Hydratropinesterase mutants with high enantioselectivity, methods for their construction and use

Abstract

This invention discloses a highly enantioselective hydrated pinol esterase mutant, its construction method, and its applications, belonging to the field of enzyme engineering technology. The hydrated pinol esterase mutant provided by this invention is obtained through a high-throughput screening method based on fluorescence conjugation from Bacillus subtilis. Bacillus subtilis The hydrated pinyl esterase was obtained by single-point or combined mutations of leucine at position 273, phenylalanine at position 314, leucine at position 362, and methionine at position 193 in its amino acid sequence. Among them, mutant M4 exhibited the best catalytic performance for racemic pinyl acetate hydrate, with an enantiomeric excess of 99.35% (1...). S 5 R The conversion rate of 1-acetic acid hydrated pinyl esterase was 83.90%. The hydrated pinyl esterase mutant provided by the invention catalyzes (1) S 5 R Pinyl acetate hydrate has high enzyme activity. ee p High value, high 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 subjected to dynamic kinetic hydrolysis resolution under the action of an esterase catalyst, and a single configuration chiral intermediate can be obtained through separation 1S , 5R A finished product of hydrated 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 high enantioselective hydrated pinol esterase mutant, wherein the hydrated pinol esterase mutant is obtained by mutating the amino acid sequence of wild-type hydrated pinol esterase pnbA shown as 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 changed 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 hydrated pinol esterase mutant is obtained by mutating the amino acid sequence of hydrated pinol esterase pnbA shown as SEQ ID NO. 1 in any of the following (1) to (4):

[0007] (1) the phenylalanine (F) at position 314 is mutated to tyrosine (Y), and the obtained single-site mutant is denoted as F314Y;

[0008] (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;

[0009] (3) the leucine (L) at position 273 is changed 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;

[0010] (4) the methionine (M) at position 193 is mutated to glycine (G), the leucine (L) at position 273 is changed 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.

[0011] 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.

[0012] In a second aspect of the present application, a gene encoding the mutant pinol hydrate esterase as described above is provided.

[0013] Preferably, the nucleotide sequence encoding the wild-type pinol hydrate esterase 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. The genes encoding the mutant pinol hydrate esterases are obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO. 5.

[0014] In a third aspect of the present application, a recombinant plasmid comprising the gene as described above is provided.

[0015] Preferably, the plasmid vector of the recombinant plasmid is pET-28a(+).

[0016] In a fourth aspect of the present application, a host cell comprising the gene or the recombinant plasmid as described above is provided.

[0017] Preferably, the host cell is a prokaryotic cell.

[0018] Preferably, the host cell is E. coli BL21 (DE3) carrying the recombinant plasmid as described above. E. coli

[0019] In a fifth aspect of the present application, a method for constructing the mutant pinol hydrate esterase with high enantioselectivity as described above is provided, which comprises the following steps: culturing the host cell as described above, inducing expression of the mutant pinol hydrate esterase, collecting the host cell, breaking and centrifuging the host cell, and separating and purifying the mutant pinol hydrate esterase from the supernatant.

[0020] In a sixth aspect of the present application, the mutant pinol hydrate esterase, the gene, the recombinant plasmid, and the host cell as described above are used for catalyzing the production of a pre-ester compound in a pinol hydrate. 1S , 5R Preferably, the pre-ester compound is racemic trans-pinol formate or racemic trans-pinol acetate.

[0021] Preferably, the pre-ester compound is racemic trans-pinol formate or racemic trans-pinol acetate.

[0022] In a seventh aspect of the present application, a method for producing (1 S , 5​R A green synthesis method for 1-pinel hydrate, wherein the method involves adding a hydrated pinol esterase mutant to a reaction system to obtain a reaction solution; and extracting the product from the reaction solution to obtain the target product (1). S , 5 R )-Pinol hydrate.

[0023] Preferably, the concentration of the hydrated pinyl esterase mutant added to the reaction solution is 0.025~0.25 mg / mL.

[0024] 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.

[0025] The beneficial effects of this invention are:

[0026] 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 5 R Pinyl acetate hydrate has high enzyme activity. ee p High value, high conversion rate, and suitable for producing high-value chiral intermediates (1 S , 5 R Application prospects of 1-hydrated pinol and similar terpene diols.

[0027] The engineered hydrated pinol esterase mutant M3 exhibited excellent reusability in a continuous flow whole-cell catalytic system, with each batch reaction completed in just 1 hour. Furthermore, after seven cycles, the conversion and enantioselectivity remained stable (exceeding 87% and 95%, respectively), highlighting its potential for green kinetic resolution (1... S , 5 R The potential applications of 1-pinel hydrate; this stepwise engineering strategy based on high-throughput screening provides an efficient solution for the engineering of stereoselectivity of carboxylesterases. Attached Figure Description

[0028] Figure 1 This is a high-throughput screening-guided enantioselectivity step engineering technology roadmap provided by the present invention;

[0029] Figure 2 This invention obtains (1) through dynamic decomposition. S , 5 R )- hydration pinol route map;

[0030] Figure 3 This is a schematic diagram of the method for detecting the activity of hydrated pinol esterase used in this invention;

[0031] Figure 4 It originates from Bacillus subtilis Bacillus subtilis Structural model of hydrated pinol esterase pnbA and its relationship with (1) S , 5 R The molecular docking results of )-pinene acetate hydrate, wherein S189-E310-H399 is the catalytic triplet, and M193, L273, F314 and L362 are the target residues for mutation in this invention;

[0032] Figure 5 This is a schematic diagram of the high-throughput screening steps for fluorescence coupling provided by the present invention;

[0033] Figure 6 The wild-type and selected superior mutants of the hydrated pinol esterase pnbA in this invention target (1) S , 5 R )-Pinyl formate hydrate and (1 R , 5 S The relative enzyme activities of 1-formate hydrate pinyl ester were compared, with wild type versus (1 S , 5 R The activity of 1-formate hydrate pinyl was used for relative enzyme activity comparison;

[0034] Figure 7 This is the result of stepwise directed evolution of the dominant mutant of hydrated pinol esterase pnbA in this invention, wherein A: M1-M4 underwent 4 rounds of stepwise site-directed mutation screening. ee p Indicates (1) S , 5 R Enantiomeric excess of 1-pinel hydrate, ConV Indicates (1) S , 5 R A: Conversion rate of 1-pinel hydrate; B: GC detection spectra of WT and M1, M2, M3, and M4 dominant mutants after reaction;

[0035] Figure 8 In the stepwise directed evolution process of this invention, the wild-type (WT) hydrated pinol esterase pnbA and the optimal mutant pair (1) in each round are used. S , 5 R Enantioselectivity of 1-pinel hydrate E The value change trend graph, where 1S5R represents (1 S , 5 R )-Hydrated pinol;

[0036] Figure 9 The hydrated pinol esterase pnbA wild-type (WT) and mutant M3 / M4 pair in this invention (1 S , 5 R )-pine acetate hydrate and (1 R , 5 S Kinetic parameters of 1-acetic acid hydrate pinyl ester;

[0037] Figure 10 This invention provides a schematic diagram and results of a continuous flow reaction catalyzed by whole-cell hydratol esterase pnbA mutant M3, where A: schematic diagram of the continuous flow reaction apparatus; B: reaction at different reuse times (1 S , 5 R )- Hydrated pinol ee p Value and conversion rate. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0039] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0040] 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.

[0041] 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.

[0042] 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).

[0043] This invention provides a high-throughput screening-guided enantioselective step engineering method to obtain a performance-enhanced target hydrated pinol esterase pnbA mutant, referring to... Figure 1For its technical route, the method takes the enzyme activity of Bacillus subtilis p-nitrobenzyl esterase (pnBA) as the starting point, successfully identifies the key positive amino acid sites that affect the stereoselectivity of the enzyme, and then gradually introduces these active sites to ultimately obtain mutants with gradually improved performance.

[0044] The culture medium involved in the following examples is as follows:

[0045] 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.

[0046] LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and 50 mg / L kanamycin.

[0047] The fluorescence coupling detection principle of hydrated pinol esterase pnbA is as shown in the following formula: Figure 1 The specific scheme is as follows:

[0048] Take (1 S , 5 R )-formic acid hydrated pinol ester or (1 R , 5 S )-formic acid hydrated pinol ester as the initial reaction substrate, which is hydrolyzed by hydrated pinol esterase pnbA in the first stage reaction to generate (1 S , 5 R )-hydrated pinol or (1 R , 5 S )-hydrated pinol, and at the same time, equimolar number of formic acid is also produced. As a byproduct, formic acid can be specifically recognized and oxidized by formate oxidase (AoFOX) in the second stage coupling reaction to form CO2 and H2O2, and H2O2 is used by horseradish peroxidase (HRP) in the third stage coupling reaction to oxidize the fluorescence substrate AR-2 to produce fluorescent resorufin-Cl. The fluorescence intensity of the fluorescence product can be quickly and sensitively detected to relatively quantitatively analyze the activity of pnbA.

[0049] Fluorescent enzyme activity detection system (100 μL): 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 formate oxidase AoFOX, 0.9 U / mL horseradish peroxidase HRP, and 0.1 mM fluorescent substrate AR-2. As Figure 3The figure shows the esterase pnbA-formate oxidase-peroxidase fluorescence coupling strategy designed by the present application for the oxidation of chiral pinacol ester substrate to chiral pinacol product, which is used for the detection of pinacol esterase pnbA enzyme activity.

[0050] The reaction was carried out at 37°C and the fluorescence was detected in real time, with an excitation wavelength of 582 nm, an emission wavelength of 602 nm, a gain of 50, a continuous shaking of 5 s, a detection of fluorescence every 1 min, and a continuous detection for 30 min.

[0051] The qualitative and quantitative analysis of the chiral ester substrate and chiral alcohol product of the catalytic reaction was carried out by gas chromatography (GC), with N2 as the carrier gas at a flow rate of 1 mL / min, an injection volume of 1 μL, and the following temperature program: initial temperature of 120°C for 0.5 min, then heating from 120°C to 195°C at a rate of 1.5°C / min, and maintaining 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 formulas:

[0052]

[0053] In the formulas, C 1S5R and C 1R5S represent the final concentrations of (1 S , 5 R )-pinacol and (1 R , 5 S )-pinacol in the reaction system at the end of the reaction, respectively, and C 1S5R-ace represents the initial concentration of (1 S , 5 R )-pinacol acetate in the reaction system at the beginning of the reaction.

[0054] Example 1: Construction of a saturated mutation library and a site-directed mutation plasmid of pinacol esterase pnbA.

[0055] According to the amino acid sequence SEQ ID NO. 1 of wild-type pinacol esterase pnbA, protein homology modeling analysis was carried out 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

[0056] Using the primers shown in Table 1, we performed whole-plasmid single-point mutation PCR with wild-type plasmid pET-28a(+)-pnbA as a template to construct an NNK saturated mutation library for key amino acid sites. Then, we mixed the mutation libraries for different sites in equal molar amounts to construct a saturated mutation library for all sites. After confirming that the sequencing was correct, we extracted and preserved the plasmid.

[0057] Table 1. NNK primers for constructing the pnbA site saturated mutant library

[0058] 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

[0059] 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.

[0060] Table 2 Primers for constructing pnbA site-directed mutagenesis

[0061] 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

[0062] The whole plasmid single-point mutation PCR system is shown in Table 3.

[0063] Table 3 PCR reaction system

[0064] 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

[0065] The whole plasmid single-point mutation PCR program is set as follows:

[0066] (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℃.

[0067] Example 2: Screening of the hydrated pinyl esterase pnbA mutant library.

[0068] 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 600When 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.

[0069] 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 Pinyl formate hydrate, 10 μL crude enzyme solution, 0.05 mg / mL formate oxidase (AoFOX), 0.9 U / mL horseradish peroxidase (HRP), 0.1 mM fluorescent substrate AR-2. The reaction was carried out at 37℃ with real-time fluorescence detection. Excitation wavelength: 582 nm, emission wavelength: 602 nm, gain: 50, continuous shaking for 5 s, fluorescence detection every 1 min, continuous detection for 30 minutes. Relative enzyme activity was calculated by calculating the rate of fluorescence growth. Results are as follows. Figure 6 As shown, dominant mutant pore sites were selected, and mutant sequence information was obtained through sequencing.

[0070] Example 3: Preparation of wild-type and mutant hydrated pinyl esterase pnbA enzyme solutions.

[0071] The mutant plasmids obtained in Example 1 were transformed into competent E. coli BL21(DE3) cells, plated, and allowed to grow overnight; then, single colonies were picked and placed in 15 mL of solution containing 0.1 mg / mL... Kan + In LB broth, incubate overnight; then transfer 1% to 1 L of medium containing 0.1 mg / mL... Kan + In 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.

[0072] The bacterial slurry was collected by centrifugation, resuspended with 40 mL of buffer (10 mM PBS, 150 mM NaCl), and then subjected to pressure disruption at 1100 Pa for 2 min. Then, the sample was centrifuged at 12000 rpm for 30 min, and the supernatant was filtered once using a filter membrane and stored on ice. Before purification, the nickel column was washed with 10 column volumes of pure water, and then washed with 10 column volumes of buffer. Then, the filtered supernatant was slowly poured into and out of the column, and the column was washed with 10 column volumes of each of the following imidazole buffers (10 mM PBS, 150 mM NaCl) containing 20 mM, 40 mM, 160 mM, and 500 mM imidazole, respectively, and the flow-through was collected. The target protein was mainly contained in the flow-through with 40 mM and 160 mM imidazole. The flow-through containing the high-purity target protein was concentrated and desalted using a desalting gravity column, and then replaced into a 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.

[0073] Example 4: Screening of pnbA advantage mutants with high enantioselectivity by gas chromatography

[0074] Based on the biochemical properties of amino acids such as volume, hydrophobicity, isoelectric point, and hydrogen bonding, the pnbA gene was subjected to directed evolution by selecting the mutant sites prepared in Example 2 that had large differences in activity for the two chiral pinacols. The mutant with the best performance in each round was used as the basis for further iterative combinatorial mutation in the next round. As shown in Table A, M1 (F314Y) was selected as the starting point for the second round of evolution in the first round of evolution. The obtained combinatorial mutant plasmids were transformed into competent cells of E. coli L21 (DE3), and plated for overnight growth. Single colonies were picked and inoculated into deep-well plates containing 500 μL of 0.1 mg / mL LB liquid medium, and incubated overnight. Then, the cells were inoculated into 1 L of deep-well plates containing 500 μL of 0.1 mg / mL LB liquid medium at a ratio of 1%, and incubated until the OD Figure 7 Kan + was about 0.8, 0.5 mM IPTG was added, and the cells were incubated at 16°C and 220 rpm for 16 hours to express the target protein. Kan + 600

[0075] ​​​4500 rpm for 15 min, and the bacterial cells were collected. After the supernatant was discarded, 100 μL of PBS buffer was added to resuspend the bacterial cells. The gas chromatography reaction system (1 mL) was prepared: 50 mM trans-pinacolyl acetate, 10 μL of resuspended bacterial cells, and 50 mM phosphate buffer (pH 7.0). After 2 h of reaction at 37 °C, the reaction was terminated with ethyl acetate, and the chiral ester substrate and chiral alcohol product were extracted. The extracted compounds were qualitatively and quantitatively analyzed by gas chromatography. The results are shown in Table 1. Figure 7 and Figure 8 As shown in Table 1, the pnbA mutant M1 (F314Y), M2 (F314Y / L362R), M3 (L273F / F314Y / L362R), and M4 (M193G / L273F / F314Y / L362R) performed best in each round of evolution, and the ee p values of the mutants were significantly improved compared to the previous generation. In particular, the ee p values of the mutants M3 (L273F / F314Y / L362R) and M4 (M193G / L273F / F314Y / L362R) reached 97.20% and 99.35%, respectively, E and the

[0076] values reached 104.66 and 482.59, respectively.

[0077] Example 5: Kinetic determination of the pnbA wild-type and mutant hydratase enzymes. V max , K m , k cat and k cat / K m The kinetic parameters were calculated by curve fitting according to Michaelis-Menten kinetics. The results are shown in Table 2. Figure 9 As shown in Table 2, the mutant M3 had the best kinetic performance, and its S , 5 R )-pinacolyl acetate k cat / K m It reached 1.03 s -1 ·mM -1 , and for (1 R , 5 S Pinyl acetate hydrate is inactive.

[0078] Example 6: Whole-cell catalytic continuous flow reaction using the engineered hydrated pinol esterase pnbA mutant M3.

[0079] 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 p The conversion rate and the conversion value remained relatively stable, ranging from 95.00% to 96.86% and from 87.05% to 89.28%, respectively.

[0080] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A highly enantioselective hydrated pinol esterase mutant, characterized in that, The hydrated pinol esterase mutant was obtained by mutating the amino acid sequence of the hydrated pinol esterase pnbA as shown in SEQ ID NO.1 as follows: The 273rd leucine (L) is changed to phenylalanine (F), the 314th phenylalanine (F) is mutated to tyrosine (Y), and the 362nd leucine (L) is mutated to arginine (R). The resulting combined mutant is denoted as L273F / F314Y / L362R.

2. The highly enantioselective hydrated pinol esterase mutant according to claim 1, characterized in that, The amino acid sequence of the mutant L273F / F314Y / L362R is SEQ ID NO.

4.

3. A gene encoding the hydrated pinyl esterase mutant according to any one of claims 1-2.

4. The gene according to claim 3, characterized in that, The nucleotide sequence encoding the wild-type hydrated pinol esterase pnbA as shown in SEQ ID NO.1 is SEQ ID NO.

6. The genes encoding the hydrated pinol esterase mutant were all obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO.

6.

5. A recombinant plasmid containing the gene as described in claim 3 or 4.

6. The recombinant plasmid according to claim 5, characterized in that, The plasmid vector for the recombinant plasmid is pET-28a(+).

7. A host cell comprising the gene as described in claim 3 or 4 or the recombinant plasmid as described in claim 5 or 6.

8. The host cell according to claim 7, characterized in that, The host cell is a prokaryotic cell.

9. The host cell according to claim 7, characterized in that, The host cell is *Escherichia coli* E. carrying the recombinant plasmid described in claim 5 or 6. coli BL21(DE3).

10. A method for constructing a highly enantioselective hydrated pinol esterase mutant as described in any one of claims 1-2, characterized in that, The method includes the following steps: culturing the host cells according to any one of claims 7-9, inducing the expression of the hydrated pinyl esterase mutant; collecting the host cells for disruption and centrifugation, and separating and purifying the hydrated pinyl esterase mutant from the supernatant.

11. The hydrated pinol esterase mutant according to any one of claims 1-2, the gene according to claim 3 or 4, the recombinant plasmid according to claim 5 or 6, and the host cell according to any one of claims 7-9 in catalysis (1 S 5 R )-Pinyl formate hydrate or (1 S , 5 R )-Pinyl acetate hydrate formation 1S , 5R Applications of hydrated pinol.

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