CN113897322A

CN113897322A - Engineering bacterium of 3-methyl-4-nitrobenzoic acid and preparation method thereof

Info

Publication number: CN113897322A
Application number: CN202110724281.5A
Authority: CN
Inventors: 邵先祥; 夏俊刚; 丛日刚; 苗华明; 翟天龙; 门晋名
Original assignee: Dijia Pharmaceutical Group Co ltd
Current assignee: Dijia Pharmaceutical Group Co ltd
Priority date: 2021-06-29
Filing date: 2021-06-29
Publication date: 2022-01-07
Anticipated expiration: 2041-06-29
Also published as: CN113897322B; CN116536228A

Abstract

The invention relates to a method for preparing 3-methyl-4-nitrobenzoic acid by using microorganisms, belonging to the technical field of biochemical engineering. The present invention provides a recombinant Escherichia coli strain which isE.coli BL21-pRSFDuet-1-L163W, the nucleotide sequence of which is shown as SEQ ID NO. 3, and the corresponding amino acid sequence of which is shown as SEQ ID NO. 4, and the application of the recombinant Escherichia coli strain in the preparation of 3-methyl-4-nitrobenzoic acid. The invention provides a process for preparing and producing 3-methyl-4-nitrobenzoic acid by a microbiological method, which overcomes the defects of a catalyst and an oxidant in the prior artHigh price or serious pollution or potential safety hazard in the production process.

Description

Engineering bacterium of 3-methyl-4-nitrobenzoic acid and preparation method thereof

Technical Field

The invention relates to a method for preparing 3-methyl-4-nitrobenzoic acid by using microorganisms, belonging to the technical field of biochemical engineering.

Background

3-methyl-4-nitrobenzoic acid is a widely used chemical product, and many important organic products are obtained by using it as a raw material, such as 4-chloroquinazoline-6-ethyl formate, 2-n-propyl-4-methyl-6-carboxybenzimidazole, 4-methyl-2-ethyl-1H-benzimidazole-6-methyl formate, 3, 4-dihydro-4-quinazolinone-6-carboxylic acid, 4-aminophenyl-1, 3-dicarboxylic acid, 4-amino-3-methyl benzoate, 4-nitrophenyl-1, 3-dicarboxylic acid, etc.; in addition, the 3-methyl-4-nitrobenzoic acid is also used in medicine and is an important intermediate for synthesizing antihypertensive drugs telmisartan and AIDS drugs.

The method for synthesizing the 3-methyl-4-nitrobenzoic acid mainly comprises an air oxidation method, a cobalt acetate catalytic oxidation method, a potassium dichromate oxidation method, a nitric acid oxidation method and the like, and the yield is about 30-86%. In the text of the synthesis of 3-methyl-4-nitrobenzoic acid by catalytic molecular oxygen oxidation, molecular oxygen is taken as an oxidant in the text of the synthesis of 3-methyl-4-nitrobenzoic acid by catalytic molecular oxygen oxidation in applied chemistry No. 2005 No. 12 Yueyaobo, Wei Yuyang and the like, a cocatalyst sodium bromide is added into a cobalt acetate catalyst system (cobalt acetate/butanone/acetic acid), and the yield of the 3-methyl-4-nitrobenzoic acid is 51 percent; in the text of "a catalyst and its application in the reaction of synthesizing 4-nitro-3-methylbenzoic acid", chinese patent CN200610107316.6 discloses a method for synthesizing 3-methyl-4-nitrobenzoic acid by catalytically oxidizing 2, 4-dimethylnitrobenzene with a catalyst comprising a transition metal oxide and an N-containing organic compound substituted with bromine or hydroxyl on N in the presence of a solvent, wherein the yield is 51%. Chinese patent CN103319347A method for synthesizing 3-methyl-4-nitrobenzoic acid by stepwise heating and indirect electrosynthesis firstly electrolytically oxidizes chromium sulfate into chromium trioxide, and then oxidizes 2, 4-dimethyl nitrobenzene into 3-methyl-4-nitrobenzoic acid by chromium trioxide; by adopting a step heating method, the conversion rate reaches 65-86%, but the catalyst and the oxidant are expensive, the post-treatment difficulty such as catalyst recovery is high, and the production cost and the environmental protection treatment cost are high; the nitric acid oxidation method has stronger oxidability and is easy to oxidize to generate a product of the double nitro, but the nitration process has higher potential safety hazard and the nitric acid has serious pollution to the environment.

The production methods of 3-methyl-4-nitrobenzoic acid reported at present are all synthesized by a chemical method, and no microbial catalysis process is reported.

Disclosure of Invention

The purpose of the invention is as follows: provides a microbiological method preparation process of 3-methyl-4-nitrobenzoic acid suitable for industrial production.

It was found in the experiment that a recombinant Escherichia coli strain for producing 5-methylpyrazine-2-carboxylic acid (which recombinant Escherichia coli strain originated from Pseudomonas putida (Pseudomonas putida) (R))Pseudomonas putida) Xylene monooxygenase XMO encoding gene of ATCC 33015xylMABADH encoding gene of benzyl alcohol dehydrogenasexylBAnd benzaldehyde dehydrogenase BZDDH coding genexylC) The 3-methyl-4-nitrobenzoic acid (compound shown in the formula 4) can be obtained by catalyzing 2, 4-dimethyl nitrobenzene serving as a substrate (compound shown in the formula 1) and oxygen serving as an oxidant under certain conditions, and the highest product quality yield is 12%. The specific experimental process is as follows: taking basic recombinant escherichia coli engineering bacteria (the strain preservation number is CGMCC NO. 14930) expressing xylene monooxygenase, benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, performing shake culture at 37 ℃ for 20h in an LB liquid culture medium, taking out bacterial liquid, centrifuging at 10000 r/m for 15 min, collecting the bacteria, and resuspending the bacteria by using a phosphate buffer solution with the volume of 1/50-1/10 and the pH value of 8 to obtain a whole-cell enzyme solution. The whole-cell enzyme liquid takes 2, 4-dimethyl nitrobenzene as a substrate to carry out catalytic reaction under certain conditions, and 3-methyl-4-nitrobenzoic acid products are generated through HPLC detection. Filtering the catalytic reaction solution by using diatomite, adding concentrated hydrochloric acid into clear solution for acid precipitation, filtering to obtain a crude product, extracting by using ethyl acetate, and carrying out rotary evaporation to obtain a product of 3-methyl-4-nitrobenzoic acid, wherein the substrate conversion rate is 12%, the product content is 98.5%, and the purity is 99.8%. The catalytic process is as follows:

。

in view of the low conversion rate of the experiment, the applicant tries to improve the recombinant escherichia coli engineering bacteria to obtain the technical scheme of the invention, which is as follows.

Utilizing an extrachromosomal evolution technology based on an error-prone PCR technology to contain benzyl alcohol dehydrogenase BADHCoding genexylBThe recombinant plasmid pRSFDuet-1-xylBAnd (3) performing directed evolution by using an error-prone PCR (polymerase chain reaction) technology as a template, and screening a large number of mutants to obtain mutant strains with obviously improved enzyme activity units. The detection shows that the mutant strain is the benzyl alcohol dehydrogenase mutant, and the mutant recombinant strain can improve the substrate conversion rate from 12% to more than 80%.

The technical scheme of the invention is as follows: a recombinant Escherichia coli strain is E.coli BL21-pRSFDuet-1-L163W, the nucleotide sequence is shown as SEQ ID NO. 3, and the corresponding amino acid sequence is shown as SEQ ID NO. 4.

The preparation method of the recombinant escherichia coli strain comprises the following steps:

the first step of recombinant Escherichia coli containing improved benzyl alcohol dehydrogenase coding gene is prepared:

containing a gene encoding benzyl alcohol dehydrogenase BADHxylBThe recombinant plasmid pRSFDuet-1-xylBAs a template, directed evolution was performed using error-prone PCR technology. According to the molecular cloning guidelines, PCR products containing linear gene fragments are obtained after PCR reactions, and these PCR products and the pRSFDuet-1 expression plasmid are subjected to double digestion (EcoRI, Not I), purification, ligation and transformation, respectively, to obtain the DNA containing the XMO encoding gene of the xylylen monooxygenasexylMAAnd benzaldehyde dehydrogenase BZDDH coding genexylCThe competent cells of basic Escherichia coli (E.coli) were plated on LB agar plates containing 50 mg/L kanamycin sulfate and cultured overnight at 37 ℃.

Secondly, selecting a single colony growing on the culture dish, inoculating the single colony in an LB liquid culture medium containing kanamycin sulfate, carrying out shaking culture at 37 ℃ for 15 h, centrifugally collecting thalli, carrying out plasmid extraction, PCR identification and double enzyme digestion identification, and carrying out induced expression on escherichia coli containing correct recombinant plasmids, wherein the specific operation is as follows: transferring the bacterial liquid into 100 mL LB liquid culture medium containing 50 mug/mL kanamycin sulfate, carrying out shaking culture at 37 ℃ for 20h, taking out the bacterial liquid, centrifuging at 10000 r/m for 15 min, collecting thalli, and carrying out resuspension on the thalli by using 1/50-1/10 volume of phosphate buffer solution with pH of 8 to obtain whole-cell enzyme liquid. The whole cell enzyme solution is used for a catalytic verification test,obtaining a recombinant escherichia coli strain with obviously improved enzyme activity, and naming the recombinant escherichia coli strainE.coli BL21-pRSFDuet-1-L163W, the nucleotide sequence is shown as SEQ ID NO. 3, and the corresponding amino acid sequence is shown as SEQ ID NO. 4.

Another discovery of the invention is that the recombinant Escherichia coli strain is applied to the preparation of 3-methyl-4-nitrobenzoic acid, and specifically comprises the following steps

Step 1, carrying out amplification culture on the recombinant escherichia coli obtained in the second step in a fermentation culture medium, and centrifuging to obtain resting cells;

in this step, the fermentation medium formula is as follows: 1.0-3.2g/L of glucose monohydrate, 2.0-4.6 g/L of yeast extract powder, 1 g/L of citric acid monohydrate, 2.5 g/L of ammonium sulfate and 14.4 g/L of disodium hydrogen phosphate dodecahydrate. And adding sterilized glucose solution in the fermentation process.

Step 2, collecting the resting cells obtained in the step 1, suspending the resting cells in a buffer solution according to a certain proportion, adding a substrate 2, 4-dimethyl nitrobenzene to obtain a catalytic reaction solution, maintaining the pH stability with an alkaline solution in the buffer solution with the temperature of 10-55 ℃ and the pH of 6.5-9.5, and generating 3-methyl-4-nitrobenzoic acid through reaction at the ventilation volume of 0.4-8L/min by using a compressed air, wherein the feeding mass ratio of the substrate to the resting cells is 1: 0.5-5. And detecting the reaction process by using HPLC, filtering, precipitating with acid, extracting and performing rotary evaporation after the reaction is finished to obtain the product 3-methyl-4-nitrobenzoic acid.

In the step, the concentration of the substrate in the reaction solution is 8-20 g/l; the mass ratio of the substrate to the resting cells is 1:0.5-5, preferably 0.8-2;

in the step, the buffer solution is phosphate buffer solution, borate buffer solution or normal saline or purified water;

in the step, the catalytic reaction temperature can be 15-50 ℃, and preferably 20-30 ℃;

in this step, the catalytic reaction pH may be 6.5 to 9.5, preferably 8.0 to 8.5;

in the step, the alkaline solution for maintaining the stable pH through the catalytic reaction is one of an ammonia water solution and a sodium hydroxide solution;

in the step, the catalytic reaction is carried out, and the introduction amount of compressed air of a 4L catalytic system is 0.4-5L/min.

Has the advantages that: the invention provides a process for preparing and producing 3-methyl-4-nitrobenzoic acid by a microbiological method, which solves the problems of high price or serious pollution of catalysts and oxidants or potential safety hazard of the production process in the prior art. Specifically, the invention solves the problems of high catalyst and price and environmental unfriendliness in a chemical method; the problems of serious pollution caused by a nitric acid oxidation method in a chemical method and potential safety hazard in a technological process are solved; the strain containing the new mutation of the benzyl alcohol dehydrogenase BADH is obtained by an in vitro evolution technology of an error-prone PCR technology, and the conversion rate of catalytic reaction can be remarkably improved; the invention realizes the process method for preparing the 3-methyl-4-nitrobenzoic acid by using the microorganism for the first time, and has the advantages of simple process steps, mild reaction conditions and environmental friendliness.

Drawings

FIG. 1. example 8 fermentation growth curves of recombinant E.coli

FIG. 2. example 8 enzyme-catalyzed reaction Curve

FIG. 3 liquid phase diagram of the product of example 8

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not indicated are those in routine experiments.

Example 1 preparation of 3-methyl-4-nitrobenzoic acid Using basic recombinant E.coli

(1) Taking out the recombinant Escherichia coli engineering bacteria (the strain preservation number is CGMCC NO.14930, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation unit address is the microorganism research institute of China academy of sciences No. 3, West Lu No.1 institute of North Cheng, Xilu, Beijing, the rising area, the preservation date is 2017, 11 months and 20 days) which are preserved at the temperature of-80 ℃ and contain the xylene monooxygenase, the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase, the Latin literature name of the classification naming of the biological material is as follows:Escherichie coli. ) On Luria-Bertani (LB) agarInoculating on the medium, followed by culturing overnight at 37 ℃;

(2) picking a monoclonal colony on an LB agar plate, inoculating the colony in an LB liquid culture medium, and then culturing at 37 ℃ overnight;

(3) taking a proper amount of bacterial liquid from an LB liquid culture medium after overnight culture, inoculating the bacterial liquid into an improved M9 liquid culture medium, culturing for 8h at 37 ℃, then inoculating the bacterial liquid into a fermentation culture medium, culturing at 37 ℃ until OD 600 is about 90, centrifugally collecting thalli, and washing twice by using normal saline to obtain resting cells; a sterilized glucose solution may be optionally added during the incubation at 37 ℃.

TABLE 1 fermentation Medium formulation

。

TABLE 2 feed Medium formulation

。

(4) Adding 4L of phosphate buffer solution with the pH value of 8 into a fermentation tank, adding 36.0g of 2, 4-dimethyl nitrobenzene into the fermentation tank, adding 100.8g of resting cells to obtain catalytic reaction solution, setting the catalytic conditions to stir at 300rpm, the temperature to 20 ℃, the ventilation volume to be 2L/min, and controlling the pH value of the catalytic reaction solution to be stabilized at about 8.0 by using ammonia water solution. Sampling every 3h, and detecting the conversion condition of the raw material by HPLC, wherein the raw material is not converted any more, and the catalysis is stopped.

(5) Filtering the catalytic reaction liquid by diatomite, adding concentrated hydrochloric acid into clear liquid for acid precipitation, filtering to obtain a crude product, extracting by ethyl acetate, and performing rotary evaporation to obtain a product, namely 3-methyl-4-nitrobenzoic acid.

The conversion rate of the substrate 2, 4-dimethyl nitrobenzene is 12%, and the obtained product is detected by HPLC: the content is 98.5 percent, and the purity is 99.8 percent.

Example 2 construction and screening of recombinant E.coli containing improved Gene encoding benzyl alcohol dehydrogenase

Using recombinant plasmid containing benzyl alcohol dehydrogenase gene as template and Primer 5.0 to design and synthesize two-end Primer: (TABLE 3), PCR products containing linear gene fragments were obtained after PCR reaction according to molecular cloning instructions using error-prone PCR techniques (materials and concentrations are shown in TABLE 4, reaction conditions are shown in TABLE 5), and these PCR products and pET28a (+) expression plasmids were subjected to double digestion (EcoRI, Not I), purification, ligation and transformation, respectively, of the XMO encoding gene already containing xylene monooxygenasexylMAAnd benzaldehyde dehydrogenase BZDDH coding genexylCThe competent cells of basic Escherichia coli (E.coli) were plated on LB agar plates containing 50 mg/L kanamycin sulfate and cultured overnight at 37 ℃.

TABLE 3 random mutation primer sequences

。

TABLE 450 μ L error-prone PCR Material System

。

TABLE 5 error-prone PCR reaction conditions

。

Picking up a single colony growing on the culture dish, inoculating the single colony in an LB liquid culture medium containing 50 mu g/mL kanamycin sulfate, carrying out shaking culture at 37 ℃ for 15 h, centrifugally collecting thalli, carrying out plasmid extraction, PCR identification and double enzyme digestion identification (detection by using agarose gel electrophoresis), naming correct recombinant plasmid as pRSFDuet-1-A-W, and carrying out induced expression on escherichia coli containing the correct recombinant plasmid, wherein the specific operation is as follows: transferring the bacterial liquid into 100 mL LB liquid culture medium containing 50 mug/mL kanamycin sulfate, carrying out shaking culture at 37 ℃ for 20h, taking out the bacterial liquid, centrifuging at 10000 r/m for 15 min, collecting thalli, and carrying out resuspension on the thalli by using 1/50-1/10 volume of phosphate buffer solution with pH of 8 to obtain whole-cell enzyme liquid. The whole-cell enzyme solution is used for a catalytic verification test to obtain a mutant strain with obviously improved enzyme activity, which is named as E.coli BL21-pRSFDuet-1-L163W, the corresponding nucleotide sequence before mutation is shown as SEQ ID NO.1, the corresponding amino acid sequence is shown as SEQ ID NO. 2, the corresponding nucleotide sequence after mutation is shown as SEQ ID NO. 3, and the corresponding amino acid sequence is shown as SEQ ID NO. 4.

EXAMPLE 3 preparation of 3-methyl-4-nitrobenzoic acid using modified recombinant E.coli (containing benzyl alcohol dehydrogenase L163W mutant)

The recombinant escherichia coli engineering bacteria of the mutant L163W containing the benzyl alcohol dehydrogenase are adopted for high-density fermentation culture, the culture mode is the same as that of the example 1, and catalysis and extraction are carried out according to the following steps:

(1) adding 4L of physiological saline with pH of 9.5 into a fermentation tank, adding 48.0g of 2, 4-dimethyl nitrobenzene and 96.0 g of resting cells into the physiological saline to obtain a catalytic reaction solution, setting the catalytic conditions of stirring at 300rpm, the temperature of 45 ℃, the ventilation quantity of 0.5L/min, and controlling the pH of the catalytic reaction solution to be stabilized at about 9.5 by using an ammonia water solution. Sampling every 3h, and detecting the conversion condition of the raw material by HPLC, wherein the raw material is not converted any more, and the catalysis is stopped.

(2) Filtering the catalytic reaction liquid through diatomite, adding concentrated hydrochloric acid into clear liquid for acid precipitation, filtering to obtain a crude product, extracting with ethyl acetate, combining organic phases, and performing rotary evaporation to obtain a product, namely 3-methyl-4-nitrobenzoic acid.

The conversion rate of the substrate 2, 4-dimethyl nitrobenzene is 80.2 percent, and the obtained product is detected by HPLC: the content is 94.8 percent, and the purity is 97.3 percent.

Example 4 preparation of 3-methyl-4-nitrobenzoic acid using modified recombinant E.coli (containing benzyl alcohol dehydrogenase L163W mutant)

(1) adding 4L of physiological saline with pH of 6.5 into a fermentation tank, adding 32.0g of 2, 4-dimethyl nitrobenzene and 160.0 g of resting cells into the physiological saline to obtain a catalytic reaction solution, setting the catalytic conditions to stir at 300rpm, the temperature to be 15 ℃, the ventilation volume to be 4L/min, and controlling the pH of the catalytic reaction solution to be stabilized at about 6.5 by using an ammonia water solution. Sampling every 3h, and detecting the conversion condition of the raw material by HPLC, wherein the raw material is not converted any more, and the catalysis is stopped.

The conversion rate of the substrate 2, 4-dimethyl nitrobenzene is 81.26%, and the obtained product is detected by HPLC: the content is 97.8 percent, and the purity is 98.4 percent.

EXAMPLE 5 preparation of 3-methyl-4-nitrobenzoic acid using modified recombinant E.coli (containing benzyl alcohol dehydrogenase L163W mutant)

(1) adding 4L of physiological saline with the pH value of 8.0 into a fermentation tank, adding 60.0g of 2, 4-dimethyl nitrobenzene and 120.0g of resting cells into the physiological saline to obtain a catalytic reaction solution, setting the catalytic conditions to stir at 300rpm, the temperature to be 30 ℃, the ventilation volume to be 2.5L/min, and controlling the pH value of the catalytic reaction solution to be stabilized at about 8.0 by using an ammonia water solution. Sampling every 3h, and detecting the conversion condition of the raw material by HPLC, wherein the raw material is not converted any more, and the catalysis is stopped.

The conversion rate of the substrate 2, 4-dimethyl nitrobenzene is 83.8 percent, and the obtained product is detected by HPLC: the content is 97.8 percent, and the purity is 99.0 percent.

Example 6 preparation of 3-methyl-4-nitrobenzoic acid using modified recombinant E.coli (containing benzyl alcohol dehydrogenase L163W mutant)

(1) adding 4L of physiological saline with the pH value of 8.5 into a fermentation tank, adding 80.0g of 2, 4-dimethyl nitrobenzene and 40.0 g of resting cells into the physiological saline to obtain a catalytic reaction solution, setting the catalytic conditions to stir at 300rpm, the temperature to 20 ℃, the ventilation volume to 3L/min, and controlling the pH value of the catalytic reaction solution to be stable at about 8.2 by using an ammonia water solution. Sampling every 3h, and detecting the conversion condition of the raw material by HPLC, wherein the raw material is not converted any more, and the catalysis is stopped.

The conversion rate of the substrate 2, 4-dimethyl nitrobenzene is 81.6 percent, and the obtained product is detected by HPLC: the content is 95.8 percent, and the purity is 99.1 percent.

Example 7 preparation of 3-methyl-4-nitrobenzoic acid using modified recombinant E.coli (containing benzyl alcohol dehydrogenase L163W mutant)

(1) adding 4L of phosphate buffer solution with the pH value of 8.5 into a fermentation tank, adding 65.6 g of 2, 4-dimethyl nitrobenzene into the fermentation tank, adding 100.0 g of resting cells into the fermentation tank to obtain catalytic reaction liquid, setting the catalytic conditions to stir at 300rpm, controlling the pH value of the catalytic reaction liquid to be stable at about 8.5 and ventilating at 3L/min, using sodium hydroxide solution to control the pH value of the catalytic reaction liquid to be stable, sampling every 3h, detecting the conversion condition of raw materials by HPLC, and stopping catalysis when the raw materials are not converted any more.

The conversion rate of the substrate 2, 4-dimethyl nitrobenzene is 86%, and the obtained product is detected by HPLC: the content is 98.0 percent, and the purity is 99.3 percent.

Example 8 preparation of 3-methyl-4-nitrobenzoic acid using modified recombinant E.coli (containing benzyl alcohol dehydrogenase L163W mutant)

(1) adding 4L of phosphate buffer solution with the pH value of 8.5 into a fermentation tank, adding 65.6 g of 2, 4-dimethyl nitrobenzene and 65.6 g of resting cells into the fermentation tank to obtain catalytic reaction liquid, setting the catalytic conditions to stir at 300rpm, the temperature to 25 ℃, the ventilation volume to 3L/min, and controlling the pH value of the catalytic reaction liquid to be stable at about 8.5 by using an ammonia water solution. Sampling every 3h, and detecting the conversion condition of the raw material by HPLC, wherein the raw material is not converted any more, and the catalysis is stopped.

The conversion rate of the substrate 2, 4-dimethyl nitrobenzene is 89%, and the obtained product is detected by HPLC: the product content is 98.5 percent, and the purity is 99.8 percent.

Example 9 preparation of 3-methyl-4-nitrobenzoic acid using modified recombinant E.coli (containing benzyl alcohol dehydrogenase L163W mutant)

(1) adding 8L of phosphate buffer solution with the pH value of 8.5 into a fermentation tank, adding 131.2g of 2, 4-dimethyl nitrobenzene and 131.2g of resting cells into the fermentation tank to obtain catalytic reaction liquid, setting the catalytic conditions to stir at 300rpm, the temperature to 30 ℃, the ventilation volume to 8L/min, and controlling the pH value of the catalytic reaction liquid to be stable at about 8.5 by using an ammonia water solution. Sampling every 3h, and detecting the conversion condition of the raw material by HPLC, wherein the raw material is not converted any more, and the catalysis is stopped.

The conversion rate of the substrate 2, 4-dimethyl nitrobenzene is 88%, and the obtained product is detected by HPLC: the content is 98.4 percent, and the purity is 99.3 percent.

EXAMPLE 10 preparation of 3-methyl-4-nitrobenzoic acid using modified recombinant E.coli (containing benzyl alcohol dehydrogenase L163W mutant)

(1) 40L of phosphate buffer solution with the pH value of 8.5 is added into a fermentation tank, 984g of 2, 4-dimethyl nitrobenzene and 984g of resting cells are added into the fermentation tank to obtain catalytic reaction liquid, the catalytic conditions are set to be stirring at 300rpm, the temperature is 30 ℃, the ventilation quantity is 30L/min, and the pH value of the catalytic reaction liquid is controlled to be stabilized at about 8.5 by using ammonia water solution. Sampling every 3h, and detecting the conversion condition of the raw material by HPLC, wherein the raw material is not converted any more, and the catalysis is stopped.

The conversion rate of the substrate 2, 4-dimethyl nitrobenzene is 89%, and the obtained product is detected by HPLC: the content is 98.5 percent, and the purity is 99.5 percent.

Sequence listing

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ctgaatgcgc ttaatccgcc agcgggttct tctatcgcga tctttggtgc cggggccgta 600

ggcctttcgg cagttatggc tgccgttgtg gcggggtgta cgaaaatcat cgttgtcgat 660

gtcaaagaga atcgccttaa attggccgat gagcttgggg cgacgcatgt gattaatgca 720

gcaagttctg atccagttga gaagattaag gaaatttgtg ctggcggcgt tccgtatgtg 780

ctcgaaacta gcggtttgcc ttcggttctt cagcaggcga tcctcagctc cgccataggt 840

ggtgagatag gtattgtagg tgcaccaccg atgggtgcca caattcccgt tgatattaat 900

tttttactgt ttaatcgtaa gcttcgaggt attgttgagg ggcaatctat ttcggatata 960

tttattccaa gattggtgga actatatcgt caagggaagt ttccatttga taaattgtta 1020

aagttctatt cctttgatga aattaatcag gcagcggagg actcggaaaa tggaataacc 1080

cttaagccag tgcttcgaat atcctaa 1107

<210>SEQ ID NO: 4

<211> 368

<212> PRT

<213>L163W

<400> 4

Met Glu Met Glu Ile Lys Ala Ala Ile Val Arg Gln Lys Asn Gly Pro

1 5 10 15

PHe Leu Leu Glu His Val Ala Leu Asn Glu Pro Ala Glu Asp Gln Val

20 25 30

Leu Val Arg Leu Val Ala Thr Gly Leu Cys His Thr Asp Leu Val Cys

35 40 45

Arg Asp Gln His Tyr Pro Val Pro Leu Pro Met Val PHe Gly His Glu

50 55 60

Gly Ala Gly Val Val Glu Arg Val Gly Ser Ala Val Lys Lys Val Gln

65 70 75 80

Pro Gly Asp His Val Val Leu Thr PHe Tyr Thr Cys Gly Ser Cys Asp

85 90 95

Ala Cys Leu Ser Gly Asp Pro Thr Ser Cys Ala Asn Ser PHe Gly Pro

100 105 110

Asn PHe Met Gly Arg Ser Val Thr Gly Glu Cys Thr Ile His Asp His

115 120 125

Gln Gly Ala Glu Val Gly Ala Ser PHe PHe Gly Gln Ser Ser PHe Ala

130 135 140

Thr Tyr Ala Leu Ser Tyr Glu Arg Asn Thr Val Lys Val Thr Lys Asp

145 150 155 160

Val Pro Trp Glu Leu Leu Gly Pro Leu Gly Cys Gly Ile Gln Thr Gly

165 170 175

Ala Gly Ser Val Leu Asn Ala Leu Asn Pro Pro Ala Gly Ser Ser Ile

180 185 190

Ala Ile PHe Gly Ala Gly Ala Val Gly Leu Ser Ala Val Met Ala Ala

195 200 205

Val Val Ala Gly Cys Thr Lys Ile Ile Val Val Asp Val Lys Glu Asn

210 215 220

Arg Leu Lys Leu Ala Asp Glu Leu Gly Ala Thr His Val Ile Asn Ala

225 230 235 240

Ala Ser Ser Asp Pro Val Glu Lys Ile Lys Glu Ile Cys Ala Gly Gly

245 250 255

Val Pro Tyr Val Leu Glu Thr Ser Gly Leu Pro Ser Val Leu Gln Gln

260 265 270

Ala Ile Leu Ser Ser Ala Ile Gly Gly Glu Ile Gly Ile Val Gly Ala

275 280 285

Pro Pro Met Gly Ala Thr Ile Pro Val Asp Ile Asn PHe Leu Leu PHe

290 295 300

Asn Arg Lys Leu Arg Gly Ile Val Glu Gly Gln Ser Ile Ser Asp Ile

305 310 315 320

PHe Ile Pro Arg Leu Val Glu Leu Tyr Arg Gln Gly Lys PHe Pro PHe

325 330 335

Asp Lys Leu Leu Lys PHe Tyr Ser PHe Asp Glu Ile Asn Gln Ala Ala

340 345 350

Glu Asp Ser Glu Asn Gly Ile Thr Leu Lys Pro Val Leu Arg Ile Ser

355 360 365

Sequence listing

<110> Dijia group of pharmaceuticals, Inc

<120> method for preparing 3-methyl-4-nitrobenzoic acid by microbiological method

<130> 20210622

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1107

<212> DNA

<213> Pseudomonas putida

<400> 1

atggaaatgg aaatcaaagc ggcaatagtt cgccaaaaaa atggcccttt cttacttgag 60

catgtagctc ttaatgagcc agctgaagat caggttctcg ttagattggt tgcaaccggg 120

ctgtgtcata cggatctggt ttgtcgcgat cagcactatc cggttccact accgatggta 180

tttgggcatg aaggggctgg tgtggttgag cgggttgggt ccgcggtcaa aaaggttcag 240

ccgggcgatc atgttgtttt gacattttat acctgcggga gctgtgatgc ttgtctttcc 300

ggagacccta ccagttgtgc aaactcattt ggccctaact ttatggggcg ctcggtaacc 360

ggggagtgca ccatccacga tcaccaaggg gcagaggtgg gagcaagctt ttttgggcag 420

tcctcctttg cgacatatgc gctatcttat gaacgcaaca ctgtgaaggt cacgaaagac 480

gtaccgcttg agctgcttgg gcctcttggt tgtggcattc aaactggcgc agggtctgtt 540

ctgaatgcgc ttaatccgcc agcgggttct tctatcgcga tctttggtgc cggggccgta 600

ggcctttcgg cagttatggc tgccgttgtg gcggggtgta cgaaaatcat cgttgtcgat 660

gtcaaagaga atcgccttaa attggccgat gagcttgggg cgacgcatgt gattaatgca 720

gcaagttctg atccagttga gaagattaag gaaatttgtg ctggcggcgt tccgtatgtg 780

ctcgaaacta gcggtttgcc ttcggttctt cagcaggcga tcctcagctc cgccataggt 840

ggtgagatag gtattgtagg tgcaccaccg atgggtgcca caattcccgt tgatattaat 900

tttttactgt ttaatcgtaa gcttcgaggt attgttgagg ggcaatctat ttcggatata 960

tttattccaa gattggtgga actatatcgt caagggaagt ttccatttga taaattgtta 1020

aagttctatt cctttgatga aattaatcag gcagcggagg actcggaaaa tggaataacc 1080

cttaagccag tgcttcgaat atcctaa 1107

<210> 2

<211> 368

<212> PRT

<213> Pseudomonas putida

<400> 2

Met Glu Met Glu Ile Lys Ala Ala Ile Val Arg Gln Lys Asn Gly Pro

1 5 10 15

Phe Leu Leu Glu His Val Ala Leu Asn Glu Pro Ala Glu Asp Gln Val

20 25 30

Leu Val Arg Leu Val Ala Thr Gly Leu Cys His Thr Asp Leu Val Cys

35 40 45

Arg Asp Gln His Tyr Pro Val Pro Leu Pro Met Val Phe Gly His Glu

50 55 60

Gly Ala Gly Val Val Glu Arg Val Gly Ser Ala Val Lys Lys Val Gln

65 70 75 80

Pro Gly Asp His Val Val Leu Thr Phe Tyr Thr Cys Gly Ser Cys Asp

85 90 95

Ala Cys Leu Ser Gly Asp Pro Thr Ser Cys Ala Asn Ser Phe Gly Pro

100 105 110

Asn Phe Met Gly Arg Ser Val Thr Gly Glu Cys Thr Ile His Asp His

115 120 125

Gln Gly Ala Glu Val Gly Ala Ser Phe Phe Gly Gln Ser Ser Phe Ala

130 135 140

Thr Tyr Ala Leu Ser Tyr Glu Arg Asn Thr Val Lys Val Thr Lys Asp

145 150 155 160

Val Pro Leu Glu Leu Leu Gly Pro Leu Gly Cys Gly Ile Gln Thr Gly

165 170 175

Ala Gly Ser Val Leu Asn Ala Leu Asn Pro Pro Ala Gly Ser Ser Ile

180 185 190

Ala Ile Phe Gly Ala Gly Ala Val Gly Leu Ser Ala Val Met Ala Ala

195 200 205

Val Val Ala Gly Cys Thr Lys Ile Ile Val Val Asp Val Lys Glu Asn

210 215 220

Arg Leu Lys Leu Ala Asp Glu Leu Gly Ala Thr His Val Ile Asn Ala

225 230 235 240

Ala Ser Ser Asp Pro Val Glu Lys Ile Lys Glu Ile Cys Ala Gly Gly

245 250 255

Val Pro Tyr Val Leu Glu Thr Ser Gly Leu Pro Ser Val Leu Gln Gln

260 265 270

Ala Ile Leu Ser Ser Ala Ile Gly Gly Glu Ile Gly Ile Val Gly Ala

275 280 285

Pro Pro Met Gly Ala Thr Ile Pro Val Asp Ile Asn Phe Leu Leu Phe

290 295 300

Asn Arg Lys Leu Arg Gly Ile Val Glu Gly Gln Ser Ile Ser Asp Ile

305 310 315 320

Phe Ile Pro Arg Leu Val Glu Leu Tyr Arg Gln Gly Lys Phe Pro Phe

325 330 335

Asp Lys Leu Leu Lys Phe Tyr Ser Phe Asp Glu Ile Asn Gln Ala Ala

340 345 350

Glu Asp Ser Glu Asn Gly Ile Thr Leu Lys Pro Val Leu Arg Ile Ser

355 360 365

<210> 3

<211> 1107

<212> DNA

<213> Artificial Sequence

<400> 3

atggaaatgg aaatcaaagc ggcaatagtt cgccaaaaaa atggcccttt cttacttgag 60

catgtagctc ttaatgagcc agctgaagat caggttctcg ttagattggt tgcaaccggg 120

ctgtgtcata cggatctggt ttgtcgcgat cagcactatc cggttccact accgatggta 180

tttgggcatg aaggggctgg tgtggttgag cgggttgggt ccgcggtcaa aaaggttcag 240

ccgggcgatc atgttgtttt gacattttat acctgcggga gctgtgatgc ttgtctttcc 300

ggagacccta ccagttgtgc aaactcattt ggccctaact ttatggggcg ctcggtaacc 360

ggggagtgca ccatccacga tcaccaaggg gcagaggtgg gagcaagctt ttttgggcag 420

tcctcctttg cgacatatgc gctatcttat gaacgcaaca ctgtgaaggt cacgaaagac 480

gtaccgtggg agctgcttgg gcctcttggt tgtggcattc aaactggcgc agggtctgtt 540

ctgaatgcgc ttaatccgcc agcgggttct tctatcgcga tctttggtgc cggggccgta 600

ggcctttcgg cagttatggc tgccgttgtg gcggggtgta cgaaaatcat cgttgtcgat 660

gtcaaagaga atcgccttaa attggccgat gagcttgggg cgacgcatgt gattaatgca 720

gcaagttctg atccagttga gaagattaag gaaatttgtg ctggcggcgt tccgtatgtg 780

ctcgaaacta gcggtttgcc ttcggttctt cagcaggcga tcctcagctc cgccataggt 840

ggtgagatag gtattgtagg tgcaccaccg atgggtgcca caattcccgt tgatattaat 900

tttttactgt ttaatcgtaa gcttcgaggt attgttgagg ggcaatctat ttcggatata 960

tttattccaa gattggtgga actatatcgt caagggaagt ttccatttga taaattgtta 1020

aagttctatt cctttgatga aattaatcag gcagcggagg actcggaaaa tggaataacc 1080

cttaagccag tgcttcgaat atcctaa 1107

<210> 4

<211> 368

<212> PRT

<213> Artificial Sequence

<400> 4

Met Glu Met Glu Ile Lys Ala Ala Ile Val Arg Gln Lys Asn Gly Pro

1 5 10 15

Phe Leu Leu Glu His Val Ala Leu Asn Glu Pro Ala Glu Asp Gln Val

20 25 30

Leu Val Arg Leu Val Ala Thr Gly Leu Cys His Thr Asp Leu Val Cys

35 40 45

Arg Asp Gln His Tyr Pro Val Pro Leu Pro Met Val Phe Gly His Glu

50 55 60

Gly Ala Gly Val Val Glu Arg Val Gly Ser Ala Val Lys Lys Val Gln

65 70 75 80

Pro Gly Asp His Val Val Leu Thr Phe Tyr Thr Cys Gly Ser Cys Asp

85 90 95

Ala Cys Leu Ser Gly Asp Pro Thr Ser Cys Ala Asn Ser Phe Gly Pro

100 105 110

Asn Phe Met Gly Arg Ser Val Thr Gly Glu Cys Thr Ile His Asp His

115 120 125

Gln Gly Ala Glu Val Gly Ala Ser Phe Phe Gly Gln Ser Ser Phe Ala

130 135 140

Thr Tyr Ala Leu Ser Tyr Glu Arg Asn Thr Val Lys Val Thr Lys Asp

145 150 155 160

Val Pro Trp Glu Leu Leu Gly Pro Leu Gly Cys Gly Ile Gln Thr Gly

165 170 175

Ala Gly Ser Val Leu Asn Ala Leu Asn Pro Pro Ala Gly Ser Ser Ile

180 185 190

Ala Ile Phe Gly Ala Gly Ala Val Gly Leu Ser Ala Val Met Ala Ala

195 200 205

Val Val Ala Gly Cys Thr Lys Ile Ile Val Val Asp Val Lys Glu Asn

210 215 220

Arg Leu Lys Leu Ala Asp Glu Leu Gly Ala Thr His Val Ile Asn Ala

225 230 235 240

Ala Ser Ser Asp Pro Val Glu Lys Ile Lys Glu Ile Cys Ala Gly Gly

245 250 255

Val Pro Tyr Val Leu Glu Thr Ser Gly Leu Pro Ser Val Leu Gln Gln

260 265 270

Ala Ile Leu Ser Ser Ala Ile Gly Gly Glu Ile Gly Ile Val Gly Ala

275 280 285

Pro Pro Met Gly Ala Thr Ile Pro Val Asp Ile Asn Phe Leu Leu Phe

290 295 300

Asn Arg Lys Leu Arg Gly Ile Val Glu Gly Gln Ser Ile Ser Asp Ile

305 310 315 320

Phe Ile Pro Arg Leu Val Glu Leu Tyr Arg Gln Gly Lys Phe Pro Phe

325 330 335

Asp Lys Leu Leu Lys Phe Tyr Ser Phe Asp Glu Ile Asn Gln Ala Ala

340 345 350

Glu Asp Ser Glu Asn Gly Ile Thr Leu Lys Pro Val Leu Arg Ile Ser

355 360 365

Claims

1. A recombinant Escherichia coli strain is characterized in that the recombinant Escherichia coli strain is E.coli BL21-pRSFDuet-1-L163W, the nucleotide sequence is shown as SEQ ID NO. 3, and the corresponding amino acid sequence is shown as SEQ ID NO. 4.

2. The method for preparing a recombinant E.coli strain according to claim 1, comprising the steps of:

containing a gene encoding benzyl alcohol dehydrogenase BADHxylBThe recombinant plasmid pRSFDuet-1-xylBAs a template, directed evolution was carried out using error-prone PCR techniques, and the PCR product and the pRSFDuet-1 expression plasmid were subjected to double digestion (EcoRI, Not I), purification, ligation and transformation, respectively, to already contain the gene encoding the XMO monooxygenase XMxylMAAnd benzaldehyde dehydrogenase BZDDH coding genexylCThe basic Escherichia coli competent cells of (1) were spread on an LB agar plate containing 50 mg/L kanamycin sulfate, and cultured overnight at 37 ℃;

secondly, selecting a single colony growing on the culture dish, inoculating the single colony in an LB liquid culture medium containing kanamycin sulfate, carrying out shaking culture at 37 ℃ for 15 h, centrifugally collecting thalli, carrying out plasmid extraction, PCR identification and double enzyme digestion identification, and carrying out induced expression on escherichia coli containing correct recombinant plasmids, wherein the specific operation is as follows: transferring the bacterial liquid into 100 mL LB liquid culture medium containing 50 mug/mL kanamycin sulfate, carrying out shaking culture at 37 ℃ for 20h, taking out the bacterial liquid, centrifuging at 10000 r/m for 15 min, collecting thalli, carrying out heavy suspension on the thalli by using a phosphate buffer solution with the pH value of 8 of the original fermentation liquid volume of 1/50-1/10 to obtain a whole cell enzyme liquid, and using the whole cell enzyme liquid for a catalytic verification test to obtain a recombinant escherichia coli strainE.coliBL21-pRSFDuet-1-L163W。

3. The recombinant Escherichia coli strain according to claim 2E.coliThe application of BL21-pRSFDuet-1-L163W in preparing 3-methyl-4-nitrobenzoic acid.

4. A method for preparing 3-methyl-4-nitrobenzoic acid is characterized by comprising the following steps:

step 1, performing amplification culture on the recombinant escherichia coli obtained in the second step of claim 2 in a fermentation culture medium, and centrifuging to obtain resting cells, wherein the formula of the fermentation culture medium is as follows: 1.0-3.2g/L of glucose monohydrate, 2.0-4.6 g/L of yeast extract powder, 1 g/L of citric acid monohydrate, 2.5 g/L of ammonium sulfate and 14.4 g/L of disodium hydrogen phosphate dodecahydrate;

step 2, collecting the resting cells obtained in the step 1, suspending the resting cells in a buffer solution according to a certain proportion, adding a substrate 2, 4-dimethyl nitrobenzene to obtain a catalytic reaction solution, maintaining the pH stability with an alkaline solution in the buffer solution with the temperature of 10-55 ℃ and the pH of 6.5-9.5, and generating 3-methyl-4-nitrobenzoic acid through reaction at the ventilation volume of 0.4-8L/min by using a compressed air, wherein the feeding mass ratio of the substrate to the resting cells is 1: 0.5-5.

5. The method for preparing 3-methyl-4-nitrobenzoic acid according to claim 4 where the fermentation in step 1 is supplemented with a sterilized glucose solution.

6. The method for preparing 3-methyl-4-nitrobenzoic acid according to claim 4 where in step 2 the substrate concentration is 8-20 g/l and the mass ratio of substrate to resting cells is 0.8-2.

7. The method for preparing 3-methyl-4-nitrobenzoic acid according to claim 4 where in step 2 the buffer is selected from the group consisting of phosphate buffer, borate buffer.

8. The method for preparing 3-methyl-4-nitrobenzoic acid according to claim 4 where in step 2 the catalytic reaction temperature is 20-30 ℃.

9. The method of claim 4, wherein the pH of step 2 is 8.0 to 8.5.