A cytochrome P450 monooxygenase mutant and its application in hydroxylation of methylpyrazine compounds

Abstract

The present invention relates to a cytochrome P450 monooxygenase mutant and its application in hydroxylation of methylpyrazine compounds, belonging to the technical field of enzyme engineering. The amino acid sequence of the cytochrome P450 monooxygenase mutant provided by the present invention is shown in SEQ ID NO.2. The cytochrome P450 monooxygenase mutant L76S / A83E provided by the present invention exhibits hydroxylation activity towards methylpyrazine substrates, and the reaction conversion rate is greater than or equal to 95%, achieving a breakthrough in the catalytic activity of cytochrome P450 monooxygenase from non-existent to existent. Therefore, the cytochrome P450 monooxygenase and its gene described in the present invention have good prospects for industrial application development.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and particularly to a cytochrome P450 monooxygenase mutant and its application in hydroxylation of methyl pyrazine compounds. Background Art

[0002] Hydroxylation reaction is an important C-H activation reaction with high atom economy, which can realize the synthesis of hydroxyl compounds with higher added value from inert C-H. Cytochrome P450 monooxygenase (P450) is known as the universal catalyst in nature and can efficiently and highly selectively catalyze the oxidation reactions of various organic compounds with complex skeletons. In recent years, more and more studies have been carried out on the catalytic C-H bond activation by P450 monooxygenase.

[0003] As a kind of aromatic compounds, pyrazine is difficult to introduce new functional groups through electrophilic substitution. When the pyrazine molecule contains a methyl side chain, the activity of the hydrogen atom on the methyl group is significantly improved, which helps to enhance the application of such molecules in the fields of drugs, materials, etc. For example, 2,5-dimethylpyrazine (DMP) is a high-value-added alkyl pyrazine compound and has important application value in the fields of food and pharmacy. As an important pharmaceutical intermediate, its methyl group can be hydroxylated to produce 5-methylpyrazine-2-methanol, and 5-methylpyrazine-2-carboxylic acid can be synthesized through further carboxylation reaction. The product 5-methylpyrazine-2-carboxylic acid (MPCA) is a beige solid crystal and has a wide range of uses in the pharmaceutical industry, mainly used for synthesizing the second-generation sulfonylurea hypoglycemic drug glipizide, the new antihypertensive drug acipimox, and the antituberculosis drug methyl 5-methylpyrazine-2-carboxylate and other drugs.

[0004] Currently, there are two methods for synthesizing 5-methylpyrazine-2-carboxylic acid: chemical synthesis method and biocatalysis method. The chemical methods mainly include four synthetic routes: intermolecular cyclization method, multi-step synthesis method of pyrazine side chain, direct oxidation method and electrochemical method, but they all have problems such as high reaction conditions, high production cost, low product yield, and environmental pollution. The biocatalysis method has the typical characteristics of green biomanufacturing such as environmental friendliness, high catalytic efficiency, and simple synthesis process, and has developed into the primary choice to replace or expand traditional chemical synthesis in the field of pharmaceutical and chemical industry, with great development prospects.

[0005] In 2012, Zheng Yuguo et al. screened a Pseudomonas putida strain from soil. Using p-xylene as the sole carbon source and inducer, they carried out the biological preparation of 5-methylpyrazine-2-carboxylic acid with this strain. Through fed-batch fermentation, the yield could reach 75.6%, the product concentration was 20.41 g / L, and the cycle was as long as 22 days. In 2017, Feng Jing et al. disclosed a method for the enzymatic production of 5-methylpyrazine-2-carboxylic acid. The enzyme was an aldehyde dehydrogenase or a fusion protein obtained by connecting tags to the N-terminus and / or C-terminus of aldehyde dehydrogenase. Using 5-methyl-2-pyrazine aldehyde as the reaction substrate and catalyzing the reaction for 12 h, the final concentration of the product 5-methylpyrazine-2-carboxylic acid was 17.02 mM. This method does not require the exogenous addition of toxic xylene for induced culture, making the reaction process simpler and having a fast mass transfer rate. In 2020, Gu Liuyan et al. cloned four genes xylM, xylA, xylB, and xylC from Pseudomonas putida (ATCC 33015) and constructed a whole-cell catalytic pathway for 5-methylpyrazine-2-carboxylic acid in Escherichia coli. Among them, xylM and xylA encode XMO, and xylB and xylC encode BADH and BLDH respectively. By regulating the copy numbers of different genes to control the expression levels of each component protein, the optimization of the reaction process was achieved, and the final yield reached 11.6 g / L. In the same year, Cao Yanli et al. selected a high-yield 2,5-DMP strain by heterologously expressing L-threonine dehydrogenases from different microbial species in Bacillus subtilis 168. On this basis, a genetically engineered strain with high 2,5-DMP production was constructed. Using L-threonine as the fermentation substrate, the yield of 2,5-DMP was as high as 616.04 mg / L after 24 h of fermentation.

[0006] In summary, the existing technologies of biocatalysis mostly use 2,5-dimethylpyrazine as the starting substrate, and synthesize 5-methylpyrazine-2-carboxylic acid through three-step catalysis by xylene monooxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. However, all known xylene monooxygenases are membrane proteins, their catalytic activity severely depends on phospholipids, and there are defects such as low energy production, high production cost, and long cycle.

[0007] Therefore, there is an urgent need to develop a production method for 5-methylpyrazine-2-carboxylic acid that is simple, green, environmentally friendly, and efficient. Summary of the Invention

[0008] To solve the defects of the existing technology, the present invention provides a cytochrome P450 monooxygenase mutant and its application in hydroxylation of methylpyrazine compounds, achieving a breakthrough in the catalytic activity of cytochrome P450 monooxygenase from scratch.

[0009] The present invention is achieved through the following technical solutions:

[0010] The first object of the present invention is to provide a cytochrome P450 monooxygenase mutant, the amino acid sequence of the cytochrome P450 monooxygenase mutant is shown in SEQ ID NO.2.

[0011] The second object of the present invention is to provide a gene encoding the cytochrome P450 monooxygenase mutant, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0012] A third object of the present invention is to provide an expression vector carrying the gene. The expression vector can be constructed by cloning the cytochrome P450 monooxygenase mutant gene into various expression vectors using conventional methods in the art. The expression vector preferably includes various conventional vectors in the art, such as commercially available plasmids, cosmids, phage, or viral vectors, and the vector is preferably the pET28a plasmid.

[0013] A fourth objective of the present invention is to provide a recombinant bacterium expressing the aforementioned cytochrome P450 monooxygenase mutant. The recombinant bacterium is prepared by transforming the aforementioned expression vector into a host cell. The host cell is any conventional host cell in the art, as long as the recombinant expression vector can stably replicate on its own and the cytochrome P450 monooxygenase mutant gene carried by the recombinant expression vector can be effectively expressed. The host cell is preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3) or Escherichia coli DH5α.

[0014] A fifth object of the present invention is to provide the use of the cytochrome P450 monooxygenase mutant in hydroxylating methylpyrazine compounds, wherein the method of the use comprises the following steps:

[0015] In a buffer solution, a methylpyrazine compound is used as a substrate and the cytochrome P450 monooxygenase mutant is used as a catalyst to carry out a hydroxylation reaction to generate corresponding alcohol substances.

[0016] In one embodiment of the present invention, the methylpyrazine compound is selected from one or more of 2-methylpyrazine, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine and 2,6-dimethylpyrazine.

[0017] In one embodiment of the present invention, the added amount of the methylpyrazine compound is 10 mmol / L to 100 mmol / L.

[0018] In one embodiment of the present invention, the pH value of the buffer solution is 7.5-8.5, preferably 8.0.

[0019] In one embodiment of the present invention, the hydroxylation reaction is carried out by glucose dehydrogenase and NADP +It is carried out in the presence of

[0020] The addition amount of the NADP + is 0.1 mmol / L to 1 mmol / L; the addition amount of the glucose dehydrogenase is 0.1 g / L to 2 g / L.

[0021] In one embodiment of the present invention, the temperature of the hydroxylation reaction is 25°C to 30°C; the time of the hydroxylation reaction is 6 h to 24 h.

[0022] In one embodiment of the present invention, the addition form of the cytochrome P450 monooxygenase mutant is a crude enzyme solution containing the cytochrome P450 monooxygenase mutant; the addition amount of the cytochrome P450 monooxygenase mutant is 1 kU / L to 10 kU / L.

[0023] The present invention has at least the following advantages:

[0024] The present invention provides a cytochrome P450 monooxygenase mutant and its application in hydroxylation of methylpyrazine compounds. The cytochrome P450 monooxygenase mutant L76S / A83E provided by the present invention shows hydroxylation activity towards methylpyrazine compounds, and the reaction conversion rate is greater than or equal to 95%, achieving a breakthrough in the catalytic activity of wild-type P450 from non-existent to existent. Therefore, the cytochrome P450 monooxygenase and its gene described in the present invention have good prospects for industrial application development. Description of the Drawings

[0025] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention and in combination with the drawings, wherein

[0026] Figure 1 shows the protein expression situation analyzed by polyacrylamide gel electrophoresis of the crude enzyme solution in Example 3 of the present invention. Detailed Embodiments

[0027] The following further illustrates the present invention in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and can implement it, but the examples given are not intended to limit the present invention.

[0028] Unless otherwise specified, the test methods in each example are carried out according to conventional methods and conditions or in accordance with the actual instructions. Unless otherwise clearly marked, the content of each component is expressed as mass / volume (w / v) content. The expression plasmid pET28a was purchased from Novagen, Shanghai; the restriction endonuclease was purchased from TaKaRa Biotechnology Co., Ltd., Dalian; the high-fidelity PCR enzyme KOD was purchased from Toyobo Biotechnology Co., Ltd., Shanghai; the competent cells of E. coli BL21(DE3), the DNA Marker, the agarose gel DNA recovery kit and the bacterial genomic DNA extraction kit were all purchased from Shanghai GeneRay Biotechnology Co., Ltd.

[0029] Example 1: Construction of Cytochrome P450 Monooxygenase L76S / A83E.

[0030] Using the cytochrome P450 monooxygenase CYP of Bacillus megaterium (accession number: P14779) as a template, the site-directed mutagenesis primers were designed as follows (SEQ ID NO.3-4):

[0031] L76S / A83E-F (SEQ ID NO.3):

[0032] 5’-TTAAGTCAAGCG AGC AAATTTGTACGTGATTTT GAA GGAGACGG-3’

[0033] L76S / A83E-R (SEQ ID NO.4):

[0034] 5’-TAACCCGTCTCC TTC AAAATCACGTACAAATTT GCT CGCTTGAC-3’

[0035] Among them, the underlined part indicates the mutated sequence.

[0036] Using the recombinant plasmid pET28a-CYP containing the cytochrome P450 monooxygenase of Bacillus megaterium as a template, whole plasmid PCR amplification was carried out. The PCR system was as follows: 1 μL of each of the upstream primer and the downstream primer (0.3 μmol / L), 1 μL of the pET28a-CYP template (0.1 μg), 5.0 μL of dNTP, Mg 2+3.0 μL, 5.0 μL of KOD Buffer, 1.0 μL of KOD enzyme, made up to 50 μL with ddH2O. The PCR amplification program is as follows: (1) Pre-denaturation at 96°C for 5 min; (2) Denaturation at 98°C for 30 s; (3) Annealing at 55°C for 30 s; (4) Extension at 68°C for 4 min 30 s; (5) Repeat steps (2)-(4) for 30 cycles; (6) Further extension at 68°C for 10 min; (7) Incubation at 10°C. After the PCR program is completed, the product is purified by agarose DNA gel electrophoresis. The full-length gene sequence containing the cytochrome P450 monooxygenase mutant enzyme L76S / A83E is obtained, verified correct by DNA sequencing, and named L76S / A83E. The nucleotide sequence of the gene is shown as SEQ ID NO.1 in the sequence listing.

[0037] The above PCR product was digested with Quick Cut DpnⅠ to remove the template. The digestion system is: 2.0 μL of DpnⅠ Buffer, 1.0 μL of DpnⅠ, 3 μL of PCR product, made up to 20 μL with ddH2O. 10 μL of the digested product was taken and transformed into competent cells of Escherichia coli BL21(DE3). Positive recombinants were screened on a kanamycin-containing resistant plate, and monoclonal colonies were picked and verified by colony PCR for positive clones. The recombinant bacteria were cultured, and the plasmid was extracted after plasmid amplification, namely the recombinant plasmid pET28a-L76S / A83E, and positive clones were verified by gene sequencing.

[0038] Example 2: Preparation of a recombinant expression transformant of cytochrome P450 monooxygenase mutant enzyme L76S / A83E.

[0039] The recombinant plasmid pET28a-L76S / A83E obtained in Example 1 was re-transformed into competent cells of Escherichia coli BL21(DE3). The transformation solution was spread on an LB plate containing kanamycin and cultured overnight at 37°C in an inverted position, and the positive recombinant transformant Escherichia coli BL21(DE3) / pET28a-L76S / A83E was obtained. Positive clones were verified by colony PCR and gene sequencing.

[0040] Example 3: Expression of cytochrome P450 monooxygenase mutant enzyme L76S / A83E.

[0041] The recombinant Escherichia coli obtained in Example 2 was inoculated into an LB medium containing kanamycin (10 g / L of peptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, pH 7.0), cultured overnight with shaking at 37°C, and inoculated into a 250 mL Erlenmeyer flask containing 40 mL of LB medium at an inoculation amount of 1% (v / v), and cultured with shaking on a shaker at 37°C and 180 rpm. When the OD of the culture broth 600When it reached 0.6, IPTG with a final concentration of 0.1 mmol / L was added as an inducer. After induction at 16 °C for 12 h, the cells were collected by centrifugation at 4 °C and 8000 r / min for 5 min. The collected cells were suspended in Tris-HCl buffer (100 mM, pH 8.0), sonicated in an ice bath, and the supernatant was collected by centrifugation, which was the crude enzyme solution of the recombinant cytochrome P450 monooxygenase mutant enzyme L76S / A83E. The obtained crude enzyme solution was analyzed by polyacrylamide gel electrophoresis for protein expression (as Figure 1 shown). As can be seen from Figure 1 , there was a high soluble expression of the protein at the target position in the supernatant, indicating that the recombinant cytochrome P450 monooxygenase mutant enzyme L76S / A83E was successfully expressed in Escherichia coli in a soluble form.

[0042] Example 4: Determination of the activity of cytochrome P450 monooxygenase.

[0043] The activity of cytochrome P450 monooxygenase was measured using a microplate reader by detecting the change in absorbance at 340 nm. The method for determining the activity of cytochrome P450 monooxygenase was as follows: In a 200 μL reaction system (100 mM Tris-HCl buffer, pH 8.0), 1 mmol / L methylpyrazine and 1 mmol / L NADPH were added, and an appropriate amount of the crude enzyme solution prepared in Example 3 was added and quickly mixed evenly, and the change in absorbance at 340 nm was detected. The activity (U) of each unit of cytochrome P450 monooxygenase was defined as the amount of enzyme required to catalyze 1 μmol of NADPH per minute under the above conditions.

[0044] Examples 5 - 8: Hydroxylation reaction of methylpyrazine catalyzed by cytochrome P450 monooxygenase mutant L76S / A83E.

[0045] In 20 mL of Tris-HCl buffer (100 mM, pH 8.0), the crude enzyme solution of the CYP mutant enzyme prepared in Example 3 was added. The dosage of the recombinant cytochrome P450 monooxygenase mutant L76S / A83E was 100 U / L (the definition of enzyme activity is shown in Example 4). The final concentration of the methylpyrazine compound was 10 mM, the dosage of glucose dehydrogenase was 2 g / L, the final concentration of glucose was 20 mM, and the final concentration of NADP + was 1 mM. The reaction was carried out at 30 °C for 24 h.

[0046] The specific analysis method for the product conversion rate is as follows: Take 250 μL of the reaction solution, inactivate it at a high temperature of 100 °C in a metal bath for 10 min, centrifuge it at a high speed of 12,000 rpm for 5 min, aspirate the supernatant and treat it with an aqueous phase filter membrane, and then analyze it using a liquid chromatograph. The chromatographic column is Diamonsil C18 (250 mm × 4.6 mm, 5 μm), the mobile phase is V(water):V(acetonitrile) = 70:30, the flow rate is 1 mL / min, the column temperature is 25 °C, the detection wavelength is 270 nm, and the injection volume is 10 μL. The results are shown in Table 1. The mutant enzyme L76S / A83E can catalyze the hydroxylation reaction of different methylpyrazine compounds, and the conversion rate reaches more than 95% after 24 h of reaction. Therefore, the mutant enzyme L76S / A83E shows good potential for industrial application.

[0047] Table 1 Hydroxylation reaction of different methylpyrazine compounds catalyzed by P450 mutant enzyme L76S / A83E

[0048]

[0049] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of the present invention.

Claims

1. A cytochrome P450 monooxygenase mutant, characterized in that, The amino acid sequence of the cytochrome P450 monooxygenase mutant is shown in SEQ ID NO.

2.

2. A gene encoding the cytochrome P450 monooxygenase mutant according to claim 1.

3. An expression vector carrying the gene according to claim 2.

4. A recombinant bacterium expressing the cytochrome P450 monooxygenase mutant according to claim 1.

5. Use of the cytochrome P450 monooxygenase mutant according to claim 1 in hydroxylation of methylpyrazine compounds, characterized in that, The method of the application comprises the following steps: In a buffer solution, using a methylpyrazine compound as a substrate and the cytochrome P450 monooxygenase mutant as a catalyst to carry out a hydroxylation reaction to generate the corresponding alcohol; The methylpyrazine compound is selected from one or more of 2-methylpyrazine, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine and 2,6-dimethylpyrazine.

6. The application according to claim 5, characterized in that, The addition amount of the methylpyrazine compound is 10 mmol / L to 100 mmol / L.

7. The application according to claim 5, wherein The hydroxylation reaction is carried out in the presence of glucose dehydrogenase and NADP + existing.

8. The application according to claim 5, characterized in that, The temperature of the hydroxylation reaction is 25°C to 30°C; the time of the hydroxylation reaction is 6 h to 24 h.

9. The application according to claim 5, wherein The addition form of the cytochrome P450 monooxygenase mutant is a crude enzyme solution containing the cytochrome P450 monooxygenase mutant; the addition amount of the cytochrome P450 monooxygenase mutant is 1 kU / L to 10 kU / L.

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