , 5 R )-pinostrobin.

[0019] ​​Preferably, the concentration of the hydrated pinyl esterase mutant added to the reaction solution is 0.025~0.25 mg / mL.

[0020] In one embodiment of the present invention, the reaction temperature is 37°C, the pH is 7.0, and the reaction time is 30~60 min.

[0021] The beneficial effects of this invention are: This invention first uses site-directed saturation mutagenesis to target bacteria derived from Bacillus subtilis. Bacillus subtilis A saturated mutant library of key amino acid sites in the active pocket of hydrated pinol esterase (WP_326228118.1, named pnbA, sequence SEQ ID NO.1) was constructed, and positive sites that could potentially affect the activity and enantioselectivity of hydrated pinol esterase were obtained through fluorescence coupling reaction and screening. Then, the positive mutant sites obtained by screening (F314, A107, L110, L273, M358, L362) catalyzed (1 S , 5 R The activity of 1-formate hydrate pinyl ester was significantly higher than that of the unmutated wild type (WT). Specifically, when the F314 site was mutated to other amino acids (especially polar residues), the enzyme activity of the mutants was significantly enhanced. Among these mutants, F314Y, F314H, L273V, and L362R showed the best activity against (1)-formate pinyl ester. S , 5 R The activity of 1-pine methyl formate hydrate is relatively (1 R , 5 S The activity ratios of 2-formate hydrated pinene were 2.9, 2.1, 2.1, and 1.8, respectively. Therefore, this invention subsequently selected site-combination mutagenesis based on pnbA-F314Y, and used gas chromatography to detect and screen the dynamic kinetic hydrolytic resolution effect of the mutants on racemic 2-acetic acid hydrated pinene, obtaining mutants M1 (F314Y), M2 (F314Y / L362R), M3 (L273F / F314Y / L362R), and M4 (M193G / L273F / F314Y / L362R) with significantly improved enantioselectivity. Specifically, mutants M1, M2, M3, and M4 exhibited significantly improved enantioselectivity. ee p The values ​​increased to 85.00%, 91.33%, 97.20%, and 99.35% respectively, while the corresponding selection rate... E The values ​​were increased to 22.27, 40.21, 104.66, and 482.59, respectively. The enantioselectivity enhancement shows that the hydrated pinyl esterase mutants disclosed in this invention, especially M3 (L273F / F314Y / L362R) and M4 (M193G / L273F / F314Y / L362R), catalyze (1... S 5R ) - high enzyme activity of pinanediol acrylate, ee p high value, high conversion rate, and production of high-value chiral intermediates (1 S , 5 R ) - application prospect of pinanediol and similar terpene diol compounds.

[0022] The engineered pinanediol esterase mutant M3 exhibits excellent reusability in a continuous flow whole-cell catalytic system, and the conversion rate and enantioselectivity value remain stable after seven cycles (more than 87% and 95%, respectively), which highlights its application potential in green kinetic resolution (1 S , 5 R ) - pinanediol; this step-by-step engineering strategy based on high-throughput screening provides an efficient solution for the stereoselective engineering of carboxylate esterases. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a high-throughput screening guided enantioselective step-by-step engineering roadmap provided by the present application; Figure 2 is a roadmap for obtaining (1 S , 5 R ) - pinanediol by kinetic resolution by the present application; Figure 3 is a schematic diagram of the pinanediol esterase activity detection method used by the present application; Figure 4 is a structural model of pinanediol esterase pnbA derived from Bacillus subtilis Bacillus subtilis and its molecular docking results with (1 S , 5 R ) - pinanediol acrylate, wherein S189-E310-H399 is the catalytic triad, and M193, L273, F314, and L362 are the target residues for mutation in the present application; Figure 5 is a schematic diagram of the fluorescence coupling high-throughput screening step provided by the present application; Figure 6 is a comparison of the relative enzyme activity of the pinanediol esterase pnbA wild type and the screened superior mutant of the present application against (1 S , 5 R ) - pinanediol formate and (1 R , 5 S ) - pinanediol formate, with the activity of the wild type against (1 S , 5 R ) - pinanediol formate being 1 for relative enzyme activity comparison; Figure 7 is the stepwise directed evolution result of the hydratropinesterase pnbA dominant mutant in the present application, wherein A: M1-M4 4 rounds of stepwise site-directed mutation screening, ee p represents the enantiomeric excess value of (1 S , 5 R )-hydratropinol, ConV represents the conversion rate of (1 S , 5 R )-hydratropinol; B: corresponding GC detection spectrum after reaction of WT and M1, M2, M3, M4 dominant mutants; Figure 8 is the wild type (WT) of hydratropinesterase pnbA and the optimal mutant pair of each round in the stepwise directed evolution process in the present application, wherein (1 S , 5 R )-hydratropinol E value trend chart, wherein 1S5R represents (1 S , 5 R )-hydratropinol; Figure 9 is the wild type (WT) of hydratropinesterase pnbA and the mutant M3 / M4 pair in the present application, wherein (1 S , 5 R )-hydratropinyl acetate and (1 R , 5 S )-hydratropinyl acetate kinetic parameters; Figure 10 is the schematic diagram and result of continuous flow reaction catalyzed by the whole cell using the mutant M3 of the engineered hydratropinesterase pnbA provided by the present application, wherein A: continuous flow reaction device schematic diagram; B: (1 S , 5 R )-hydratropinol ee p value and conversion rate under different repeated use times. DETAILED DESCRIPTION

[0024] The present application will be further described in detail below with reference to examples, so that those skilled in the art can implement the present application according to the description.

[0025] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0027] The Escherichia coli used in this invention E. coli BL21(DE3) competent cells were purchased from Beijing TransGen Biotech Co., Ltd.; the wild-type plasmid of hydrated pinyl esterase pnbA based on the pET-28a(+) plasmid vector and the primers for constructing the site-directed mutant plasmid were synthesized by Beijing Qingke Biotechnology Co., Ltd.; the molecular biology reagents used, such as DNA polymerase, restriction endonuclease, DpnI, and recombinase, were purchased from Takara; the trans-acetic acid hydrate pinyl ester, (1 S , 5 R )-Pinyl formate hydrate and (1 R , 5 S Pinyl formate hydrate was provided by Professor Chen Jianzhong of Shanghai Jiao Tong University; the fluorescent substrate AR-2 was provided by Professor Liu Fang of Guangzhou University of Chinese Medicine; and other biochemical reagents were purchased from Aladdin Biochemical Technology Co., Ltd.

[0028] The PCR instrument used was purchased from Thermo Fisher Scientific, and the microplate reader used was a BioTek Synergy H1; the high-performance gas chromatograph used was an Agilent 8860 GC System, the detector used was a flame ionization detector (FID), and the chiral column used was an Agilent Cyclodex-B (60 m × 0.25 mm × 0.25 μm).

[0029] This invention provides a high-throughput screening-guided enantioselective step engineering method to obtain a target hydrated pinol esterase pnbA mutant with improved performance, referring to... Figure 1 This method uses Bacillus subtilis p-nitrobenzyl esterase (pnBA), which has poor enzyme activity, as its starting point. It successfully identifies key positive amino acid sites that affect the stereoselectivity of the enzyme. Then, by gradually introducing these active sites, mutants with progressively improved performance are finally obtained.

[0030] The culture media involved in the following examples are as follows: LB solid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 15 g / L agar and 50 mg / L kanamycin.

[0031] LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl and 50 mg / L kanamycin.

[0032] The principle of the fluorescence coupling detection of the hydrated pinol esterase pnbA is as follows Figure 1 The specific scheme is as follows: The (1 S , 5 R )-formic acid hydrated pinol ester or (1 R , 5 S )-formic acid hydrated pinol ester is used as the initial reaction substrate, and after being hydrolyzed by the hydrated pinol esterase pnbA in the first-stage reaction, (1 S , 5 R )-hydrated pinol or (1 R , 5 S )-hydrated pinol is generated, and at the same time, an equimolar amount of formic acid is also generated. As a by-product, the formic acid can be specifically recognized and oxidized by the formic acid oxidase (AoFOX) in the second-stage coupling reaction to form CO2 and H2O2, and the H2O2 is used by the horseradish peroxidase (HRP) in the third-stage coupling reaction to oxidize the fluorescence substrate AR-2 to generate the fluorescent resorufin-Cl. The fluorescence intensity of the fluorescence product can be detected to quickly and sensitively perform relative quantitative analysis on the pnbA activity.

[0033] The fluorescence enzyme activity detection system (100 μL) is as follows: 50 mM (1 S , 5 R )-formic acid hydrated pinol ester or (1 R , 5 S )-formic acid hydrated pinol ester, 0.01 mg / mL wild-type or mutant hydrated pinol esterase pnbA, 0.05 mg / mL formic acid oxidase AoFOX, 0.9 U / mL horseradish peroxidase HRP, and 0.1 mM fluorescence substrate AR-2. As shown in Figure 3 The fluorescence coupling strategy of the esterase pnbA-formic acid oxidase-peroxidase for the chiral hydrated pinol product is designed according to the present application, which is used for the detection of the enzyme activity of the hydrated pinol esterase pnbA.

[0034] The reaction is carried out at 37°C, and the fluorescence is detected in real time, the excitation wavelength is 582 nm, the emission wavelength is 602 nm, the gain is 50, the continuous vibration plate is 5 s, the fluorescence is detected once every 1 min, and the continuous detection is 30 minutes.

[0035] The chiral ester substrate and chiral alcohol product of the catalytic reaction were qualitatively and quantitatively analyzed by gas chromatography (GC) method, the carrier gas N2 flow rate was set to 1 mL / min, the injection amount was 1 μL, and the temperature program was set as follows: the initial temperature was 120°C and maintained for 0.5 min, then the temperature was raised from 120°C to 195°C at a rate of 1.5°C / min, and maintained at 195°C for 5 min. The enantiomeric excess value of the product ee p , the conversion rate and the enantiomeric ratio E were calculated according to the following formula: In the formula, C 1S5R and C 1R5S respectively represent the final concentrations of (1 S , 5 R )-pinol hydrate and (1 R , 5 S )-pinol hydrate in the reaction system at the end of the reaction, and C 1S5R-ace represents the initial concentration of (1 S , 5 R )-pinol hydrate in the reaction system at the beginning of the reaction.

[0036] Example 1: Construction of the pnbA site saturation mutation library and site-directed mutation plasmid of pinol esterase.

[0037] According to the amino acid sequence SEQ ID NO. 1 of wild-type pinol esterase pnbA, protein homology modeling analysis was performed on the SWISS-MODEL website, and the simulated structure was then subjected to molecular docking (AutoDock 4.2.6 software) with amino donor substrates and amino acceptor substrates, respectively, to analyze the substrate binding pocket and screen the key amino acid residues G105, G106, A107, F108, L110, A190, M193, I270, F271, L273, F275, F314, M358, L362, F363 According to the primers shown in Table 1, the wild-type plasmid pET-28a(+)-pnbA was used as a template for full-plasmid single-point mutation PCR to construct the NNK saturation mutation library of the key amino acid sites, and the mutation libraries of different sites were mixed in equimolar amounts to construct the saturation mutation library of all sites. After sequencing, the plasmid was extracted and stored.

[0038] Table 1 NNK primers for constructing the pnbA site saturation mutation library Primer name Number Sequence 5'-3' pnbA-G105-F SEQ ID NO. 7 GGATTCATNNKGGCGCGTTTTATCTGGGTGC pnbA-G105-R SEQ ID NO. 8 AACGCGCCMNNATGAATCCATACCATAACCGGCAG pnbA-G106-F SEQ ID NO. 9 TTCATGGCNNKGCGTTTTATCTGGGTGCAGGT pnbA-G106-R SEQ ID NO. 10 TAAAACGCMNNGCCATGAATCCATACCATAACCGG pnbA-A107-F SEQ ID NO. 11 CATGGCGGCNNKTTTTATCTGGGTGCAGGTTCCG pnbA-A107-R SEQ ID NO. 12 CAGATAAAAMNNGCCGCCATGAATCCATACC pnbA-F108-F SEQ ID NO. 13 GCGGCGCGNNKTATCTGGGTGCAGGTTCCGAG pnbA-F108-R SEQ ID NO. 14 CCCAGATAMNNCGCGCCGCCATGAATC pnbA-Y110-F SEQ ID NO. 15 GCGTTTTATNNKGGTGCAGGTTCCGAGCC pnbA-Y110-R SEQ ID NO. 16 CCTGCACCMNNATAAAACGCGCCGCCATGAA pnbA-A190-F SEQ ID NO. 17 GTGAATCTNNKGGGGGCATGAGTATCGCTG pnbA-A190-R SEQ ID NO. 18 ATGCCCCCMNNAGATTCACCGAAGACGGTAAC pnbA-M193-F SEQ ID NO. 19 CTGGGGGCNNKAGTATCGCTGCGCTGCTAG pnbA-M193-R SEQ ID NO. 20 GCGATACTMNNGCCCCCAGCAGATTCACC pnbA-I270-F SEQ ID NO. 21 GAAAACNNKTTCCAACTGTTCTTCCAGCCGGC pnbA-I270-R SEQ ID NO. 22 CAGTTGGAAMNNGTTTTCTTTTTCGGCGATACGCAG pnbA-F271-F SEQ ID NO. 23 GAAAACATTNNKCAACTGTTCTTCCAGCCGGC pnbA-F271-R SEQ ID NO. 24 GAACAGTTGMNNAATGTTTTCTTTTTCGGCGATACGC pnbA-L273-F SEQ ID NO. 25 TTTTCCAANNKTTCTTCCAGCCGGCACTAGAC pnbA-L273-R SEQ ID NO. 26 TGGAAGAAMNNTTGGAAAATGTTTTCTTTTTCGGCG pnbA-F275-F SEQ ID NO. 27 CAACTGTTCNNKCAGCCGGCACTAGACCC pnbA-F275-R SEQ ID NO. 28 GCCGGCTGMNNGAACAGTTGGAAAATGTTTTC pnbA-F314-F SEQ ID NO. 29 GGTATCTCNNKTTCACCCCCGATAGCGATGTG pnbA-F314-R SEQ ID NO. 30 GGGGTGAAMNNGAGATACCCTTCATCCCGT pnbA-M358-F SEQ ID NO. 31 CCAGATGNNKACCGATCTGCTGTTTTGGCG pnbA-M358-R SEQ ID NO. 32 AGATCGGTMNNCATCTGGATCTGTGATTCCAGGC pnbA-L362-F SEQ ID NO. 33 CCGATCTGNNKTTTTGGCGCCCAGCAGTG pnbA-L362-R SEQ ID NO. 34 CGCCAAAAMNNCAGATCGGTCATCATCTGG pnbA-F363-F SEQ ID NO. 35 CCGATCTGCTGNNKTGGCGCCCAGCAGTG pnbA-F363-R SEQ ID NO. 36 GGGCGCCAMNNCAGCAGATCGGTCATCATC Using the primers shown in Table 2, and with the wild-type plasmid pET-28a(+)-pnbA as a template, a full plasmid single-point mutation PCR was performed to construct a recombinant plasmid encoding the corresponding mutant gene. After the sequence was confirmed to be correct, the plasmid was extracted and stored.

[0039] Table 2 Primers for constructing pnbA site-directed mutagenesis Primer name No. Sequence 5'-3' pnbA-F314A-F SEQ ID NO. 37 GGTATCTCGCATTCACCCCCGATAGCGATGTG pnbA-F314A-R SEQ ID NO. 38 GGGGTGAATGCGAGATACCCTTCATCCCGT pnbA-F314T-F SEQ ID NO. 39 GGTATCTCACCTTCACCCCCGATAGCGATGTG pnbA-F314T-R SEQ ID NO. 40 GGGGTGAAGGTGAGATACCCTTCATCCCGT pnbA-F314Y-F SEQ ID NO. 41 GGTATCTCGGCTTCACCCCCGATAGCGATGTG GGTATCTCTATTTCACCCCCGATAGCGATGTG pnbA-F314Y-R SEQ ID NO. 42 GGGGTGAAATAGAGATACCCTTCATCCCGT pnbA-F314N-F SEQ ID NO. 43 GGTATCTCAACTTCACCCCCGATAGCGATGTG pnbA-F314N-R SEQ ID NO. 44 GGGGTGAAGTTGAGATACCCTTCATCCCGT pnbA-F314E-F SEQ ID NO. 45 GGTATCTCGAATTCACCCCCGATAGCGATGTG pnbA-F314E-R SEQ ID NO. 46 GGGGTGAATTCGAGATACCCTTCATCCCGT pnbA-F314H-F SEQ ID NO. 47 GGTATCTCCACTTCACCCCCGATAGCGATGTG pnbA-F314H-R SEQ ID NO. 48 GGGGTGAAGTGGAGATACCCTTCATCCCGT pnbA-L362Y-F SEQ ID NO. 49 CCGATCTGTATTTTTGGCGCCCAGCAGTG pnbA-L362Y-R SEQ ID NO. 50 CGCCAAAAATACAGATCGGTCATCATCTGG pnbA-L362F-F SEQ ID NO. 51 CCGATCTGTTCTTTTGGCGCCCAGCAGTG pnbA-L362F-R SEQ ID NO. 52 CGCCAAAAGAACAGATCGGTCATCATCTGG pnbA-L362D-F SEQ ID NO. 53 CCGATCTGGACTTTTGGCGCCCAGCAGTG pnbA-L362D-R SEQ ID NO. 54 CGCCAAAAGTCCAGATCGGTCATCATCTGG pnbA-L362K-F SEQ ID NO. 55 CCGATCTGAAATTTTGGCGCCCAGCAGTG pnbA-L362K-R SEQ ID NO. 56 CGCCAAAATTTCAGATCGGTCATCATCTGG pnbA-L362E-F SEQ ID NO. 57 CCGATCTGGAATTTTGGCGCCCAGCAGTG pnbA-L362E-R SEQ ID NO. 58 CGCCAAAATTCCAGATCGGTCATCATCTGG pnbA-L362R-F SEQ ID NO. 59 CCGATCTGCGCTTTTGGCGCCCAGCAGTG pnbA-L362R-R SEQ ID NO. 60 CGCCAAAAGCGCAGATCGGTCATCATCTGG pnbA-L273Y-F SEQ ID NO. 61 TTTTCCAATATTTCTTCCAGCCGGCACTAGAC pnbA-L273Y-R SEQ ID NO. 62 TGGAAGAAATATTGGAAAATGTTTTCTTTTTCGGCG pnbA-L273F-F SEQ ID NO. 63 TTTTCCAATTCTTCTTCCAGCCGGCACTAGAC pnbA-L273F-R SEQ ID NO. 64 TGGAAGAAGAATTGGAAAATGTTTTCTTTTTCGGCG pnbA-L273D-F SEQ ID NO. 65 TTTTCCAAGACTTCTTCCAGCCGGCACTAGAC pnbA-L273D-R SEQ ID NO. 66 TGGAAGAAGTCTTGGAAAATGTTTTCTTTTTCGGCG pnbA-L273K-F SEQ ID NO. 67 TTTTCCAAAAATTCTTCCAGCCGGCACTAGAC pnbA-L273K-R SEQ ID NO. 68 TGGAAGAATTTTTGGAAAATGTTTTCTTTTTCGGCG pnbA-L273E-F SEQ ID NO. 69 TTTTCCAAGAATTCTTCCAGCCGGCACTAGAC pnbA-L273E-R SEQ ID NO. 70 TGGAAGAATTCTTGGAAAATGTTTTCTTTTTCGGCG pnbA-L273R-F SEQ ID NO. 71 TTTTCCAACGCTTCTTCCAGCCGGCACTAGAC pnbA-L273R-R SEQ ID NO. 72 TGGAAGAAGCGTTGGAAAATGTTTTCTTTTTCGGCG pnbA-L273Y-F SEQ ID NO. 73 TTTTCCAATATTTCTTCCAGCCGGCACTAGAC pnbA-M193G-F SEQ ID NO. 74 CTGGGGGCGGCAGTATCGCTGCGCTGCTAG pnbA-M193G-R SEQ ID NO. 75 GCGATACTGCCGCCCCCAGCAGATTCACC pnbA-M193A-F SEQ ID NO. 76 CTGGGGGCGCAAGTATCGCTGCGCTGCTAG pnbA-M193A-R SEQ ID NO. 77 GCGATACTTGCGCCCCCAGCAGATTCACC pnbA-M193L-F SEQ ID NO. 78 CTGGGGGCCTGAGTATCGCTGCGCTGCTAG pnbA-M193L-R SEQ ID NO. 79 GCGATACTCAGGCCCCCAGCAGATTCACC The whole plasmid single-point mutation PCR system is shown in Table 3.

[0040] Table 3 PCR reaction system Reagent Amount used Final concentration PrimeSTAR Max Premix (2x) 25 μL 1× Primer F 10 ~ 15 pmol 0.2 ~ 0.3 μM Primer R 10 ~ 15 pmol 0.2 ~ 0.3 μM Template plasmid < 200 ng Sterilized ultrapure water Supplemented to a total volume of 50 μL The whole plasmid single-point mutation PCR program is set as follows: (1) Denature at 98℃ for 10 seconds; (2) Anneal at 60℃ for 10 seconds; (3) Extend at 72℃ for 1 minute; (4) Repeat steps (1) to (3) for 35 cycles; (5) Extend at 72℃ for 5 minutes; (6) Store the amplified product at 4℃.

[0041] Example 2: Screening of the hydrated pinyl esterase pnbA mutant library.

[0042] like Figure 5 The diagram illustrates a high-throughput screening step for fluorescence-coupled immunosorbent assays (FISA) to screen for mutation sites that could potentially affect the activity and enantioselectivity of hydrated pinol esterases. Specifically, the saturated mutant library of all sites of pnbA obtained in Example 1 was electroporated into competent E. coli BL21(DE3) cells, plated, and allowed to grow overnight; then, single colonies were picked and placed in a solution containing 500 μL of 0.1 mg / mL... Kan + Incubate overnight in LB liquid medium in deep-well plates; then transfer 1% of the medium into 500 μL of 0.1 mg / mL solution. Kan + In deep-well plates of LB liquid medium, cultured until OD 600 When the concentration was approximately 0.8, 0.5 mM IPTG was added, and the mixture was cultured at 16°C and 220 rpm for 16 hours to express the target protein.

[0043] Centrifuge at 4500 rpm for 15 min to collect bacterial cells, discard the supernatant, and add 100 μL of lysis buffer to extract the crude enzyme solution. Prepare the fluorescent enzyme activity detection system (100 μL): 50 mM (1 S , 5 R ) - or (1 R , 5 S) - formic acid hydratpinol ester, 10 μL crude enzyme solution, 0.05 mg / mL formic acid oxidase AoFOX, 0.9 U / mL horseradish peroxidase HRP, 0.1 mM fluorescent substrate AR-2. Reaction at 37°C and real-time detection of fluorescence, excitation wavelength: 582 nm, emission wavelength: 602 nm, gain: 50, continuous shaking for 5 s, detection of fluorescence every 1 min, continuous detection for 30 min. The relative enzyme activity was calculated by calculating the rate of fluorescence increase, and the results are shown in Figure 6 and the superior mutant well sites were selected, and the mutant sequence information was obtained by sequencing.

[0044] Example 3: Preparation of wild type and mutant enzyme solution of hydratpinol esterase pnbA

[0045] The mutant plasmids obtained in Example 1 were respectively transformed into competent cells of Escherichia coli BL21 (DE3), and plated and grown overnight; then single colonies were respectively picked and placed in 15 mL LB liquid medium containing 0.1 mg / mL ampicillin, and cultured overnight; then transferred into 1 L LB liquid medium containing 0.1 mg / mL ampicillin at a ratio of 1%, and cultured until the OD Kan + was about 0.8, 0.5 mM IPTG was added, and the target protein was expressed at 16°C and 220 rpm for 16 hours. Kan + 600 was about 0.8, 0.5 mM IPTG was added, and the target protein was expressed at 16°C and 220 rpm for 16 hours.

[0046] The bacterial slurry was centrifuged to collect the bacterial slurry, resuspended with 40 mL buffer (10 mM PBS, 150 mM NaCl), and then pressure-crushed, with the treatment condition being 1100 Pa for 2 min; then centrifuged at 12000 rpm for 30 min, and then filtered once with a filter membrane, and placed on ice for standby; before purification, the nickel column was first washed with 10 column volumes of pure water, and then washed with 10 column volumes of buffer, and then the filtered supernatant was slowly poured in and flowed out, and 10 column volumes of imidazole buffer (10 mM PBS, 150 mM NaCl) containing 20 mM, 40 mM, 160 mM and 500 mM were used in turn to wash the nickel column and collect the flow-through liquid, and the target protein was mainly in the flow-through liquid with imidazole concentrations of 40 mM and 160 mM; the flow-through liquid containing high-purity target protein was then concentrated and desalted by a desalting gravity column, and then replaced into 10 mM PBS buffer; finally, the enzyme solution was adjusted to a concentration of 5 mg / mL (containing 30% glycerol), and stored in a refrigerator at -20°C for standby.

[0047] Example 4: Screening of pnbA superior mutant with high enantioselectivity by gas chromatography ​Selecting mutant sites from Example 2 that showed significant differences in activity between the two chiral pinene acetate hydrates, the pnbA gene was subjected to directed evolution based on biochemical characteristics such as amino acid volume, hydrophobicity, isoelectric point, and hydrogen bonding. The mutants with the best performance in each round were used as the basis for further iterative combination mutations in the next round. For example... Figure 7 As shown in Figure A, M1 (F314Y) was selected as the starting point for the second round of evolution in the first round. The obtained combined mutant plasmids were transformed into competent E. coli L21 (DE3) cells, plated, and allowed to grow overnight. Single colonies were then picked and placed in a solution containing 500 μL of 0.1 mg / mL... Kan + Incubate overnight in LB liquid medium in deep-well plates; then transfer at a 1% ratio to 1 L of 500 μL 0.1 mg / mL solution. Kan + In deep-well plates of LB liquid medium, cultured until OD 600 When the concentration was approximately 0.8, 0.5 mM IPTG was added, and the mixture was cultured at 16°C and 220 rpm for 16 hours to express the target protein.

[0048] Bacterial cells were collected by centrifugation at 4500 rpm for 15 min. The supernatant was discarded, and the bacterial suspension was resuspended in 100 μL of PBS buffer. A gas chromatography reaction system (1 mL) was prepared: 50 mM trans-pinene acetate hydrate, 10 μL of resuspended bacterial suspension, and 50 mM phosphate buffer (pH=7.0). After reacting at 37℃ for 2 h, the reaction was terminated with ethyl acetate, and the chiral ester substrate and chiral alcohol product were extracted. The extracted compounds were then qualitatively and quantitatively analyzed by gas chromatography. The results are as follows: Figure 7 and Figure 8 As shown, the hydrated pinol esterase pnbA mutants M1 (F314Y), M2 (F314Y / L362R), M3 (L273F / F314Y / L362R), and M4 (M193G / L273F / F314Y / L362R) performed optimally in each round of evolution. ee p The values ​​all showed a significant improvement compared to the previous generation, especially for mutants M3 (L273F / F314Y / L362R) and M4 (M193G / L273F / F314Y / L362R). ee p The values ​​reached 97.20% and 99.35% respectively. E The values ​​reached 104.66 and 482.59 respectively.

[0049] Example 5: Kinetic determination of wild-type and mutant pinyl esterase pnbA.

[0050] Kinetic parameters of wild-type hydrated pinol esterase pnbA and mutants M3 and M4 prepared in Example 4 were determined by gas chromatography. Each 1 mL reaction system contained 0.1 mg / mL of wild-type pnbA purified enzyme or 0.05 mg / mL of mutant purified enzyme, 50 mM PBS (pH 7.0), and substrate concentrations at different gradients (0–200 mM). After incubation at 37°C and 180 rpm for 30 minutes in a shaker, the substrate and product concentrations were determined by GC. All measurements were repeated three times. Kinetic parameters ( V max , K m , k cat and k cat / K m The results were obtained through curve fitting based on Michael dynamics. Figure 9 As shown, mutant M3 exhibits the best enzyme activity mechanical properties, with the best resistance to (1) S , 5 R )-Pinyl acetate hydrate k cat / K m It reached 1.03 s -1 ·mM -1 , and for (1 R , 5 S Pinyl acetate hydrate is inactive.

[0051] Example 6: Whole-cell catalytic continuous flow reaction using the engineered hydrated pinol esterase pnbA mutant M3.

[0052] The mutant M3 (L273F / F314Y / L362R), as determined in Example 5, was selected for a continuous flow reaction based on whole-cell catalysis. This continuous flow apparatus is as follows: Figure 10 As shown in Figure A, the column consists of two vertically connected columns. The column packing material forms a sandwich-like structure, with the middle layer containing a mixture of whole mutant M3 cells and diatomaceous earth. Racemic trans-pinene acetate hydrate was dissolved in PBS (pH 7.0) to a final concentration of 100 mM. Before the reaction, the continuous flow apparatus was washed three times with PBS, and then 20 mL of substrate solution was slowly pumped in at a constant flow rate using a peristaltic pump. The reaction was stopped after 1 hour, and the column was washed three more times with PBS. The product was collected and analyzed by GC. The results are shown below. Figure 10 As shown in Figure B, after seven consecutive repeated reactions using this apparatus, the enantioselectivity of the corresponding products is ( ee pThe values of the conversion rate and the selectivity remained relatively stable, and were maintained at 95.00% to 96.86% and 87.05% to 89.28%, respectively.

[0053] Although embodiments of the present application have been disclosed in connection with the above description and the accompanying drawings, it should be understood that they are not intended to limit the application to the details described therein, but rather that they are intended to cover all modifications and alternatives falling within the scope of the application as defined by the appended claims and their equivalents.

Claims

1. A high enantioselectivity hydrated pinanol esterase mutant, characterized in that, The hydrated pinanol esterase mutant is obtained by mutating the amino acid sequence of wild-type hydrated pinanol esterase pnbA shown in SEQ ID NO. 1 in the following combinations of one or more of the following ways: mutating methionine (M) at position 193 to glycine (G), mutating leucine (L) at position 273 to phenylalanine (F), mutating phenylalanine (F) at position 314 to tyrosine (Y), and mutating leucine (L) at position 362 to arginine (R).

2. The high enantioselective hydrated pinanol esterases mutant according to claim 1, characterized in that, The hydrated pinanol esterase mutant is obtained by mutating the amino acid sequence of hydrated pinanol esterase pnbA shown in SEQ ID NO. 1 in any of the following (1) - (4) ways: (1) mutating phenylalanine (F) at position 314 to tyrosine (Y) to obtain a single-site mutant denoted F314Y; (2) mutating phenylalanine (F) at position 314 to tyrosine (Y) and mutating leucine (L) at position 362 to arginine (R) to obtain a combined mutant denoted F314Y / L362R; (3) mutating leucine (L) at position 273 to phenylalanine (F), mutating phenylalanine (F) at position 314 to tyrosine (Y), and mutating leucine (L) at position 362 to arginine (R) to obtain a combined mutant denoted L273F / F314Y / L362R; (4) mutating methionine (M) at position 193 to glycine (G), mutating leucine (L) at position 273 to phenylalanine (F), mutating phenylalanine (F) at position 314 to tyrosine (Y), and mutating leucine (L) at position 362 to arginine (R) to obtain a combined mutant denoted M193G / L273F / F314Y / L362R.

3. The high enantioselective hydrated pinanol esterases mutant according to claim 2, characterized in that, The amino acid sequences of the mutants F314Y, F314Y / L362R, L273F / F314Y / L362R, and M193G / L273F / F314Y / L362R are SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, respectively.

4. A gene encoding the hydrated pinanol esterase mutant of any one of claims 1-3.

5. The gene according to claim 4, characterized in that, The nucleotide sequence encoding the wild-type hydrated pinanol esterase pnbA shown in SEQ ID NO. 1 is SEQ ID NO. 6, and the genes encoding the hydrated pinanol esterase mutants are all obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO.

5.

6. A recombinant plasmid comprising the gene of claim 4 or 5.

7. The recombinant plasmid of claim 6, wherein, The plasmid vector of the recombinant plasmid is pET-28a(+).

8. A host cell comprising the gene of claim 4 or 5 or the recombinant plasmid of claim 6 or 7.

9. The host cell of claim 8, wherein, The host cell is a prokaryotic cell.

10. The host cell of claim 8, wherein, The host cell is E. coli carrying the recombinant plasmid of claim 6 or 7. Escherichia coli BL21(DE3).

11. A method for constructing a high enantioselective hydrated pinanol esterase mutant according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: culturing the host cell of any one of claims 8-10, inducing expression of the hydrated pinanol esterase mutant, collecting the host cell, breaking it up, centrifuging, and separating and purifying the hydrated pinanol esterase mutant from the supernatant.

12. Use of a mutant pinol hydratase according to any one of claims 1 to 3, a gene according to claim 4 or 5, a recombinant plasmid according to claim 6 or 7, a host cell according to any one of claims 8 to 10 for catalysing the production of a pre-ester compound. 1S , 5R - pinol hydrate.

13. Use according to claim 12, characterized in that, The pre-ester compound is racemic trans-pinol formate hydrate or racemic trans-pinol acetate hydrate.

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