An engineered bacterium of 3-methyl-4-nitrobenzoic acid, a preparation method and application thereof

Abstract

The application relates to a method for preparing 3-methyl-4-nitrobenzoic acid by using microorganisms and belongs to the technical field of bio-chemical engineering. E. coli BL21-pRSFDuet-1-L163W, the nucleotide sequence of which is shown as SEQ ID NO: 3, the corresponding amino acid sequence is shown as SEQ ID NO: 4, and the application of the recombinant E. coli strain in the preparation of 3-methyl-4-nitrobenzoic acid. The application provides a microbial method for preparing 3-methyl-4-nitrobenzoic acid, which overcomes the problems of high price or serious pollution of catalysts and oxidants or safety hazards in the production process in the prior art.

Description

Technical Field

[0001] This invention relates to a method for preparing 3-methyl-4-nitrobenzoic acid using microorganisms, belonging to the field of biochemical technology. Background Technology

[0002] 3-Methyl-4-nitrobenzoic acid is a widely used chemical product, and many important organic products are obtained from it, such as ethyl 4-chloroquinazoline-6-carboxylate, 2-n-propyl-4-methyl-6-carboxybenzimidazole, methyl 4-methyl-2-ethyl-1H-benzimidazole-6-carboxylate, 3,4-dihydro-4-quinazolinone-6-carboxylic acid, 4-aminophenyl-1,3-dicarboxylic acid, methyl 4-amino-3-methylbenzoate, and 4-nitrophenyl-1,3-dicarboxylic acid. In addition, 3-methyl-4-nitrobenzoic acid is also used in medicine and is an important intermediate in the synthesis of the antihypertensive drug telmisartan and AIDS drugs.

[0003] The main methods for synthesizing 3-methyl-4-nitrobenzoic acid include air oxidation, cobalt acetate catalytic oxidation, potassium dichromate oxidation, and nitric acid oxidation, with yields ranging from approximately 30% to 86%. In the article "Catalytic Oxidation for the Synthesis of 3-Methyl-4-nitrobenzoic Acid" published in the December 2005 issue of *Applied Chemistry*, Yue Caibo, Wei Yunyang, et al. used molecular oxygen as the oxidant and added sodium bromide as a co-catalyst in a cobalt acetate catalyst system (cobalt acetate / butanone / acetic acid), achieving a 3-methyl-4-nitrobenzoic acid yield of 51%. Chinese patent CN200610107316.6, "A Catalyst and Its Application in the Synthesis of 4-nitro-3-methylbenzoic Acid," used a catalyst composed of a transition metal oxide and an N-containing organic compound with bromine or hydroxyl substitution on N to catalytically oxidize 2,4-dimethylnitrobenzene to synthesize 3-methyl-4-nitrobenzoic acid under solvent conditions, achieving a yield of 51%. Chinese patent CN103319347A, "Method for Indirect Electrosynthesis of 3-Methyl-4-nitrobenzoic Acid by Step-by-Step Heating Method," firstly, chromium sulfate is electrolytically oxidized to chromium trioxide, which is then used to oxidize 2,4-dimethylnitrobenzene to 3-methyl-4-nitrobenzoic acid. By employing a step-by-step heating method, the conversion rate reaches 65%-86%, but the catalyst and oxidant are expensive, and the post-processing, such as catalyst recovery, is difficult, resulting in high production and environmental treatment costs. The nitric acid oxidation method has strong oxidizing power and easily oxidizes to produce dinitro products, but the nitration process poses high safety risks, and nitric acid causes serious environmental pollution.

[0004] All reported methods for producing 3-methyl-4-nitrobenzoic acid are chemical synthesis methods, and no reports have been found of microbial catalytic processes. Summary of the Invention

[0005] Purpose of the invention: To provide a microbial process suitable for industrial production of 3-methyl-4-nitrobenzoic acid.

[0006] The experiment found that the recombinant Escherichia coli strain used to produce 5-methylpyrazine-2-carboxylic acid (this strain was recombined from Pseudomonas putida) Pseudomonas putida The XMO encoding gene of xylene monooxygenase in ATCC 33015 xylMA BADH gene encoding benzyl alcohol dehydrogenase xylB and the gene encoding benzaldehyde dehydrogenase BZDHH xylC A novel enzyme culture was developed that can catalyze the reaction of 2,4-dimethylnitrobenzene (compound of formula 1) with oxygen as an oxidant under certain conditions to produce 3-methyl-4-nitrobenzoic acid (compound of formula 4), with a product yield of up to 12%. The specific experimental procedure is as follows: Recombinant Escherichia coli engineered bacteria (culture preservation number: CGMCCNO.14930) expressing xylene monooxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase were cultured in LB broth at 37°C with shaking for 20 h. The bacterial culture was then collected by centrifugation at 10000 r / m for 15 min. The cells were resuspended in phosphate buffer (pH 8) at 1 / 50-1 / 10 of the original fermentation broth volume to obtain a whole-cell enzyme solution. This whole-cell enzyme solution, using 2,4-dimethylnitrobenzene as a substrate, under certain conditions, catalyzed the reaction, and HPLC analysis showed the formation of 3-methyl-4-nitrobenzoic acid. The catalytic reaction solution was filtered through diatomaceous earth, and the supernatant was precipitated with concentrated hydrochloric acid and filtered again to obtain the crude product. This crude product was extracted with ethyl acetate and rotary evaporated to obtain 3-methyl-4-nitrobenzoic acid. The substrate conversion rate was 12%, the product content was 98.5%, and the purity was 99.8%. The catalytic process is shown below: Given the low conversion rate in the above experiments, the applicant attempted to improve the above-mentioned recombinant Escherichia coli engineered bacteria, and obtained the technical solution of this invention, as follows.

[0007] Using an in vitro evolutionary technique based on error-prone PCR, a gene containing the BADH encoding benzyl alcohol dehydrogenase was selected. xylB The recombinant plasmid pRSFDuet-1- xylB Using a template, error-prone PCR was employed for directed evolution, and after extensive screening, mutant strains with significantly increased enzyme activity units were obtained. The mutant strains were found to have mutated benzyl alcohol dehydrogenase, and the mutated recombinant strains were able to increase substrate conversion from 12% to over 80%.

[0008] The technical solution of this invention is: a recombinant Escherichia coli strain, which is... E. coliBL21-pRSFDuet-1-L163W, its nucleotide sequence is shown in SEQ ID NO: 3, and its corresponding amino acid sequence is shown in SEQ ID NO: 4.

[0009] The method for preparing the recombinant Escherichia coli strain of the present invention includes the following steps: Step 1: Preparation of recombinant E. coli containing the modified benzyl alcohol dehydrogenase encoding gene: Contains the gene encoding benzyl alcohol dehydrogenase BADH xylB The recombinant plasmid pRSFDuet-1- xylB Using this as a template, error-prone PCR technology was employed for directed evolution. According to *Molecular Cloning: A Laboratory Manual*, specifically, after the PCR reaction, PCR products containing linear gene fragments were obtained. These PCR products and the pRSFDuet-1 expression plasmid were then subjected to double digestion (EcoRI, Not I), purification, ligation, and transformation into a plasmid containing the xylene monooxygenase XMO encoding gene. xylMA and the gene encoding benzaldehyde dehydrogenase BZDHH xylC The basic Escherichia coli competent cells were plated on LB agar plates containing 50 mg / L kanamycin sulfate and incubated overnight at 37°C.

[0010] The second step involved picking single colonies grown on the culture dishes and inoculating them into LB broth containing kanamycin sulfate. The cultures were incubated at 37°C with shaking for 15 h. The bacterial cells were then collected by centrifugation for plasmid extraction, PCR identification, and double enzyme digestion identification. The *E. coli* strains containing the correct recombinant plasmid were induced to express the enzyme. Specifically, the bacterial culture was transferred to 100 mL of LB broth containing 50 μg / mL kanamycin sulfate and incubated at 37°C with shaking for 20 h. The bacterial culture was then collected by centrifugation at 10,000 r / m for 15 min. The cells were resuspended in phosphate buffer (pH 8) at 1 / 50–1 / 10 of the original fermentation broth volume to obtain a whole-cell enzyme solution. This whole-cell enzyme solution was used for catalytic verification experiments, yielding a recombinant *E. coli* strain with significantly enhanced enzyme activity, named... E. coli BL21-pRSFDuet-1-L163W, its nucleotide sequence is shown in SEQ ID NO: 3, and its corresponding amino acid sequence is shown in SEQ ID NO: 4.

[0011] Another discovery of this invention is the application of the recombinant Escherichia coli strain in the preparation of 3-methyl-4-nitrobenzoic acid, specifically comprising the following steps. Step 1. The recombinant E. coli obtained in Step 2 above is amplified and cultured in a fermentation medium, and then centrifuged to obtain resting cells; In this step, the fermentation medium formula is as follows: glucose monohydrate 1.0-3.2 g / L, yeast extract 2.0-4.6 g / L, citric acid monohydrate 1 g / L, ammonium sulfate 2.5 g / L, and disodium hydrogen phosphate dodecahydrate 14.4 g / L. Sterilized glucose solution is added during fermentation.

[0012] Step 2. Collect the resting cells obtained in Step 1, suspend them in a buffer solution at a certain ratio, and add the substrate 2,4-dimethylnitrobenzene to obtain a catalytic reaction solution. Maintain pH stability with an alkaline solution in a buffer solution at a temperature of 10–55℃ and a pH of 6.5–9.5, with a compressed air flow rate of 0.4–8 L / min. The reaction produces 3-methyl-4-nitrobenzoic acid. The mass ratio of substrate to resting cells is 1:0.5–5. Monitor the reaction progress using HPLC. After the reaction is complete, filter, acid precipitate, extract, and rotary evaporate to obtain the product 3-methyl-4-nitrobenzoic acid.

[0013] In this step, the substrate concentration in the reaction solution is 8-20 g / L; the mass ratio of substrate to resting cells is 1:0.5-5, preferably 0.8-2; In this step, the buffer solution is a phosphate buffer solution, a borate buffer solution, physiological saline, or purified water; In this step, the catalytic reaction temperature can be 15-50℃, preferably 20-30℃; In this step, the pH of the catalytic reaction can be 6.5–9.5, preferably 8.0–8.5; In this step, the alkaline solution used to maintain pH stability during the catalytic reaction is either an ammonia solution or a sodium hydroxide solution. In this step, the catalytic reaction is carried out with a compressed air flow rate of 0.4-5 L / min for a 4L catalytic system.

[0014] Beneficial Effects: This invention provides a microbial process for the production of 3-methyl-4-nitrobenzoic acid, overcoming the problems of expensive catalysts and oxidants, severe pollution, or safety hazards in existing technologies. Specifically, this invention solves the problems of expensive catalysts and environmental unfriendliness in chemical methods; it also solves the problems of severe pollution and safety hazards in the nitric acid oxidation method; through error-prone PCR technology and in vitro enzyme evolution, a strain containing a novel BADH mutation of benzyl alcohol dehydrogenase is obtained, which can significantly improve the conversion rate of the catalytic reaction; this invention is the first to realize a process for the preparation of 3-methyl-4-nitrobenzoic acid using microorganisms, and the process steps are simple, the reaction conditions are mild, and it is environmentally friendly. Attached Figure Description

[0015] Figure 1 Example 8: Fermentation growth curve of recombinant Escherichia coli Figure 2 Example 8: Enzyme Catalytic Reaction Curve Figure 3 Liquid phase spectrum of the product in Example 8 Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are those in conventional experiments.

[0017] Example 1. Preparation of 3-methyl-4-nitrobenzoic acid using basic recombinant Escherichia coli (1) The recombinant *Escherichia coli* engineered bacteria containing xylene monooxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase, preserved at -80℃ (culture preservation number: CGMCC NO.14930, depository: China General Microbiological Culture Collection Center, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, deposit date: November 20, 2017). The Latin name for the classification and nomenclature of the biological material of this invention is: Escherichie coli ), inoculated on Luria-Bertani (LB) agar medium, and then incubated overnight at 37°C; (2) Pick a single colony from the LB agar plate and inoculate it into LB liquid medium, then incubate at 37°C overnight; (3) Take an appropriate amount of bacterial culture from the LB liquid medium after overnight culture, inoculate it into the modified M9 liquid medium, and culture it at 37°C for 8 hours. Then inoculate it into the fermentation medium and culture it at 37°C until the OD 600 is about 90. Centrifuge to collect the bacterial cells, wash them twice with physiological saline to obtain resting cells. Sterile glucose solution can be added as needed during the culture at 37°C.

[0018] (4) Add 4L of phosphate buffer solution with pH 8 to the fermenter, then add 36.0g of 2,4-dimethylnitrobenzene and 100.8g of resting cells to obtain the catalytic reaction solution. Set the catalytic conditions to 300rpm stirring, 20℃ temperature, and 2L / min aeration rate. Use ammonia solution to control the pH of the catalytic reaction solution to be stable at around 8.0. Take samples every 3 hours for HPLC to detect the conversion of the raw materials. Stop catalysis when the conversion of the raw materials stops.

[0019] (5) After the catalytic reaction solution is filtered through diatomaceous earth, the clear liquid is precipitated with concentrated hydrochloric acid and filtered to obtain the crude product. It is extracted with ethyl acetate and rotary evaporated to obtain the product 3-methyl-4-nitrobenzoic acid.

[0020] The substrate 2,4-dimethylnitrobenzene was converted at a rate of 12%, and the resulting product was analyzed by HPLC and found to have a content of 98.5% and a purity of 99.8%.

[0021] Example 2. Construction and screening of recombinant Escherichia coli containing an improved benzyl alcohol dehydrogenase encoding gene Using a recombinant plasmid containing the benzyl alcohol dehydrogenase gene as a template, and primers at both ends designed and synthesized using Primer 5.0 (Table 3), error-prone PCR was performed (materials and concentrations are shown in Table 4, and reaction conditions are shown in Table 5). Following the instructions in *Molecular Cloning: A Laboratory Manual*, PCR products containing linear gene fragments were obtained. These PCR products and the pET28a(+) expression plasmid were then subjected to double digestion (EcoRI, Not I), purification, ligation, and transformation into a plasmid containing the xylene monooxygenase XMO encoding gene. xylMA and the gene encoding benzaldehyde dehydrogenase BZDHH xylC The basic Escherichia coli competent cells were plated on LB agar plates containing 50 mg / L kanamycin sulfate and incubated overnight at 37°C.

[0022] Single colonies grown on the above culture dishes were inoculated into LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C with shaking for 15 h. The bacterial cells were collected by centrifugation for plasmid extraction, PCR identification, and double enzyme digestion identification (using agarose gel electrophoresis). The correct recombinant plasmid was named pRSFDuet-1-AW. E. coli containing the correct recombinant plasmid were induced to express the plasmid. The specific operation was as follows: the above bacterial solution was transferred to 100 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C with shaking for 20 h. The bacterial solution was then collected by centrifugation at 10000 r / m for 15 min. The bacterial cells were resuspended in phosphate buffer with pH 8 at 1 / 50-1 / 10 of the original fermentation broth volume to obtain whole cell enzyme solution. The whole-cell enzyme solution was used for catalytic verification experiments, and a mutant strain with significantly improved enzyme activity was obtained, named E. coli BL21- pRSFDuet-1-L163W. The corresponding nucleotide sequence before mutation is shown in SEQ ID NO:1, and the corresponding amino acid sequence is shown in SEQ ID NO:2. The corresponding nucleotide sequence after mutation is shown in SEQ ID NO:3, and the corresponding amino acid sequence is shown in SEQ ID NO:4.

[0023] Example 3. Preparation of 3-methyl-4-nitrobenzoic acid using improved recombinant Escherichia coli (containing benzyl alcohol dehydrogenase L163W mutant) High-density fermentation was carried out using recombinant Escherichia coli engineered bacteria L163W containing benzyl alcohol dehydrogenase, using the same culture method as in Example 1, and catalysis and extraction were performed according to the following steps: (1) Add 4L of physiological saline with a pH of 9.5 to the fermenter, then add 48.0g of 2,4-dimethylnitrobenzene and 96.0g of resting cells to obtain the catalytic reaction solution. The catalytic conditions are set as follows: stirring at 300rpm, temperature at 45℃, and aeration rate at 0.5L / min. The pH of the catalytic reaction solution is controlled to be stable at around 9.5 using ammonia solution. Samples are taken every 3 hours for HPLC analysis to detect the conversion of the raw materials. Catalysis is stopped when the conversion of the raw materials ceases.

[0024] (2) The catalytic reaction solution was filtered through diatomaceous earth, the clear liquid was precipitated with concentrated hydrochloric acid, filtered to obtain crude product, extracted with ethyl acetate, the organic phases were combined, and rotary evaporated to obtain product 3-methyl-4-nitrobenzoic acid.

[0025] The substrate 2,4-dimethylnitrobenzene was converted at a rate of 80.2%, and the resulting product, as determined by HPLC, had a content of 94.8% and a purity of 97.3%.

[0026] Example 4. Preparation of 3-methyl-4-nitrobenzoic acid using improved recombinant Escherichia coli (containing benzyl alcohol dehydrogenase L163W mutant) High-density fermentation was carried out using recombinant Escherichia coli engineered bacteria L163W containing benzyl alcohol dehydrogenase, using the same culture method as in Example 1, and catalysis and extraction were performed according to the following steps: (1) Add 4L of physiological saline with a pH of 6.5 to the fermenter, then add 32.0g of 2,4-dimethylnitrobenzene and 160.0g of resting cells to obtain the catalytic reaction solution. Set the catalytic conditions to 300rpm stirring, 15℃ temperature, and 4L / min aeration rate. Use ammonia solution to control the pH of the catalytic reaction solution to be stable at around 6.5. Take samples every 3 hours for HPLC to detect the conversion of the raw materials. Stop catalysis when the raw materials no longer undergo conversion.

[0027] (2) The catalytic reaction solution was filtered through diatomaceous earth, the clear liquid was precipitated with concentrated hydrochloric acid, filtered to obtain crude product, extracted with ethyl acetate, the organic phases were combined, and rotary evaporated to obtain product 3-methyl-4-nitrobenzoic acid.

[0028] The substrate 2,4-dimethylnitrobenzene was converted at a rate of 81.26%, and the resulting product, as determined by HPLC, had a content of 97.8% and a purity of 98.4%.

[0029] Example 5. Preparation of 3-methyl-4-nitrobenzoic acid using improved recombinant Escherichia coli (containing benzyl alcohol dehydrogenase L163W mutant) High-density fermentation was carried out using recombinant Escherichia coli engineered bacteria L163W containing benzyl alcohol dehydrogenase, using the same culture method as in Example 1, and catalysis and extraction were performed according to the following steps: (1) Add 4L of physiological saline with pH 8.0 to the fermenter, then add 60.0g of 2,4-dimethylnitrobenzene and 120.0g of resting cells to obtain the catalytic reaction solution. Set the catalytic conditions to 300rpm stirring, 30℃ temperature, and 2.5L / min aeration rate. Use ammonia solution to control the pH of the catalytic reaction solution to be stable at around 8.0. Take samples every 3 hours for HPLC to detect the conversion of the raw materials. Stop catalysis when the raw materials no longer undergo conversion.

[0030] (2) The catalytic reaction solution was filtered through diatomaceous earth, the clear liquid was precipitated with concentrated hydrochloric acid, filtered to obtain crude product, extracted with ethyl acetate, the organic phases were combined, and rotary evaporated to obtain product 3-methyl-4-nitrobenzoic acid.

[0031] The substrate 2,4-dimethylnitrobenzene was converted to 83.8%, and the resulting product, as determined by HPLC, had a content of 97.8% and a purity of 99.0%.

[0032] Example 6. Preparation of 3-methyl-4-nitrobenzoic acid using improved recombinant Escherichia coli (containing benzyl alcohol dehydrogenase L163W mutant) High-density fermentation was carried out using recombinant Escherichia coli engineered bacteria L163W containing benzyl alcohol dehydrogenase, using the same culture method as in Example 1, and catalysis and extraction were performed according to the following steps: (1) Add 4L of physiological saline with a pH of 8.5 to the fermenter, then add 80.0g of 2,4-dimethylnitrobenzene and 40.0g of resting cells to obtain the catalytic reaction solution. Set the catalytic conditions to 300rpm stirring, 20℃ temperature, and 3L / min aeration rate. Use ammonia solution to control the pH of the catalytic reaction solution to be stable at around 8.2. Take samples every 3 hours for HPLC to detect the conversion of the raw materials. Stop catalysis when the raw materials no longer undergo conversion.

[0033] (2) The catalytic reaction solution was filtered through diatomaceous earth, the clear liquid was precipitated with concentrated hydrochloric acid, filtered to obtain crude product, extracted with ethyl acetate, the organic phases were combined, and rotary evaporated to obtain product 3-methyl-4-nitrobenzoic acid.

[0034] The substrate 2,4-dimethylnitrobenzene was converted at a rate of 81.6%, and the resulting product, as determined by HPLC, had a content of 95.8% and a purity of 99.1%.

[0035] Example 7. Preparation of 3-methyl-4-nitrobenzoic acid using improved recombinant Escherichia coli (containing benzyl alcohol dehydrogenase L163W mutant) High-density fermentation was carried out using recombinant Escherichia coli engineered bacteria L163W containing benzyl alcohol dehydrogenase, using the same culture method as in Example 1, and catalysis and extraction were performed according to the following steps: (1) Add 4L of phosphate buffer solution with pH 8.5 to the fermenter, add 65.6 g of 2,4-dimethylnitrobenzene and 100.0 g of resting cells to obtain the catalytic reaction solution. Set the catalytic conditions to 300 rpm stirring, 25℃ temperature, and 3 L / min aeration rate. Use sodium hydroxide solution to control the pH of the catalytic reaction solution to be stable at around 8.5. Take samples every 3 hours for HPLC to detect the conversion of raw materials. Stop catalysis when the raw materials no longer undergo conversion.

[0036] (2) The catalytic reaction solution was filtered through diatomaceous earth, the clear liquid was precipitated with concentrated hydrochloric acid, filtered to obtain crude product, extracted with ethyl acetate, the organic phases were combined, and rotary evaporated to obtain product 3-methyl-4-nitrobenzoic acid.

[0037] The substrate 2,4-dimethylnitrobenzene was converted at a rate of 86%, and the resulting product, as determined by HPLC, had a content of 98.0% and a purity of 99.3%.

[0038] Example 8. Preparation of 3-methyl-4-nitrobenzoic acid using improved recombinant Escherichia coli (containing benzyl alcohol dehydrogenase L163W mutant) High-density fermentation was carried out using recombinant Escherichia coli engineered bacteria L163W containing benzyl alcohol dehydrogenase, using the same culture method as in Example 1, and catalysis and extraction were performed according to the following steps: (1) Add 4L of phosphate buffer solution with pH 8.5 to the fermenter, then add 65.6 g of 2,4-dimethylnitrobenzene and 65.6 g of resting cells to obtain the catalytic reaction solution. Set the catalytic conditions to 300 rpm stirring, 25℃ temperature, and 3L / min aeration rate. Use ammonia solution to control the pH of the catalytic reaction solution to be stable at around 8.5. Take samples every 3 hours for HPLC to detect the conversion of the raw materials. Stop catalysis when the raw materials no longer undergo conversion.

[0039] (2) The catalytic reaction solution was filtered through diatomaceous earth, the clear liquid was precipitated with concentrated hydrochloric acid, filtered to obtain crude product, extracted with ethyl acetate, the organic phases were combined, and rotary evaporated to obtain product 3-methyl-4-nitrobenzoic acid.

[0040] The substrate 2,4-dimethylnitrobenzene was converted to 89%, and the resulting product was analyzed by HPLC: the product content was 98.5% and the purity was 99.8%.

[0041] Example 9. Preparation of 3-methyl-4-nitrobenzoic acid using improved recombinant Escherichia coli (containing benzyl alcohol dehydrogenase L163W mutant) High-density fermentation was carried out using recombinant Escherichia coli engineered bacteria L163W containing benzyl alcohol dehydrogenase, using the same culture method as in Example 1, and catalysis and extraction were performed according to the following steps: (1) Add 8L of phosphate buffer solution with pH 8.5 to the fermenter, then add 131.2g of 2,4-dimethylnitrobenzene and 131.2g of resting cells to obtain the catalytic reaction solution. Set the catalytic conditions to 300rpm stirring, 30℃ temperature, and 8L / min aeration rate. Use ammonia solution to control the pH of the catalytic reaction solution to be stable at around 8.5. Take samples every 3 hours for HPLC to detect the conversion of the raw materials. Stop catalysis when the raw materials no longer undergo conversion.

[0042] (2) The catalytic reaction solution was filtered through diatomaceous earth, the clear liquid was precipitated with concentrated hydrochloric acid, filtered to obtain crude product, extracted with ethyl acetate, the organic phases were combined, and rotary evaporated to obtain product 3-methyl-4-nitrobenzoic acid.

[0043] The substrate 2,4-dimethylnitrobenzene was converted to 88% purity. The resulting product, as determined by HPLC, had a content of 98.4% and a purity of 99.3%.

[0044] Example 10. Preparation of 3-methyl-4-nitrobenzoic acid using improved recombinant Escherichia coli (containing benzyl alcohol dehydrogenase L163W mutant) High-density fermentation was carried out using recombinant Escherichia coli engineered bacteria L163W containing benzyl alcohol dehydrogenase, using the same culture method as in Example 1, and catalysis and extraction were performed according to the following steps: (1) Add 40 L of phosphate buffer solution with pH 8.5 to the fermenter, then add 984 g of 2,4-dimethylnitrobenzene and 984 g of resting cells to obtain the catalytic reaction solution. The catalytic conditions are set as follows: stirring at 300 rpm, temperature at 30℃, and aeration rate at 30 L / min. The pH of the catalytic reaction solution is controlled to be stable at around 8.5 with ammonia solution. Samples are taken every 3 hours for HPLC to detect the conversion of the raw materials. Catalysis is stopped when the raw materials no longer undergo conversion.

[0045] (2) The catalytic reaction solution was filtered through diatomaceous earth, the clear liquid was precipitated with concentrated hydrochloric acid, filtered to obtain crude product, extracted with ethyl acetate, the organic phases were combined, and rotary evaporated to obtain product 3-methyl-4-nitrobenzoic acid.

[0046] The substrate 2,4-dimethylnitrobenzene was converted to 89%, and the resulting product was analyzed by HPLC and found to have a content of 98.5% and a purity of 99.5%.

Claims

1. A method for preparing a recombinant Escherichia coli strain, characterized in that, Includes the following steps: 1) The modified benzyl alcohol dehydrogenase encoding gene was ligated and transformed into basic E. coli competent cells containing the xylMA encoding gene of xylene monooxygenase XMO and the xylC encoding gene of benzaldehyde dehydrogenase BZDHH. The cells were then plated on LB agar plates containing 50 mg / L kanamycin sulfate and incubated overnight at 37°C. The nucleotide sequence of the modified benzyl alcohol dehydrogenase encoding gene is shown in SEQ ID NO:

3. 2) The bacterial culture was transferred to 100 mL of LB liquid medium containing 50 μg / ml kanamycin sulfate and cultured at 37℃ with shaking for 20 h. The bacterial culture was then collected by centrifugation at 10000 r / min for 15 min. The bacterial cells were resuspended in phosphate buffer with pH 8 at 1 / 50-1 / 10 of the original fermentation broth volume to obtain whole cell enzyme solution. This whole cell enzyme solution was used for catalytic verification test and a recombinant Escherichia coli strain with obvious activity was obtained.

2. The preparation method according to claim 1, characterized in that, The amino acid sequence of the improved benzyl alcohol dehydrogenase is shown in SEQ ID NO:

4.

3. A method for preparing 3-methyl-4-nitrobenzoic acid, characterized in that, Includes the following steps: Step 1: The recombinant Escherichia coli obtained in claim 1 is amplified and cultured in a fermentation medium, and the cells are centrifuged to obtain resting cells. The fermentation medium is formulated as follows: glucose monohydrate 1.0-3.2 g / L, yeast extract 2.0-4.6 g / L, citric acid monohydrate 1 g / L, ammonium sulfate 2.5 g / L, and disodium hydrogen phosphate dodecahydrate 14.4 g / L. Step 2: Collect the resting cells obtained in Step 1, suspend them in a buffer solution at a certain ratio, and add the substrate 2,4-dimethylnitrobenzene. React in a buffer solution at a temperature of 15-45℃ and a pH of 6.5-9.5, with the pH maintained by an alkaline solution and a compressed air flow rate of 0.4-8 L / min to obtain a catalytic reaction solution. The mass ratio of the substrate to the resting cells is 1:0.5-5. Step 3: After the catalytic reaction solution is filtered through diatomaceous earth, the clear liquid is precipitated with concentrated hydrochloric acid, filtered to obtain crude product, extracted with ethyl acetate, the organic phases are combined, and rotary evaporated to obtain the product 3-methyl-4-nitrobenzoic acid.

4. The preparation method according to claim 3, characterized in that, In step 1, a sterilized glucose solution is added during the fermentation process.

5. The preparation method according to claim 3, characterized in that, In step 2, the substrate concentration in the reaction solution is 8-20 g / L, and the mass ratio of substrate to resting cells is 1:0.8-2.

6. The preparation method according to claim 3, characterized in that, The buffer solution mentioned in step 2 is selected from either a phosphate buffer solution or a borate buffer solution.

7. The preparation method according to claim 3, characterized in that, The catalytic reaction temperature in step 2 is 20-30℃.

8. The preparation method according to claim 3, characterized in that, The pH value in step 2 is 8.0–8.

5.

9. The preparation method according to claim 3, characterized in that, The alkaline solution in step 2 is either an ammonia solution or a sodium hydroxide solution.

10. The preparation method according to claim 3, characterized in that... The compressed air supply rate is 0.4-5 L / min.

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