Biological catalysis method for synthesizing 4-acetyl-2-methyl benzoic acid based on 4-acetyl-2-methyl cyanophenyl

Abstract

The invention relates to the technical field of organic synthesis, in particular to a biological catalysis method for synthesizing 4-acetyl-2-methyl benzoic acid based on 4-acetyl-2-methyl cyanophenyl. Free or immobilized nitrilase is mostly adopted in a biological catalysis method, and the conversion rate of 4-acetyl-2-methylbenzonitrile is poor. In order to solve the technical problems, the invention provides the biological catalysis method for synthesizing the 4-acetyl-2-methyl benzoic acid based on the 4-acetyl-2-methyl cyanophenyl, and the method comprises the following steps: by taking the 4-acetyl-2-methyl cyanophenyl as a reaction substrate, resting cells of Rhodococcus sp. CCZU10-1 as a catalyst and a deep eutectic solvent (DES) as a cosolvent, synthesizing the 4-acetyl-2-methyl benzoic acid on the basis of the 4-acetyl-2-methyl cyanophenyl. A phosphoric acid buffer solution is used as a reaction solvent, 4-acetyl-2-methyl benzoic acid is obtained through catalytic stirring hydrolysis reaction, the reaction condition is mild, DES is green and low in toxicity and biodegradable, the substrate conversion rate is high (gt, 95%), the product selectivity is good, the process environment is friendly, and the green chemical principle is met.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and specifically to a biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile. Background Art

[0002] 4-Acetyl-2-methylbenzoic acid, as an important organic synthesis intermediate, is widely used in pharmaceuticals, pesticides, and fine chemicals. Its traditional synthesis mainly relies on the chemical catalytic hydrolysis of 4-acetyl-2-methylbenzonitrile. However, chemical methods typically require strong acid / base conditions, high temperature, and high pressure, resulting in poor selectivity, numerous byproducts, and environmental unfriendliness. In recent years, biocatalysis technology has attracted attention in the field of nitrile hydrolysis due to its advantages such as high selectivity and mild reaction conditions.

[0003] Existing biocatalytic methods mostly employ free or immobilized nitrile hydrolases, but enzymatic processes still face the following technical bottlenecks: Substrate solubility limitation: 4-acetyl-2-methylbenzonitrile is a hydrophobic compound with low solubility in aqueous phase. Organic solvents (such as DMSO and methanol) are required to aid solubility, but organic solvents can easily lead to enzyme inactivation. Insufficient enzyme stability: Nitrile hydrolases are easily affected by pH, temperature and substrate inhibition during the reaction, leading to a decrease in catalytic efficiency; Poor strain adaptability: The natural microbial resources that produce nitrile hydrolases are limited, and most strains require complex induction conditions, which limits their industrial application.

[0004] Deep eutectic solvents (DES), as a novel type of green solvent, are formed by non-covalent bonds between hydrogen bond acceptors (such as betaine) and hydrogen bond donors (such as organic acids). They are characterized by low toxicity, biodegradability, and high structural designability. Previous studies have attempted to apply DES to biocatalytic systems, but its compatibility with microbial cells and its mechanism of influence on enzyme activity remain unclear.

[0005] To address the aforementioned problems, this invention provides a method based on Rhodococcus. Rhodococcus A biocatalytic method using resting sp. CCZU10-1 cells. This strain, after induction under specific conditions, can efficiently express nitrile hydrolases, achieving efficient conversion of 4-acetyl-2-methylbenzonitrile in a DES-phosphate buffer system. By optimizing the DES composition (betaine-acetic acid molar ratio 1:2), reaction pH (7.0-7.5), and induction conditions (caprolactam induction), substrate solubility, enzyme stability, and reaction efficiency were significantly improved, providing a new pathway for the green biotransformation of hydrophobic nitrile compounds. Summary of the Invention

[0006] A problem with existing technologies is that biocatalytic methods often employ free or immobilized nitrile hydrolases, resulting in poor conversion rates of 4-acetyl-2-methylbenzonitrile. To address these issues, this invention provides a biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid from 4-acetyl-2-methylbenzonitrile, using 4-acetyl-2-methylbenzonitrile as the reaction substrate. Rhodococcus sp Using resting cells of CCZU10-1 as a catalyst, deep eutectic solvent (DES) as a co-solvent, and phosphate buffer solution as a reaction solvent, a catalytic stirring hydrolysis reaction was carried out to obtain 4-acetyl-2-methylbenzoic acid. The DES was a mixture formed by anhydrous betaine and acetic acid.

[0007] Preferably, the pH of the phosphate buffer solution is 7.0-7.5.

[0008] Preferably, the DES is a mixture of anhydrous betaine and acetic acid in a molar ratio of 1:2.

[0009] Preferably, Rhodococcus sp The method for preparing resting CCZU10-1 cells is as follows: (1) The activated Rhodococcus sp CCZU10-1 strain was inoculated into seed culture medium for pre-culture to obtain inoculum solution; (2) The seed culture was inoculated into the fermentation medium and cultured under aerobic conditions. During the culture, an inducer was added to induce protein expression. After the culture was completed, the protein was collected. Rhodococcus sp Resting cells of CCZU10-1.

[0010] Preferably, in step (1) Rhodococcus sp The method for activating CCZU10-1 strain includes the following steps: Will Rhodococcus sp CCZU10-1 strain was inoculated onto slant agar and cultured at 30°C for at least 48 hours to obtain activated strain. Rhodococcus sp CCZU10-1 strain; the composition of the slant culture medium is as follows: The ingredients are: glucose 5 g / L, peptone 5 g / L, yeast extract 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, NaCl 1 g / L, MgSO4 0.2 g / L, and water as the solvent.

[0011] Preferably, the fermentation medium has the following composition: The ingredients were: glucose 10 g / L, peptone 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, NaCl 1 g / L, MgSO4 0.2 g / L, and water as the solvent.

[0012] Preferably, the inducing agent is caprolactam.

[0013] Preferably, the DES content in the entire reaction system is 12-25 wt.%.

[0014] The present invention has the following beneficial effects: The present invention provides a biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile, which has the following significant advantages: (1) High selectivity, mild conditions and environmental friendliness: the core lies in the utilization of Rhodococcus sp. Using CCZU10-1 resting cells as a biocatalyst, this method utilizes the natural catalytic activity of microbial enzymes (nitrile hydrolases) to selectively hydrolyze 4-acetyl-2-methylbenzonitrile to the target product 4-acetyl-2-methylbenzoic acid under mild conditions (aqueous phase, neutral pH). Compared with traditional chemical hydrolysis methods (which often require strong acids / bases, high temperature and high pressure), this method has mild reaction conditions, low energy consumption, few by-products, and is environmentally friendly, avoiding the use of toxic reagents and the generation of large amounts of wastewater and waste residue. (2) Effectively solves the problem of poor substrate water solubility and improves reaction efficiency: A novel deep eutectic solvent (DES) formed by anhydrous betaine and acetic acid (molar ratio 1:2) is introduced as a co-solvent. This DES is low in toxicity, biodegradable, and low in cost, and can significantly improve the solubility of the hydrophobic substrate 4-acetyl-2-methylbenzonitrile in phosphate buffer solution (pH=7.0-7.5). This effectively solves the key problem of substrate mass transfer limitation in biocatalysis, promotes substrate-enzyme contact, and greatly improves reaction rate and conversion efficiency. (3) DES concentration optimization to balance solubility and biocompatibility: By controlling the mass percentage of DES in the entire reaction system to 12-25 wt.% (preferably 15 wt.%), the optimal balance between effectively improving substrate solubility and minimizing negative impact on cell activity / stability is achieved. This preferred concentration can maximize the solubilizing advantage of DES while ensuring that the catalyst (resting cells) maintains high catalytic activity. (4) High yield of target product and relatively simple purification: Thanks to the highly selective biocatalysis and the assistance of DES, this method can obtain the target product 4-acetyl-2-methylbenzoic acid with high yield and high purity. The reaction system is relatively pure, which is conducive to the subsequent separation and purification of the product. Attached Figure Description

[0015] Figure 1 This is a high-performance liquid chromatogram of 4-acetyl-2-methylbenzonitrile.

[0016] Figure 2This is a high-performance liquid chromatogram of 4-acetyl-2-methylbenzoic acid obtained in Example 1. DETAILED DESCRIPTION

[0017] The present invention will be described in detail below with reference to embodiments. However, it should be understood that the following embodiments are merely illustrative examples of implementation of the present invention and are not intended to limit the scope of the present invention.

[0018] The present invention uses Rhodococcus sp The CCZU10-1 strain belongs to the prior art and has been disclosed in Chinese invention patent CN102250802A. It is a nitrile hydrolase strain obtained by the inventors of this invention from soil near Changzhou University's Baiyun Campus and Changzhou Science and Education City through primary screening, secondary screening, and isolation and purification. Identification using 16S rDNA and physicochemical characteristics confirmed that this strain is *Rhodococcus*. Rhodococcus sp. Number Rhodococcus sp. CCZU10-1. This strain was deposited on May 25, 2011, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCCNo.4911.

[0019] The technical parameters for liquid chromatography analysis in this invention are as follows: Thermo Fisher Scientific high-performance liquid chromatograph, stainless steel packed column, C18 packing material, 0.25m in length, 4.6mm in inner diameter, flow rate 1ml / min, column temperature 30℃, mobile phase is a mixture of anhydrous methanol and 0.1wt% formic acid aqueous solution at a volume ratio of 55:45, and injection volume is 10μL.

[0020] Example 1

[0021] A biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile, the specific steps of which are as follows: The collected 0.1g Rhodococcus spResting CCZU10-1 cells were added to 1 mL of a 100 mg / mL solution of 4-acetyl-2-methylbenzonitrile diluted to volume with phosphate buffer (pH 7.0). A binary DES solution, consisting of anhydrous betaine (CAS No.: 107-43-7) and acetic acid in a molar ratio of 1:2, was then added to the reaction system. The amount of the binary DES solution added to the reaction system was 15 wt.% of the total volume. The mixture was then stirred at 30°C and 160 rpm for 16 h. After the reaction, the content of 4-acetyl-2-methylbenzoic acid in the reaction solution was determined. 1 mL of the reaction solution was centrifuged at 8000 rpm, and 50 μL of the supernatant was diluted 20-fold with 950 μL of anhydrous methanol. Liquid chromatography analysis was then performed. The results showed that the peak time of 4-acetyl-2-methylbenzonitrile was 10.1 min (as per the instructions). Figure 1 As shown in the figure), the elution time of 4-acetyl-2-methylbenzoic acid is 8.2 min (as per the instructions). Figure 2 As shown in the figure, the concentration of 4-acetyl-2-methylbenzoic acid in the test sample was 96.8 mmol / L. It can be calculated that the molar conversion rate of 4-acetyl-2-methylbenzonitrile in the reaction system was 96.8%.

[0022] The Rhodococcus sp The method for preparing resting CCZU10-1 cells is as follows: (1) Rhodococcus sp CCZU10-1 strain was inoculated onto slant agar and cultured at 30°C for 48 hours to obtain activated strain. Rhodococcus sp CCZU10-1 strain; the composition of the slant culture medium is as follows: Glucose 5g / L, peptone 5g / L, yeast extract 5g / L, KH2PO4 1g / L, K2HPO4 1g / L, NaCl 1g / L, MgSO4 0.2g / L, solvent: water; (2) The activated Rhodococcus sp CCZU10-1 bacterial strain was inoculated into seed culture medium for pre-culture (30℃, 16h) to obtain the inoculum solution; the composition of the seed culture medium is as follows: Glucose 5 g / L, peptone 5 g / L, yeast extract 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, NaCl 1 g / L, MgSO4 0.2 g / L, solvent: water; (3) The seed culture was inoculated into the fermentation medium and cultured under aerobic conditions (30℃, 48h). During the culture, caprolactam was added to induce protein expression. After the culture was completed, resting cells were collected. The composition of the fermentation medium is as follows: The ingredients were: glucose 10 g / L, peptone 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, NaCl 1 g / L, MgSO4 0.2 g / L, and water as the solvent.

[0023] Example 2 is the same as Example 1, except that the amount of binary DES solution added to the reaction system in Example 2 accounts for 25 wt.% of the total amount of the reaction system. The concentration of 4-acetyl-2-methylbenzoic acid in the test sample is 87 mmol / L. It can be calculated that the molar conversion rate of 4-acetyl-2-methylbenzonitrile in the reaction system is 87%.

[0024] Example 3 is the same as Example 1, except that the amount of binary DES solution added to the reaction system in Example 3 accounts for 10 wt.% of the total amount of the reaction system. The concentration of 4-acetyl-2-methylbenzoic acid in the test sample is 75.4 mmol / L. It can be calculated that the molar conversion rate of 4-acetyl-2-methylbenzonitrile in the reaction system is 75.4%.

[0025] Comparative Example 1 is the same as Example 1, except that no binary DES solution was added in Comparative Example 1. The concentration of 4-acetyl-2-methylbenzoic acid in the test sample was 45.2 mmol / L. It was calculated that the molar conversion rate of 4-acetyl-2-methylbenzonitrile in the reaction system was 45.2%.

[0026] Comparative Example 2 is the same as Example 1, except that it uses a binary DES solution of choline chloride and ethylene glycol in a molar ratio of 1:2 instead of the binary DES solution of anhydrous betaine and acetic acid in a molar ratio of 1:2 in Example 1. The concentration of 4-acetyl-2-methylbenzoic acid in the test sample was 37 mmol / L, and the molar conversion rate of 4-acetyl-2-methylbenzonitrile in the reaction system was calculated to be 37%.

[0027] Comparative Example 3 is the same as Example 1, except that in Comparative Example 3, the collected 0.1g... Rhodococcus spResting CCZU10-1 cells were added to 1 mL of a 100 mg / mL solution of 4-acetyl-2-methylbenzonitrile diluted with phosphate buffer (pH 7.5). A binary DES solution, consisting of anhydrous betaine and acetic acid in a 1:2 molar ratio, was then added to the reaction system. The amount of the binary DES solution added to the reaction system was 15 wt.% of the total reaction volume. The mixture was stirred at 30 °C and 160 rpm for 16 h. After the reaction, the content of 4-acetyl-2-methylbenzoic acid in the reaction solution was determined. 1 mL of the reaction solution was centrifuged at 8000 rpm, and 50 μL of the supernatant was collected and diluted 20-fold with 950 μL of anhydrous methanol. The solution was then analyzed by liquid chromatography. The concentration of 4-acetyl-2-methylbenzoic acid in the test sample was 82.4 mmol / L. Calculations show that the molar conversion rate of 4-acetyl-2-methylbenzonitrile in the reaction system was 82.4%.

[0028] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile, characterized in that, Using 4-acetyl-2-methylbenzonitrile as the reaction substrate, Rhodococcus sp Using resting cells of CCZU10-1 as a catalyst, a deep eutectic solvent as a co-solvent, and a phosphate buffer solution as a reaction solvent, a catalytic stirring hydrolysis reaction was carried out to obtain 4-acetyl-2-methylbenzoic acid (DES), which is a mixture of anhydrous betaine and acetic acid.

2. The biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile according to claim 1, characterized in that, The pH of the phosphate buffer solution is 7.0-7.

5.

3. The biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile according to claim 1, characterized in that, The DES is a mixture of anhydrous betaine and acetic acid in a molar ratio of 1:

2.

4. The biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile according to claim 1, characterized in that, Rhodococcus sp The method for preparing resting CCZU10-1 cells is as follows: (1) The activated Rhodococcus sp CCZU10-1 strain was inoculated into seed culture medium for pre-culture to obtain inoculum solution; (2) The seed culture was inoculated into the fermentation medium and cultured under aerobic conditions. During the culture, an inducer was added to induce protein expression. After the culture was completed, resting cells were collected.

5. The biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile according to claim 4, characterized in that, In step (1) Rhodococcus sp The method for activating CCZU10-1 strain includes the following steps: Will Rhodococcus sp CCZU10-1 strain was inoculated onto slant agar and cultured at 30°C for at least 48 hours to obtain activated strain. Rhodococcus sp CCZU10-1 strain; the composition of the slant culture medium is as follows: The ingredients are: glucose 5 g / L, peptone 5 g / L, yeast extract 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, NaCl 1 g / L, MgSO4 0.2 g / L, and water as the solvent.

6. The biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile according to claim 4, characterized in that, The fermentation medium consists of the following components: The ingredients are: glucose 10 g / L, peptone 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, NaCl 1 g / L, MgSO4 0.2 g / L, and water as the solvent.

7. The biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile according to claim 4, characterized in that, The inducing agent is caprolactam.

8. The biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile according to claim 4, characterized in that, The seed culture medium consists of the following components: glucose 5 g / L, peptone 5 g / L, yeast extract 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, NaCl 1 g / L, MgSO4 0.2 g / L, and water as the solvent.

9. The biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile according to claim 1, characterized in that, The DES content in the entire reaction system is 12-25 wt.%.

10. The biocatalytic method for synthesizing 4-acetyl-2-methylbenzoic acid based on 4-acetyl-2-methylbenzonitrile according to claim 9, characterized in that, The DES content in the entire reaction system is 15 wt.%.

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