Epoxide hydrolase mutant with high enantioselectivity and application thereof

Abstract

The invention discloses an epoxy hydrolase mutant with high enantioselectivity and application of the epoxy hydrolase mutant in preparation of an (R)-tebuconazole epoxy intermediate, and belongs to the field of biological catalysis. An epoxy hydrolase mutant RtEHY361F with improved enantioselectivity is obtained through site-directed mutagenesis of epoxy hydrolase, 10 mM rac-tebuconazole is subjected to hydrolytic kinetic resolution under the conditions that the temperature is 25 DEG C and the pH value is 7.0, compared with a wild type, the ees of the epoxy hydrolase mutant to a rac-tebuconazole epoxy intermediate is improved from 76.8% to 99.7%, the yield is improved from 33.1% to 34.25%, and the yield is improved from 33.1% to 34.25%. It is indicated that the mutant RtEHY361F has higher selectivity on (R)-tebuconazole in the catalysis process, so that generation of by-products is effectively reduced, the chiral purity is improved, and the mutant RtEHY361F has a high application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to an epoxide hydrolase mutant and an application thereof, which realizes the high-selective resolution of a racemic tebuconazole epoxy intermediate. Background Art

[0002] The efficient preparation of chiral epoxy intermediates is a key step in the synthesis of optically pure pesticides and drugs, but their enantioselective synthesis still faces major challenges. Although a variety of epoxy hydrolases with industrial application prospects have been found in nature, the actual production and application of existing enzymes still face many limitations, such as narrow substrate spectrum and insufficient operational stability. Au The catalytic efficiency of EH2 for o-nitrophenyl ethylene oxide ( k cat / K m = 0.533 mM⁻¹s⁻¹) was significantly lower than its activity towards phenylethylene oxide ( k cat / K m = 2.765 mM⁻¹s⁻¹), and the enzyme activity is rapidly lost at high temperatures (>40 °C). In addition, the catalytic activity of natural EHs towards non-natural substrates (such as epoxides containing heterocyclic or long-chain substituents) is generally low ( ee <50%), which limits its application in the synthesis of complex chiral molecules.

[0003] To address the technical bottlenecks of existing epoxide hydrolases (EHs), molecular engineering of these enzymes has become an important means to overcome these bottlenecks and develop new, highly efficient enzymes. Strategies for molecular engineering of epoxide hydrolases (EHs) primarily fall into three categories: directed evolution, rational design, and semi-rational design. These strategies optimize the enzyme's catalytic efficiency, stereoselectivity, and stability through diverse approaches, providing important tools for the biosynthesis of industrial-scale chiral compounds.

[0004] Previous studies have found that epoxide hydrolase from Rhodosporidium toruloides Rt While EHs demonstrate catalytic potential, their enantioselectivity and catalytic efficiency still require optimization. To better adapt epoxide hydrolases for industrial production, this study enhanced their catalytic performance through directed evolution, providing theoretical support for the development of green and efficient chiral synthesis technologies. This not only expands the potential for EHs in industrial catalysis but also offers a new strategy for the large-scale production of chiral pesticides, which is of great significance for promoting green chemical synthesis and sustainable development. Summary of the Invention

[0005] In order to solve the problem of insufficient enantioselectivity of existing epoxide hydrolases (EHs), the purpose of the present invention is to provide an epoxide hydrolase mutant with high catalytic efficiency and high enantioselectivity and its application.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows: The present invention first provides an epoxide hydrolase mutant Rt EH Y361F , the epoxide hydrolase mutant Rt EH Y361F The invention is to mutate the tyrosine Y at position 361 of the epoxide hydrolase derived from Rhodosporidium toruloides to phenylalanine F, and the obtained mutant contains the amino acid sequence shown in SEQ ID NO: 1.

[0007] The present invention provides a gene encoding the above amino acid sequence, wherein the epoxide hydrolase mutant Rt EH Y361F The gene is a mutant gene constructed using the epoxide hydrolase gene (GenBank ID: AAN32662.1) from Rhodosporidium toruloides as a template.

[0008] The present invention also provides a mutant comprising the above-mentioned epoxide hydrolase Rt EH Y361F Or a recombinant vector comprising the above encoding gene, and a recombinant strain comprising the above recombinant vector.

[0009] Preferably, the host cell of the above recombinant strain is Escherichia coli.

[0010] The present invention further provides the above-mentioned epoxide hydrolase mutant Rt EH Y361F A method for enantioselectively hydrolyzing a racemic tebuconazole epoxy intermediate is provided, wherein cells expressing the epoxide hydrolase shown in SEQ ID NO: 1 are added to a system containing racemic tebuconazole epoxy hydrolase, and under appropriate reaction conditions, ( R )-tebuconazole epoxy intermediate.

[0011] The reaction is catalyzed in a reaction system with a pH of 5.5 to 9.0 and a reaction temperature of 15 to 30°C.

[0012] In the reaction system, the chiral resolution reaction of racemic tebuconazole epoxy intermediate at a concentration of 5-100 mM was catalyzed by 60-200 mg / ml of epoxide hydrolase mutant whole cell suspension to obtain ee >99% high enantiomeric purity ( R )-tebuconazole epoxy intermediate.

[0013] Beneficial effects of the present invention: The present invention obtains a mutant Y361F with improved enantioselectivity by site-directed mutagenesis of epoxide hydrolase. Compared with the wild-type epoxide hydrolase, the enantioselectivity of the mutant Y361F is improved by site-directed mutagenesis of the epoxide hydrolase. Rt EH Y361F Whole-cell kinetic separation of 10 mM rac -tebuconazole epoxy intermediate, after 2.5h reaction, can obtain ( R )-tebuconazole epoxy intermediate enantiomeric excess ee The yield is 99.7% ee s The method for preparing a single enantiomer of tebuconazole provided by the present invention is green and environmentally friendly, has a simple process, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 (A) recombinant bacteria E.coli / Rteh Split rac -Reaction process of tebuconazole epoxy intermediate; (B) Recombinant bacteria E.coli / Rteh Y361F Split rac -Reaction process of tebuconazole epoxy intermediate. DETAILED DESCRIPTION

[0015] The present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0016] The high performance liquid chromatography used in the following examples of the present invention is as follows: Normal phase HPLC chromatographic conditions: 1260 Infinity II Agilent high performance liquid chromatograph, using a chiral Chiralcel OD-H column (250 mm × 4.6 mm), the column temperature was 30 °C, the flow rate was 0.6 mL / min, the mobile phase was n-hexane / isopropanol (90:10, v / v), and the UV detector was monitored at 220 nm; ( S )-and( R The retention times of the epoxide intermediates of )-tebuconazole were 6.8 min and 7.6 min, respectively.

[0017] Reverse-phase HPLC chromatographic conditions: Agilent 1260 Infinity II HPLC, ProntoSIL C18 column (150×4.6 mm), column temperature of 30°C, flow rate of 0.8 mL / min, mobile phase of methanol:water (90:10, v / v), UV detector monitoring at 220 nm, retention times of tebuconazole epoxy intermediate and its vicinal diol product were 9.29 min and 5.30 min, respectively.

[0018] Definition of enzyme activity unit: Under the assay conditions, the amount of wet bacteria required to consume 1 μmol of tebuconazole epoxy intermediate per minute was defined as 1 epoxide hydrolase activity unit (U).

[0019] Calculation formula: (1) ; (2) ; (3) Whole cell specific activity (U·g -1 ) is calculated as shown in formula (1), where C0 is rac- Initial concentration of tebuconazole epoxy intermediate (mM), v is the reaction volume (L), c is the conversion rate (%), t is the reaction time (min), and m is the mass of wet cells (g). ee s The calculation formula is shown in (2), where R s and S s Respectively represent ( R The peak areas of the (S)-tebuconazole epoxy intermediates are usually expressed as the enantiomeric ratio. E ) to evaluate, E The higher the value, the higher the enantioselectivity, and its calculation formula is as shown in (3).

[0020] Example 1: Construction and inducible expression of epoxide hydrolase mutants The vector with the mutant gene was amplified by one-step PCR of the whole plasmid. First, the mutant primers were designed and synthesized. The upstream primer was Y361F-F (TACCC GGGCG AGATC TTCTG CCCTG CAGAA CGGG) and the downstream primer was a universal primer with the sequence (GCCTT ACTGG TTAGC AGAAT G). The PCR product was used Dpn I enzyme digestion to remove the template plasmid. E.coli BL21 (DE3) competent cells were plated on LB solid medium containing kanamycin resistance, and after overnight culture, transformants were selected and cultured for sequencing to confirm the base sequence. A mutant with the amino acid sequence of SEQ ID NO: 1 was obtained.

[0021] Example 2: Preparation of Epoxide Hydrolase Mutant Whole-Cell Catalyst The mutant was inoculated (inoculation volume of 1%) into 2 ml of LB medium containing kanamycin at a final concentration of 50 mg / L, cultured at 37°C overnight with shaking, and then inoculated into a conical flask containing 100 ml of LB medium at a 1% (v / v) inoculation volume and cultured until the OD 600 When the concentration reached 0.6-0.8, IPTG was added as an inducer with a final concentration of 0.5 mM, and the cells were induced at 20 °C for 8 h. The cells were collected by centrifugation to obtain the mutant whole-cell wet cells.

[0022] Example 3: Determination of specific activity and enantioselectivity of epoxide hydrolase original enzyme At 25 °C, 150 mg / mL E. coli / Rteh The bacterial suspension was mixed with 100 mM potassium phosphate buffer (pH 7.0), preheated for 5 min, and then added to a final concentration of 10 mM rac After 10 min of oscillation reaction, 100 μL of the reaction solution was added to 900 μL of methanol, mixed thoroughly, and filtered through a 0.22 μm organic filter membrane for HPLC analysis.

[0023] The results showed that the epoxide hydrolase of bacteria E.coli / Rteh right rac The enzyme activity of the tebuconazole epoxide intermediate was 1.32 U / g bacterial cells.

[0024] Example 4: Determination of enzyme activity of epoxide hydrolase mutant strains The mutant whole-cell wet bacteria were prepared into a 200 mg / ml bacterial suspension using potassium phosphate buffer (pH 7.0, 100 mM). The reaction system was as follows: 375 μL of bacterial suspension was added to a 2 mL EP tube containing 100 μL of potassium phosphate buffer and mixed, incubated in a 25 ℃ water bath for 5 min, and 25 μL of pentoconazole epoxy intermediate mother solution (final concentration was 20 mM) was added to start catalysis. The reaction was carried out in a constant temperature shaker at 25 ℃ and 1000 rpm for 10 minutes, 100 μL of sample was taken and added to 900 μL of methanol, mixed, passed through a 0.22 μm organic filter membrane, and analyzed by reverse phase HPLC chromatography. The results showed that the mutant Y361F was not sensitive to the hydroxyl radical of Y361F. rac The enzyme activity of the tebuconazole epoxy intermediate was 1.27 U / g, which was not significantly changed compared with the original enzyme.

[0025] Example 5: Comparison of enantioselectivity of the original enzyme and mutants of epoxide hydrolase The reaction system was as follows: 1350 μL of bacterial suspension (final concentration 150 mg / mL) was added to a 2 mL EP tube containing 360 μL of potassium phosphate buffer and mixed thoroughly. The mixture was incubated in a 25°C water bath for 5 minutes. Catalysis was initiated by adding 90 μL of tebuconazole epoxy intermediate mother solution (final concentration 10 mM). The reaction was carried out in a constant temperature shaking reactor at 25°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. The sample was then analyzed by HPLC for residual ( R )-and( S )-tebuconazole epoxy intermediate concentration, ee s and c Draw the reaction progress curve (results as Figure 1 ).

[0026] turn out, Rt EH Y361F In the catalytic 10 mM rac -Tebuconazole process no obvious by-products are produced, among which ( S )-tebuconazole epoxy intermediate was preferentially hydrolyzed, and the conversion rate reached 68.5% at 150 min. ee s It was further improved to 99.7%, showing excellent chiral separation performance. Rt The final reaction of EH under the same conditions for 180 min was ee s Only 76.8%.

[0027] 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 coding 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 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, characterized in that The host cell of the recombinant bacteria is Escherichia coli.

6. A method for enantioselectively hydrolyzing a racemic tebuconazole 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 tebuconazole epoxy intermediate, and catalyzing the reaction to prepare ( R )-tebuconazole epoxy intermediate.

7. The method for enantioselectively hydrolyzing a racemic tebuconazole epoxy intermediate by using an epoxide hydrolase according to claim 6, wherein: The catalytic reaction is carried out in a reaction system with a pH of 5.5-9.0 and a reaction temperature of 15-30°C.

8. The method for enantioselectively hydrolyzing racemic tebuconazole epoxy intermediate using epoxide hydrolase according to claim 6, characterized in that: A whole cell suspension of the recombinant bacteria is added to a system containing a racemic tebuconazole epoxy intermediate, wherein the concentration of the whole cell suspension is 60-200 mg / mL, and the concentration of the racemic tebuconazole epoxy intermediate is 5-100 mmol / L.

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