Epoxy hydrolase mutant and application thereof in asymmetric synthesis of (S)-flutriafol epoxy intermediate

Abstract

The invention discloses an epoxy hydrolase mutant and application thereof in asymmetric synthesis of an (S)-flutriafol epoxy intermediate, and belongs to the technical field of enzyme engineering. According to the invention, the epoxy hydrolase mutant RpEH336W / L360F with improved enantioselectivity is obtained through site-directed mutagenesis of epoxy hydrolase. Compared with a wild type, the enantioselectivity of the epoxy hydrolase mutant RpEH336W / L360F on a rac-flutriafol epoxy intermediate is remarkably improved. Compared with an expensive chiral chromatographic separation method, a novel method which is environment-friendly, simple and convenient in process flow and low in production cost is provided for synthesis and application of the flutriafol single enantiomer, and the method has a relatively high application prospect.

Description

Technical Field

[0001] The present invention relates to an epoxide hydrolase mutant and its application in asymmetric synthesis ( S The invention discloses an application of the invention in the epoxy intermediate of )-flutriafol, and belongs to the technical field of enzyme engineering. Background Art

[0002] As chiral pesticides are widely used in agricultural production, the differences in biological activity, toxicity and environmental fate between enantiomers of chiral pesticides have attracted more and more attention (Dong et al. , 2016). Flutriafol is a typical chiral triazole fungicide with the advantages of broad fungicide spectrum, high efficiency, low toxicity and long lasting effect. It is widely used to control powdery mildew and stripe rust of wheat, powdery mildew of strawberry, false smut and sheath blight of rice, etc. Studies have shown that there are differences in the acute toxicity of flutriafol and its enantiomers to non-target macrodaphnia, among which ( R )-flutriafol toxicity is rac -1.56 times of flutriafol, which is ( S )-4.65 times of flutriafol. The order of activity of flutriafol and its enantiomers against target wheat stripe rust is ( S )-flutriafol>( R )-flutriafol> rac -flutriafol, the activity multiples were not much different, and all three showed good fungicidal activity against wheat stripe rust (Tao Yan, 2015, Master's thesis, Chinese Academy of Agricultural Sciences). Zhang et al Discover( S )-flutriafol is preferentially degraded in cucumber, ( R ) form of enrichment. On the contrary, ( R )-enantiomer degrades faster in tomatoes than its antipodes (Zhang et al. , 2015). Therefore, selecting high-efficiency and low-toxic single enantiomers according to different target crops is of great significance for improving biological activity and reducing pesticide residues.

[0003] At present, the preparation of single enantiomers of flutriafol is mainly carried out by high performance liquid chromatography asymmetric resolution or precious metal catalyst synthesis (Ceng et al. , 2016; Chang et al. , 2018), but this method has the disadvantages of high cost and low yield, making it difficult to achieve industrial production and large-scale application. The enantiopure flutriafol epoxy intermediate and its hydrolysis product, the flutriafol vicinal diol intermediate, are important intermediates for the synthesis of chiral flutriafol (Chang et al., 2008). Epoxide hydrolases (EHs) are a class of hydrolases that can catalyze the stereoselective ring-opening hydrolysis of epoxides, retaining single-configuration epoxides and generating corresponding vicinal diols. They are ideal biocatalysts for the synthesis of chiral epoxide intermediates. Asymmetric catalysis mediated by bioenzymatic methods is one of the effective ways to obtain optically active monomers of some triazole fungicides. However, the large steric hindrance of the epoxide intermediate of triazole makes it difficult to achieve its highly stereoselective synthesis by chemical and biological methods. Previous studies have found that an epoxide hydrolase can catalyze large steric hindrance epoxide substrates, but the stereoselectivity is poor (CN 117143934 A). In order to better apply epoxide hydrolases to the industrial production of enantiopure epoxides and vicinal diols, R It is necessary to optimize the structure and catalytic properties of EH. Summary of the invention

[0004] The first object of the present invention is to provide an asymmetric synthesis ( S )-Epoxide hydrolase mutants of the epoxide intermediate of flutriafol.

[0005] In one embodiment of the present invention, the epoxide hydrolase mutant R EH H336W / L360F The gene is R. paludigena The mutant gene was constructed using the epoxide hydrolase gene from JNU001 (GenBank accession number: MK748445) as a template.

[0006] The epoxide hydrolase mutant R EH H336W / L360F Yes R. paludigena The amino acid at position 336 of the epoxide hydrolase from JNU001 mutated from histidine to tryptophan, and the amino acid at position 360 mutated from leucine to phenylalanine.

[0007] In one embodiment of the present invention, the mutant contains the amino acid sequence shown in SEQ ID NO:1.

[0008] The second object of the present invention is to provide a method for improving the enantioselectivity of epoxide hydrolase, wherein the epoxide hydrolase shown in SEQ ID NO: 1 or a cell expressing the epoxide hydrolase shown in SEQ ID NO: 1 is added to a system containing a racemic flutriafol epoxide intermediate, and under suitable reaction conditions, ( S )-flutriafol epoxy intermediate and ( R )-flutriafol vicinal diol intermediate.

[0009] The reaction is catalyzed in a reaction system with a pH of 6.5-8.0 and a reaction temperature of 20-35°C.

[0010] The concentration of the recombinant bacterial epoxide hydrolase mutant in the reaction system was 25-200 mg / mL based on the mass concentration of the wet bacteria, and the concentration of the racemic flutriafol epoxide intermediate substrate was 20-100 mmol / L.

[0011] In the reaction system, the dosage of the substrate racemic fluconazole epoxy intermediate and the recombinant bacterial wet cells is 0.1-5 mmol:1 g.

[0012] In one embodiment of the present invention, a mutant enzyme having both improved enantioselectivity and catalytic activity is used. Rpeh H336W / L360F , a highly enantiopure trifloxylidene epoxide ( ee = 97.5%), providing a new greener and more environmentally friendly method for the synthesis of single enantiomers of flutriafol.

[0013] The present invention also provides the above-mentioned epoxide hydrolase mutant, recombinant bacteria E.coli / Rpeh H336W / L360F Application in the preparation of chiral epoxy intermediate products.

[0014] The present invention also provides application of the method in the fields of chiral pesticides, chiral drugs, etc.

[0015] Beneficial effects of the present invention: The present invention obtains a mutant H336W / L360F with improved enantioselectivity by site-directed mutagenesis of epoxide hydrolase. Compared with the wild type, H336W / L360F has an improved enantioselectivity. rac - Flutriafol epoxy intermediate E Value increased from 5.3 to 30.5, for rac -The enantioselectivity of the flutriafol epoxide intermediate was increased by 5.8 times. R EH H336W / L360F Whole-cell kinetic resolution 20 mmol L -1 of rac -flutriazole epoxy intermediate, after 2 h of reaction, ( S )-Enantiomeric excess of flutriafol epoxy intermediate ee The yields were 97.6% ee s and 43.1%. Compared with the expensive chiral chromatography separation method, this method provides a new method for the synthesis of single enantiomers of flutriafol that is environmentally friendly, simple in process flow, and low in production cost, and has a high application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Recombinant bacteria E.coli / Rpeh Split rac -Reaction process of flutriafol epoxy intermediate; Figure 2 Recombinant bacteria E.coli / Rpeh H336W / L360F Split rac -Reaction process of flutriafol epoxy intermediate; Figure 3 Recombinant bacteria E.coli / Rpeh H336W / L360F Split rac -HPLC spectrum of fixed-point sampling of flutriafol epoxy intermediate. DETAILED DESCRIPTION

[0017] Mutant naming method: The mutant is represented by "the amino acid replaced by the original amino acid position". For example, H336W means that the amino acid at position 336 is replaced by Trp from His, and the position number corresponds to R Amino acid sequence of EH.

[0018] Whole Cell Specific Viability and Enantioselectivity Assays: Add 100 μL of 200 mg / mL recombinant bacterial suspension (to a final concentration of 40 mg / mL wet bacteria) and 350 μL of potassium phosphate buffer (100 mM, pH 7.0) to a 2 mL EP tube, preheat at 30°C for 5 min; add 50 μL of 200 mM racemic cyclohexane epoxy intermediate (to a final concentration of 20 mM), react for 10 min, take 100 μL and add 1 mL of ethyl acetate to extract, dry the organic phase over anhydrous magnesium sulfate, pass through a 0.22 μm organic membrane, and perform normal phase HPLC chromatography analysis. Normal phase HPLC chromatography conditions: 1260 Infinity II Agilent high performance liquid chromatograph, using a chiral Chiralcel OD-H column (250 mm×4.6 mm), the detection column temperature is 30°C, the flow rate is 0.6 mL / min, the mobile phase is n-hexane / isopropanol (90:10, v / v), the UV detector is monitored at 220 nm, ( R )-and( S The retention times of the epoxide intermediates of )-flutriafol were 7.27 min and 8.05 min, respectively; R )-and( S The retention times of the )-vicinal diol intermediates were 13.52 min and 14.67 min, respectively.

[0019] Definition of enzyme activity unit: Under the assay conditions, the amount of wet bacteria required to consume 1 μmol of trifloxylidene epoxide intermediate per minute is defined as 1 epoxide hydrolase activity unit (U).

[0020] Calculation formula: (1) ; (2) ; (3) Whole cell specific activity (U·g -1 ) is calculated as shown in formula (1), where C0 is rac -The initial concentration of flutriafol epoxy intermediate, v is the reaction volume, c Catalysis for whole cells rac -Conversion rate of flutriafol epoxide intermediate, t is the reaction time, m is the total cell mass. ee s and products ee p The calculation formula (2) shows that R s and S s Respectively represent ( R )-flutriafol epoxy intermediate and ( S )-flutriafol epoxy intermediate peak area, R p and S p express( R )-flutriafol vicinal diol intermediate and ( S )-flutriazole vicinal diol intermediate peak area. Enantioselectivity of enzymes is usually expressed as enantiomeric ratio. E ) to evaluate, E The higher the value, the higher the enantioselectivity, and its calculation formula is as shown in (3). Example 1 Construction of epoxide hydrolase double mutant

[0021] The SanPrep column plasmid DNA small-scale extraction kit (purchased from Shanghai Biotechnology) was used to extract DNA from the laboratory collection. E. coli BL21(DE3) / pET-28a- Rpeh The plasmid was extracted from the PCR product and used as a template. The upstream primers were L360F-F (TGTCCAACTTTCCCGACGAGTTCTTTACGCC) and H3363W-F (5'-3'TGCTCTCAATCCTTGGTTCGGCACCTTCCT), and the downstream primer was pET28-2254-F (5'-3'GCCTTACTGGTTAGCAGAATG). The plasmid was used as a template for two-step PCR amplification of the whole plasmid. The PCR product was added to 1 μL Dpn IThe restriction endonuclease was incubated at 37℃ for 3-4 h to digest the template plasmid. E. coli BL21(DE3) competent cells were plated on LB solid plates containing Kan resistance and cultured at 37°C for 12-16 h. A single colony was picked for culture and sequencing to confirm the base sequence. The mutant enzyme with the amino acid sequence of SEQ ID NO: 1 was obtained. R Compared with EH, the amino acid at position 336 mutated from histidine to tryptophan, and the amino acid at position 360 mutated from leucine to phenylalanine.

[0022] SEQ ID NO:1 E.coli / Rpeh H336W / L360F Amino acid sequence MAAHSFTAPPAPYNIDFAPQVDDLHRRLDAARWPGQDVVPDDVDYGEHGAFGLGAGPSLALMKELAQEWRGQDQKQLQDHLNSYKNYRVEIEGLDIHFLHYPS ARADAFPLILCHGWPGGYHEFLHVLERLTEPKDQGSRAFHVVVPSMPGYAFSSPPKTAKWGMEDTARVFDKLMTGLGYVKYAAQGGDWGSITARCLGSLHKEN CVAVHLNFCPVPPPPFPLNMFNPRTLLDWMPRFVLPDERRAKLERGVAYIERGSSYYAMQNLTPRTPAYGLNDSPIGLAWIGEKMIPGIDKAVKHPNATLNREALFTTLSIYWFTGSIGSSFLPYALNPWFGTFLVSPRHHLPNFALSNFPDEFFTPEERDARRTGNLRWYKDAEDGGHFAALEKPEVFAEHVREAMGVLLSNQA. Example 2 Preparation of Epoxide Hydrolase Double Mutant Whole Cell Catalyst

[0023] The mutant enzyme expression vector was inoculated (inoculation volume was 1%) into 2 mL of LB medium containing 1% kanamycin and cultured overnight at 37°C and 220 rpm. 1 mL of the culture solution was transferred into 100 mL of LB medium containing 1% kanamycin and cultured until OD 600 When the concentration is 0.6-0.8, add 40 μL IPTG (500 mmol·L -1 ) to a final concentration of 0.2 mmol·L -1 The recombinant bacteria were collected by centrifugation after induction at 20°C for 8 h. racThe enzyme activity of the epoxide intermediate of -flutriafol was 4.8 U / g bacterial cells. Example 3

[0024] Whole-cell catalysis of the enzyme before and after mutation rac -Comparison of enzyme activities of flutriafol epoxide intermediates

[0025] The whole-cell wet bacteria of the recombinant bacteria were prepared into a 200 mg / mL bacterial suspension using potassium phosphate buffer (pH 7.0, 100 mmol / L). The reaction system is as follows: add 100 μL of bacterial suspension to a 2 mL EP tube containing 350 μL of potassium phosphate buffer and mix well, incubate in a 30°C water bath for 5 min, add 50 μL of trifloxylidene epoxide intermediate mother solution (final concentration is 20 mmol / L) to start catalysis. React in a constant temperature shaking reactor at 30°C and 1000 rpm for 10 min, take 50 μL of sample and extract with 1 mL of ethyl acetate, dry the organic phase over anhydrous magnesium sulfate, and analyze the sample by HPLC. Appropriately adjust the reaction time and bacterial suspension concentration to control the conversion rate to no more than 15%. The results showed that H336WL360F had a good effect on rac -The whole cell specific activity of flutriafol epoxide intermediate increased from 4.8 U·g -1 to 5.6 U·g -1 . Example 4

[0026] Whole-cell catalysis of the enzyme before and after mutation rac Comparison of enantioselectivity of flutriafol epoxy intermediates

[0027] The whole-cell wet bacteria of the recombinant bacteria were prepared into a 200 mg / mL bacterial suspension using potassium phosphate buffer (pH 7.0, 100 mmol / L). The reaction system was as follows: 270 μL of bacterial suspension was added to a 2 mL EP tube containing 1350 μL of potassium phosphate buffer and mixed, incubated in a 30°C water bath for 5 min, and 180 μL of trifloxylidene epoxide intermediate mother solution (final concentration was 20 mmol / L) was added to start catalysis. The reaction was carried out in a constant temperature shaking reactor at 30°C and 1000 rpm. Samples were taken at fixed points, 50 μL of the sample was extracted with ethyl acetate, the organic phase was dried over anhydrous magnesium sulfate, and the sample was determined by HPLC for residual ( R )-flutriafol epoxy intermediate and ( S )-concentration of flutriafol epoxy intermediate, ee s , ee p and c Draw the reaction progress curve (results such as Figure 1 and 2 ).

[0028] Through for R EH H336W / L360F Initial concentration for whole-cell kinetic resolution: 20 mmol L -1 of rac - Regular sampling and monitoring of flutriafol epoxy intermediates revealed that R EH H336WL360F Whole cell catalysis rac -No obvious by-products were observed during the hydrolysis of the flutriafol epoxy intermediate, among which ( R )-flutriafol epoxy intermediate is preferentially hydrolyzed by catalysis ( Figure 2 ), the concentration increased from the initial 10 mmol L -1 After 2 h of reaction, the concentration quickly dropped to 0.1 mmol L -1 , which is only 1.1% of the initial concentration. S The yield of )-flutriafol epoxy intermediate was 43.1%, ee s 97.5% ( Figure 3 ). At the same time, when the conversion rate c Measured at about 50% E Compared with the wild type, H336W / L360F rac - Flutriafol epoxy intermediate E Value increased from 5.3 to 30.5, for rac -The enantioselectivity of the flutriafol epoxide intermediate was increased by 5.8 times.

[0029] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention, all of which belong to the scope of protection of the present invention and should be based on the definition of the claims.

Claims

1. An epoxide hydrolase mutant, It is characterized in that The amino acid sequence of the epoxide hydrolase mutant is shown in SEQ ID NO:

1.

2. A coding gene, It is characterized in that The encoding gene encodes the epoxide hydrolase mutant according to claim 1.

3. An expression vector, It is characterized in that The expression vector contains the epoxide hydrolase mutant according to claim 1 or the coding gene according to claim 2.

4. A recombinant bacterium, It is characterized in that The recombinant bacterium contains the epoxide hydrolase mutant according to claim 1 or the encoding gene according to claim 2 or the expression vector according to claim 3.

5. The recombinant bacterium according to claim 4, It is characterized in that The host cell of the recombinant bacteria is Escherichia coli.

6. A method for enantioselectively hydrolyzing a racemic flutriafol epoxy intermediate using an epoxide hydrolase, It is characterized in that The method comprises adding the epoxide hydrolase mutant of claim 1 or the recombinant bacteria of claim 3 to a system containing a racemic flutriafol epoxide intermediate, and catalyzing a reaction to prepare ( S )-flutriafol epoxy intermediate and ( R )-flutriafol vicinal diol intermediate.

7. The method for enantioselectively hydrolyzing the racemic flutriafol epoxy intermediate by epoxide hydrolase according to claim 6, It is characterized in that The catalytic reaction is carried out in a reaction system with a pH of 6.5-8.0 and a reaction temperature of 20-35°C.

8. The method for enantioselectively hydrolyzing a racemic flutriafol epoxy intermediate by an epoxide hydrolase according to claim 6, It is characterized in that The concentration of epoxide hydrolase in the reaction system was 25-200 mg / mL based on the mass concentration of the wet cells of the recombinant bacteria, and the concentration of the racemic flutriafol epoxide intermediate substrate was 20-100 mmol / L.

9. Use of the epoxide hydrolase mutant according to claim 1 in the preparation of a chiral epoxy intermediate product.

Images