An epoxy hydrolase mutant and its use in asymmetric synthesis of (s)-paraflutolan epoxy intermediate

Abstract

The present invention discloses an epoxide hydrolase mutant and its application in asymmetric synthesis ( S The present invention relates to the application of )-flutriafol epoxy intermediates in the field of enzyme engineering technology. The present invention obtains an epoxide hydrolase mutant with improved enantioselectivity by site-directed mutagenesis of epoxide hydrolase. Rp EH H336W / L360F Compared with the wild type, the epoxide hydrolase mutant Rp EH H336W / L360F right rac The enantioselectivity of the flutriafol epoxy intermediate is significantly improved. Compared with expensive chiral chromatography separation methods, this method provides an environmentally friendly, simple process, and low-cost method for the synthesis of single enantiomers of flutriafol, which has great application prospects.

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 field of enzyme engineering technology. 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. Flutriafol is a typical chiral triazole fungicide with a broad spectrum of activity, high efficacy, low toxicity, and a long-lasting effect. It is widely used to control powdery mildew and stripe rust in wheat, powdery mildew in strawberries, and false smut and sheath blight in rice. Studies have shown that flutriafol and its enantiomers differ in their acute toxicity to non-target Daphnia magna. R )-flutriafol toxicity is rac -1.56 times that of flutriafol, which is ( S )-flutriafol is 4.65 times more active than 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). 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 a single enantiomer with high efficiency and low toxicity according to different target crops is of great significance for improving biological activity and reducing pesticide residues.

[0003] Currently, the preparation of single enantiomers of flutriafol is mainly based on 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. Enantiopure flutriafol epoxy intermediate and its hydrolysis product 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 a single configuration of epoxides and generating the 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 epoxy intermediate of triacontanol makes it difficult to achieve high stereoselective synthesis by chemical and biological methods. Previous studies have found that an epoxide hydrolase can catalyze the reaction of large steric hindrance epoxide substrates, but with poor stereoselectivity (CN 117143934 A). In order to better apply epoxide hydrolases to the industrial production of enantiopure epoxides and vicinal diols, Rp It is very 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 )-flutriafol epoxy intermediate epoxide hydrolase mutant.

[0005] In one embodiment of the present invention, the epoxide hydrolase mutant Rp 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 Rp EH H336W / L360F It will R. paludigena The amino acid at position 336 of the epoxide hydrolase derived 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 epoxy 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 epoxide hydrolase mutant in the reaction system was 25-200 mg / mL based on the wet bacterial mass concentration, and the concentration of the racemic flutriafol epoxide intermediate substrate was 20-100 mmol / L.

[0011] In the reaction system, the usage ratio of the substrate racemic flutriafol 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 , obtained highly enantiopure trifloxetine 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 applications of the method in the fields of chiral pesticides, chiral medicines, and the like.

[0015] Beneficial effects of the present invention:

[0016] 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 enhanced enantioselectivity. rac - Flutriafol epoxy intermediate E Value increased from 5.3 to 30.5, for rac -The enantioselectivity of the flutriafol epoxy intermediate was increased by 5.8 times. Rp EH H336W / L360F Whole-cell kinetic resolution of 20 mmol L -1 of rac -flutriafol epoxy intermediate, after 2 h of reaction, can obtain ( S )-Enantiomeric excess of flutriafol epoxy intermediate ee and yields were 97.6% ee s 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 great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Recombinant bacteria E.coli / Rpeh Split rac -Reaction progress of flutriafol epoxy intermediate;

[0018] Figure 2 Recombinant bacteria E.coli / Rpeh H336W / L360F Split rac -Reaction progress of flutriafol epoxy intermediate;

[0019] Figure 3 Recombinant bacteria E.coli / Rpeh H336W / L360F Split rac -HPLC spectrum of fixed-point sampling of flutriafol epoxy intermediate. DETAILED DESCRIPTION

[0020] Mutant naming method:

[0021] 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 His to Trp. The position number corresponds to Rp Amino acid sequence of EH.

[0022] Whole-cell specific viability and enantioselectivity assays:

[0023] To a 2 mL EP tube, 100 μL of a 200 mg / mL recombinant bacterial suspension (to a final concentration of 40 mg / mL wet cells) and 350 μL of potassium phosphate buffer (100 mM, pH 7.0) were added and preheated at 30°C for 5 min. 50 μL of 200 mM racemic trifloxetine epoxide intermediate (to a final concentration of 20 mM) was added and allowed to react for 10 min. 100 μL of the solution was then extracted with 1 mL of ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate and passed through a 0.22 μm organic membrane for normal phase HPLC analysis. Normal phase HPLC conditions: Agilent 1260 Infinity II HPLC using a chiral Chiralcel OD-H column (250 mm × 4.6 mm), a column temperature of 30°C, a flow rate of 0.6 mL / min, and a mobile phase of n-hexane / isopropanol (90:10, v / v). The UV detector was 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.

[0024] Definition of enzyme activity unit: Under the assay conditions, the amount of wet bacteria required to consume 1 μmol of trifloxetine epoxide intermediate per minute is defined as 1 epoxide hydrolase activity unit (U).

[0025] Calculation formula:

[0026] (1)

[0027] ; (2)

[0028] ; (3)

[0029] 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 Whole-cell catalysis rac -Conversion rate of trifloxetine epoxy intermediate, t is the reaction time, m is the total cell mass. ee s and products ee p The calculation formula (2) is shown below, where 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 The enantiomeric ratio is usually used to measure the enantiomeric selectivity of enzymes. E ) to evaluate, E The higher the value, the higher the enantioselectivity, and its calculation formula is as shown in (3).

[0030] Example 1 Construction of Epoxide Hydrolase Double Mutant

[0031] The plasmid DNA was extracted from the laboratory using the SanPrep column-based small-scale plasmid DNA extraction kit (purchased from Shanghai Bioengineering). E. coli BL21(DE3) / pET-28a- RpehThe 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 entire plasmid. The PCR product was added to 1 μL Dpn I The template plasmid was digested by restriction endonuclease incubated at 37℃ for 3~4 hours. E. coli BL21 (DE3) competent cells were plated on LB solid plates containing Kan resistance and cultured at 37 ° C for 12-16 h. Single colonies were 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. Rp 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.

[0032] SEQ ID NO: 1

[0033] E.coli / Rpeh H336W / L360F Amino acid sequence

[0034] MAAHSFTAPPAPYNIDFAPQVDDLHRRLDAARWPGQDVVPDDVDYGEHGAFGLGAGPSLALMKELAQEWRGQDQKQLQDHLNSYKNYRVEIEGLDIHFLHYPS ARADAFPLILCHGWPGGYHEFLHVLERLTEPKDQGSRAFHVVVPSMPGYAFSSPPKTAKWGMEDTARVFDKLMTGLGYVKYAAQGGDWGSITARCLGSLHKEN CVAVHLNFCPVPPPPFPLNMFNPRTLLDWMPRFVLPDERRAKLERGVAYIERGSSYYAMQNLTPRTPAYGLNDSPIGLAWIGEKMIPGIDKAVKHPNATLNREALFTTLSIYWFTGSIGSSFLPYALNPWFGTFLVSPRHHLPNFALSNFPDEFFTPEERDARRTGNLRWYKDAEDGGHFAALEKPEVFAEHVREAMGVLLSNQA.

[0035] Example 2 Preparation of Epoxide Hydrolase Double Mutant Whole-Cell Catalyst

[0036] 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 to 100 mL of LB medium containing 1% kanamycin and cultured until the 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 After induction at 20℃ for 8 hours, the recombinant bacteria were collected by centrifugation. rac The enzyme activity of the epoxide intermediate of flutriafol was 4.8 U / g bacterial cells. Example 3

[0037] Whole-cell catalysis of the enzyme before and after mutation rac Comparison of enzyme activities of flutriafol epoxy intermediates

[0038] 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: 100 μL of bacterial suspension was added to a 2 mL EP tube containing 350 μL of potassium phosphate buffer and mixed, incubated in a 30°C water bath for 5 min, and 50 μL of triacontanol epoxy intermediate mother liquor (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 for 10 min, 50 μL of sample was extracted with 1 mL of ethyl acetate, the organic phase was dried over anhydrous magnesium sulfate, and the sample was analyzed by HPLC. The reaction time and bacterial suspension concentration were appropriately adjusted to control the conversion rate to no more than 15%. The results showed that H336WL360F had a high conversion rate to 15%. rac The whole cell specific activity of flutriafol epoxy intermediate increased from 4.8 U·g -1 to 5.6 U·g -1 . Example 4

[0039] Whole-cell catalysis of the enzyme before and after mutation rac Comparison of enantioselectivity of flutriafol epoxy intermediates

[0040] 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 well. The mixture was incubated in a 30°C water bath for 5 min, and 180 μL of triacontadine epoxy 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, and 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 )-flutriafol epoxy intermediate concentration, ee s 、 ee p and c Draw the reaction progress curve (results as Figure 1 and 2 ).

[0041] Through for Rp EH H336W / L360F The initial concentration for whole-cell kinetic resolution was 20 mmol L -1 of rac - Regular sampling and monitoring of flutriafol epoxy intermediates revealed that Rp EH H336WL360F Whole-cell catalysis rac -No obvious by-products were generated during the hydrolysis of the flutriafol epoxy intermediate, among which ( R )-flutriafol epoxy intermediate is preferentially catalytically hydrolyzed ( Figure 2 ), the concentration increased from the initial 10 mmol L -1 After 2 h of reaction, the concentration quickly decreased 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 around 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 epoxy intermediate was improved by 5.8 times.

[0042] 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 these changes and modifications fall within the scope of protection of the present invention and should be based on the definition of the claims.

Claims

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

1.

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

3. An expression vector, characterized in that The expression vector contains the coding gene according to claim 2.

4. A recombinant bacterium, characterized in that The recombinant bacteria contains the coding gene according to claim 2 or the expression vector according to claim 3.

5. The recombinant bacterium according to claim 4, 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, characterized in that: The method comprises adding the epoxide hydrolase mutant of claim 1 or the recombinant bacterium of claim 3 to a system containing a racemic flutriafol epoxy intermediate, and catalyzing the reaction to prepare ( S )-flutriafol epoxy intermediate and ( R )-flutriafol vicinal diol intermediate.

7. The method for enantioselectively hydrolyzing a racemic flutriafol epoxy intermediate using an epoxide hydrolase according to claim 6, wherein: 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 using an epoxide hydrolase according to claim 6, wherein: The concentration of epoxide hydrolase in the reaction system was 25-200 mg / mL based on the wet cell mass of the recombinant bacteria, and the concentration of the racemic flutriafol epoxide intermediate substrate was 20-100 mmol / L.

9. An epoxide hydrolase mutant as claimed in claim 1 is prepared by using racemic flutriafol epoxy intermediate as substrate ( S )-flutriafol epoxy intermediate and ( R )-flutriafol vicinal diol intermediates.

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