Epoxide hydrolase mutant with improved activity and thermal stability and application thereof

Abstract

The invention discloses an epoxide hydrolase mutant with improved activity and thermal stability and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. On one hand, the invention provides the epoxide hydrolase mutant with improved activity and thermal stability, and on the other hand, the invention provides application of the epoxide hydrolase mutant in preparation of meso-dihydroxysuccinic acid or salt thereof. According to the invention, alanine at the 142 site of the wild epoxide hydrolase is mutated into cysteine or tryptophan at the 178 site of the wild epoxide hydrolase is mutated into glutamine, so that the activity and thermal stability of the enzyme are greatly improved. Through a genetic engineering technology, some genetically engineered bacteria expressing the mutant epoxide hydrolase disclosed by the invention are obtained, and the meso-dihydroxysuccinic acid is produced by utilizing the genetically engineered bacteria, so that the activity of cells is greatly improved, the service life of the cells is greatly prolonged, and the requirements of current industrial application are met.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to an epoxide hydrolase mutant with improved activity and thermal stability and its application. Background Art

[0002] Epoxide hydrolases (EHs; EC 3.3.2.3) are widely present in nature and can function without cofactors. They are known to stereoselectively add water molecules to the epoxy ring of epoxides to produce optically active vicinal diols. They are widely present in organisms such as animals, plants, and microorganisms. EHs from microbial sources are increasingly recognized as biocatalysts for the preparation of enantiopure epoxides and vicinal diols, and they form an important group of enantiopure compounds in the chemical and pharmaceutical industries.

[0003] Epoxide hydrolase can hydrolyze epoxy succinic acid into dihydroxy succinic acid, which is the key enzyme for the synthesis of dihydroxy succinic acid. Most of the reported EHs so far are from bacteria. The stereoselective specificities of EHs from different sources are different, and their conversion products can be dextrorotatory L(+)-dihydroxy succinic acid, levorotatory D(-)-dihydroxy succinic acid, or meso(±)-dihydroxy succinic acid respectively, which are the three optical isomers of dihydroxy succinic acid.

[0004] Dihydroxy succinic acid is a white crystalline organic acid with the chemical formula C 4 H 6 O 6 , which exists in nature, especially in fruits such as grapes, and is widely used in industries such as food, medicine, chemical engineering, and light industry. There are two asymmetric carbon atoms in the dihydroxy succinic acid molecule, so there are three optical isomers. Among them, meso-dihydroxy succinic acid is relatively rare and is mainly used as an anti-caking agent in the form of ferrous dihydroxy succinate, and is also used in snow removal, preparation of baking powder, and drugs. However, the reported EHs that produce meso-dihydroxy succinic acid have poor thermal stability.

[0005] With the development of protein engineering technology and molecular biology, the use of directed evolution and rational / semi-rational design methods to artificially evolve and modify enzyme molecules has become a research hotspot in the current enzyme engineering field. Among them, error-prone PCR, DNA shuffling, semi-rational design, rational design, etc. have become common methods in enzyme molecule modification, greatly accelerating the protein evolution process. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for an epoxide hydrolase mutant with improved activity and thermal stability and its application.

[0007] The present invention is specifically implemented by the following technical solutions: In the first aspect of the present invention, an epoxide hydrolase mutant with improved activity and thermal stability is provided. This epoxide hydrolase mutant is obtained by performing single-site mutation on the 142nd or 178th position of the amino acid sequence of the wild-type epoxide hydrolase shown in SEQ ID No.1; the single-site mutation is to mutate alanine at the 142nd position to cysteine, or to mutate tryptophan at the 178th position to glutamine.

[0008] Furthermore, this epoxide hydrolase mutant is one of the following: 1) Mutate alanine at the 142nd position of the amino acid sequence of the wild-type epoxide hydrolase shown in SEQ ID No.1 to cysteine, and the amino acid sequence of this epoxide hydrolase mutant is as shown in SEQ ID No.2; 2) Mutate tryptophan at the 178th position of the amino acid sequence of the wild-type epoxide hydrolase shown in SEQ ID No.1 to phenylalanine, and the amino acid sequence of this epoxide hydrolase mutant is shown in SEQ ID No.3.

[0009] In the second aspect of the present invention, a gene encoding the above epoxide hydrolase mutant is provided.

[0010] In the third aspect of the present invention, a recombinant vector containing the above encoding gene is provided.

[0011] In the fourth aspect of the present invention, a host cell containing the above recombinant vector is provided.

[0012] In the fifth aspect of the present invention, the above epoxide hydrolase mutant is provided for use in the preparation of meso-dihydroxysuccinic acid or its salt.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Through rational design, site-directed mutagenesis, enzyme kinetics and thermal stability determination techniques, some epoxide hydrolase mutants are obtained, whose amino acid sequences are as shown in SEQ ID No.2 or SEQ ID No.3 and are encoded by the nucleotide sequences shown in SEQ ID No.5 or SEQ ID No.6.

[0014] 2. Through genetic engineering techniques, a new recombinant vector is obtained, which contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No.5 or SEQ ID No.6.

[0015] 3. By genetic engineering technology, a new host cell was obtained, which contains a recombinant vector. The recombinant vector contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No.5 or SEQ ID No.6, and this host cell can express a mutant epoxide hydrolase.

[0016] 4. In the present invention, the alanine at the 142nd position of the wild-type epoxide hydrolase was mutated to cysteine or the tryptophan at the 178th position was mutated to glutamine, which greatly improved the enzyme activity and thermal stability.

[0017] 5. By genetic engineering technology, some genetically engineered bacteria expressing the mutant epoxide hydrolase of the present invention were obtained. Using these genetically engineered bacteria to produce meso-dihydroxysuccinic acid greatly improved the cell activity and service life, meeting the requirements of current industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 SDS-PAGE diagrams of the purification of wild-type and mutant epoxide hydrolases. M represents the protein molecular weight standard, which are 180 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa, 10 kDa from top to bottom; lane 1 is the wild-type epoxide hydrolase; lane 2 is the mutant protein A142C; lane 3 is the mutant protein S178Q.

[0019] Figure 2 Enzyme activity results of wild-type and mutant epoxide hydrolases.

[0020] Figure 3 Thermal stability results of wild-type and mutant epoxide hydrolases. DETAILED DESCRIPTION OF THE INVENTION

[0021] In the following examples of the present invention, the relevant detection methods are as follows: (1) Method for enzyme activity assay: Add 100 μL of 0.8 M substrate trans - epoxy succinic acid sodium solution (pH 8.0) and an appropriate amount of pH 8.0 Tris - HCl buffer into a 1.5 mL EP tube, incubate at 37 °C for 10 min, add an appropriate amount of purified enzyme solution, and make the final reaction system 1 mL. React at 37 °C for 30 min, and terminate the reaction by boiling water bath for 5 min. Centrifuge the boiled reaction solution at 1000 rpm for 2 min, take the supernatant, dilute it with distilled water, inject it into a sample bottle through a 0.22 μm water membrane, and perform high - performance liquid chromatography to detect and analyze the product concentration. Under the above conditions, the definition of enzyme activity: The amount of enzyme that produces 1 μmol of dihydroxy succinic acid per minute at 37 °C is defined as one enzyme activity unit (U), and the number of enzyme activity units per milligram of protein is defined as specific activity, expressed as U / mg.

[0022] (2) Detection method for dihydroxy succinic acid content: The chromatographic column is Chirex 3126(D) - penicillanmine (50 nicilla), the mobile phase is 88% of 1 mM copper acetate and 50 mM ammonium acetate aqueous solution (adjusted to pH 4.5 with glacial acetic acid) and 12% isopropanol, the detection wavelength is UV280 nm, the column temperature is 60 °C, the flow rate is 1 mL / min, and the injection volume is 10 μL.

[0023] (3) Detection method for protein concentration: It is determined using a modified Bradford protein concentration assay kit (Sangon Biotech (Shanghai) Co., Ltd.).

[0024] The following further illustrates the present invention in combination with embodiments. It should be noted that these embodiments are only used to explain the present invention, rather than limiting the scope of the present invention.

[0025] Example 1: Synthesis of epoxide hydrolase gene and construction of genetically engineered bacteria According to the coding gene of epoxide hydrolase from Pseudomonas koreensis (GenBank accession number WP_151550555), Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the gene sequence, whose nucleotide sequence is shown in SEQ ID No.4, and add 6 histidine tags at the N - terminus of the protein. Using Nco I and BamH I restriction endonuclease sites, insert it into the pET - 15b(+) vector, and transform Escherichia coli BL21(DE3). Subsequently, a genetically engineered bacterium containing the epoxide hydrolase gene of Pseudomonas koreensis was obtained from Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequence shown in SEQ ID No.4 is translated to obtain the wild - type epoxide hydrolase shown in SEQ ID No.1.

[0026] Example 2: Rational design for screening and constructing epoxide hydrolase mutants (1) The three-dimensional structure of the epoxy succinic acid hydrolase was simulated using Swiss-Model. The b-factor of the enzyme was analyzed using HOTSOPTWIZARE v3.1 software, and the stability and molecular dynamics simulation calculations of the enzyme mutants were performed in combination with Discovery Studio 2019 client to screen suitable mutants. Among a large number of mutants, two mutants were A142C and S178Q.

[0027] (2) The upstream and downstream primers of the mutants were synthesized by Youkang Biotechnology. The primers used were A142C and S178Q, and their nucleotide sequences were two reverse complementary sequences shown in SEQ ID NO.7 and SEQ ID No.8. The reaction conditions were: pre-denaturation at 94°C for 1 min, denaturation at 98°C for 15 s, annealing and extension at 64°C for 5 min 30 s, for a total of 30 cycles. After PCR, 1% agarose gel electrophoresis was performed.

[0028] (3) The PCR products with obvious bands verified by agarose gel electrophoresis were treated with Dpn I restriction endonuclease to remove the methylated templates to reduce the growth of false-positive bacteria. The enzyme digestion reaction system was prepared by mixing 42 μL of the PCR product, 3 μL of DpnI, and 5 μL of 10×QuickCut Buffer. After gently mixing and briefly centrifuging, the digestion was carried out at 37°C for 30 min.

[0029] (4) The digested fragments were recovered according to the operating steps of the SanPrep column-type PCR product purification kit (Sangon Biotech (Shanghai) Co., Ltd.) to remove various enzyme proteins, primers, etc. in the reaction solution. The concentration of the recovered sample was measured using a micro-spectrophotometer.

[0030] (5) Add 10 μL of the PCR product to every 100 μL of competent cells. Gently flick the tube wall to mix the competent cells and the PCR product evenly, and then let it stand on ice for 30 min. After heat shock in a 42 °C water bath for 90 s, quickly place it in an ice bath and let it stand for 5 min. Note to reduce shaking during the transfer process. Add 800 μL of LB liquid medium without antibiotics (10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, pH 7.0) to the EP tube, mix well, and then incubate it on a shaker at 37 °C and 200 rpm for 1 h. Pipette 100 μL of the recovery solution and spread it evenly on the LB solid medium (10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, 15 g / L agar, pH 7.0) containing ampicillin (final concentration 100 μg / mL). Invert the plate and culture it in an incubator at 37 °C for 16 h.

[0031] (6) Pick a single colony grown on the plate and inoculate it into 5 mL of LB liquid medium containing ampicillin (final concentration 100 μg / mL). Culture it at 37 °C and 220 rpm for 16 h, and send it to Youkang Sequencing Company for bacterial liquid sequencing using the universal primer T7 of pET15b(+). The nucleotide sequence of primer T7 is as shown in SEQ ID No.9. Preserve the successfully sequenced mutant bacteria.

[0032] Two mutant strains were thus obtained. Among them, in mutant A142C, the codon (GCA) encoding alanine at position 142 mutated to the codon (ACG) encoding cysteine, and its nucleotide sequence is as shown in SEQ ID No.5. In mutant S178Q, the codon (AGT) encoding tryptophan at position 178 mutated to the codon (GTC) of glutamine, and its nucleotide sequence is as shown in SEQ ID No.6.

[0033] Example 3: Expression and Purification of Wild-Type and Mutant Epoxide Hydrolases The engineered bacteria in Example 1, and the glycerol bacteria of the A142C and S178Q mutants in Example 2 were streaked on an LB solid medium containing 100 μg / mL ampicillin resistance and cultured at 37 °C for 16 h. Well-grown single colonies were picked and placed in 5 mL of LB liquid medium, and ampicillin with a final concentration of 100 μg / mL was added. They were cultured at 37 °C and 220 rpm for 8 h to obtain the seed solution. The seed solution was inoculated into 1 L of LB liquid medium containing 1% lactose for induction expression according to an inoculation amount of 1%, and the induction conditions were 30 °C, 200 rpm, and the induction time was 16 h. After the fermentation was completed, the culture medium was removed by centrifugation at 4 °C and 5000 rpm for 10 min. The centrifuged cells were washed with 100 mL of 1% sodium chloride and then resuspended in 100 mL of lysis buffer (20 mmol / L Tris-HCl, 0.5 mol / L NaCl, pH 8.0). They were ultrasonically disrupted in an ice bath for 30 min. The disrupted cell suspension was centrifuged at 4 °C and 12,000 rpm for 15 min, and the supernatant was taken and loaded onto a Ni-NTA affinity column (purchased from Sangon Biotech (Shanghai) Co., Ltd.). The impurity components were washed away with the washing buffer (20 mmol / L Tris-HCl, 0.5 mol / L NaCl, pH 8.0), and the target protein was eluted with the elution buffer (20 mmol / L Tris-HCl, 0.5 mol / L NaCl, 20 / 50 / 200 mmol / L imidazole, pH 8.0). The collected enzyme solution was ultrafiltered thoroughly in a buffer of 20 mmol / L Tris-HCl, 0.5 mol / L NaCl, pH 8.0 at 4 °C, glycerol was added to a final concentration of 50%, and it was stored at -20 °C. The purification effect was detected by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). As Figure 1 shown, lane 1 is the wild-type epoxide hydrolase, lane 2 is the mutant protein A142C, and lane 3 is the mutant protein S178Q, indicating that the bands of the three proteins after purification are single and consistent with the theoretical molecular weight.

[0034] Example 4: Activity and Thermal Stability Analysis of Wild-Type and Mutant Epoxide Hydrolases The wild-type epoxide hydrolase, mutant epoxide hydrolase A142C, and mutant epoxide hydrolase S178Q purified in Example 3 were assayed for enzyme activity at 37 °C according to the above detection method. The results showed (see Figure 2) Under the condition of 37 °C, the specific activity of the mutant epoxide hydrolase was significantly higher than that of the wild-type epoxide hydrolase. The specific activities of the mutant epoxide hydrolases A142C and S178Q were 193.3 U / mg and 194.4 U / mg respectively, and their activities were 1.3 times that of the wild-type epoxide hydrolase (147.8 U / mg). This indicates that the mutation of the 142nd and 178th amino acids in the epoxide hydrolase can significantly improve the activity of the wild-type epoxide hydrolase.

[0035] The wild-type epoxide hydrolase, mutant epoxide hydrolase A142C, and mutant epoxide hydrolase S178Q purified in Example 3 were respectively placed at 50 °C for 0, 15, 30, 45, 60, 75, and 90 min, and then under the condition of 37 °C, the activities of the wild-type and mutant epoxide hydrolases were measured according to the above detection method, and the residual activity was calculated based on the control group. The control group was: these three epoxide hydrolases were directly assayed at 37 °C without incubation at 50 °C (i.e., placed at 50 °C for 0 min), and the corresponding enzyme activity was defined as 100%. The results are shown (see Figure 3 )

[0036] After the wild-type epoxide hydrolase was incubated at 50 °C for 15 min, the residual activity was 67.7%. Subsequently, the residual activity showed a rapid decline. After incubation at 50 °C for 60 min, only 3.3% of the activity remained. After incubation at 50 °C for 75 min, the activity was completely lost. However, for the mutant epoxide hydrolases A142C and S178Q, after incubation at 50 °C for 15 min, the residual activities were 82.3% and 84.5% respectively. Subsequently, the rate of decline in residual activity was significantly slower than that of the wild type. After incubation at 50 °C for 60 min, the residual activities were still 51.8% and 31.7% respectively. The half-lives (the time required for the enzyme activity to decrease to half of the initial enzyme activity) of mutant A142C and mutant S178Q at 50 °C were approximately 61 min and 47 min respectively, which were increased by 106.8% and 59.3% respectively compared to the half-life (29.5 min) of the wild-type epoxide hydrolase. This indicates that the temperature stability of the mutant epoxide hydrolase is significantly improved compared to the wild-type epoxide hydrolase. The mutant epoxide hydrolases described in the present invention have the amino acid sequences shown in SEQ ID NO.2 or SEQ ID NO.3, and they are obtained by expressing a recombinant vector containing the nucleotide sequence shown in SEQ ID NO.5 or SEQ ID NO.6 in a host cell; the recombinant vector of the present invention contains a DNA molecule with the nucleotide sequence shown in SEQ ID NO.5 or SEQ ID NO.6; the host cell of the present invention contains the recombinant vector, and the recombinant vector contains a DNA molecule with the main nucleotide sequence shown in SEQ ID NO.5 or SEQ ID NO.6.

[0037] Example 5: Preparation of meso-dihydroxysuccinic acid or its salt using the wild-type and mutant genetically engineered bacterial cells of the present invention The method for producing meso-dihydroxysuccinic acid or its salt in the present invention is as follows: in a solution of trans-epoxysuccinic acid or its salt, epoxide hydrolase (wild-type and / or mutant) and / or genetically engineered bacteria (wild-type and / or mutant) are added for an enzymatic reaction to generate meso-dihydroxysuccinic acid or its salt. The trans-epoxysuccinate involved in the present invention is a salt formed by trans-epoxysuccinic acid and various cations, and the cations include but are not limited to ammonium ions, potassium ions, sodium ions, calcium ions, etc. The following is an example: 1 g each of the wild-type and two mutant genetically engineered bacterial cells A142C and S178Q induced by isopropylthio-β-D-galactoside were respectively taken and reacted with shaking at 37 °C for 8 h in 1000 ml of 1 mol / L disodium trans-epoxysuccinate solution, and CaCl 2An aqueous solution was filtered and the precipitate was washed with water. Then, it was acid-hydrolyzed with sulfuric acid, refined by anion and cation exchange columns, concentrated, crystallized, and dried to obtain 145.2 g, 147.3 g, and 147.1 g of meso-dihydroxysuccinic acid respectively. During the transformation process, the conversion rates of wild-type and mutant genetically engineered bacteria were detected. When the conversion rate reached 95%, the wild-type genetically engineered bacteria, mutant A142C, and mutant S178Q required 6 h, 2.5 h, and 3 h respectively. Since the enzyme activities and thermal stabilities of these mutant genetically engineered bacteria are higher than those of the wild-type genetically engineered bacteria, their transformation time is significantly shortened and the transformation efficiency is significantly improved. The mutant genetically engineered bacteria described in the present invention contain a recombinant vector, and the recombinant vector contains a DNA molecule with a main nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO.5. The main nucleotide sequence encodes a protein shown in SEQ ID NO.2 or SEQ ID NO.3, and this protein has epoxide hydrolase activity.

Claims

1. A mutant of an epoxide hydrolase with improved activity and thermal stability, characterized in that: The epoxide hydrolase mutant is obtained by performing a single-site mutation at position 142 or position 178 of the amino acid sequence of the wild-type epoxide hydrolase shown in SEQ ID No. 1; the single-site mutation is to mutate the alanine at position 142 to cysteine, or to mutate the tryptophan at position 178 to glutamine.

2. The epoxide hydrolase mutant with improved activity and thermal stability according to claim 1, characterized in that: The epoxide hydrolase mutant is one of the following: 1) The alanine at position 142 of the amino acid sequence of the wild-type epoxide hydrolase shown in SEQ ID No. 1 is mutated to cysteine, and the amino acid sequence of the epoxide hydrolase mutant is shown in SEQ ID No. 2; 2) The tryptophan at position 178 of the amino acid sequence of the wild-type epoxide hydrolase shown in SEQ ID No. 1 is mutated to phenylalanine. The amino acid sequence of the epoxide hydrolase mutant is shown in SEQ ID No.

3.

3. A gene encoding the epoxide hydrolase mutant according to claim 1 or 2.

4. A recombinant vector containing the coding gene according to claim 3.

5. A host cell containing the recombinant vector according to claim 4.

6. Use of the epoxide hydrolase mutant according to claim 1 or 2 in the preparation of meso-dihydroxysuccinic acid or its salt.

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