An enzyme-catalyzed process for the production of high purity (S)-nornicotine from myosmine

Abstract

The application discloses an enzyme catalysis method for generating high-purity (S)-nornicotine from meso. The method comprises the following steps: (1) taking meso as raw material, and generating racemic (R,S)-nornicotine by using immobilized enzyme catalysis; (2) taking (R)-nornicotine in the racemic (R,S)-nornicotine generated in the step (1) as raw material, and generating meso by using immobilized enzyme catalysis; and high-purity (S)-nornicotine is obtained by continuously circulating the above steps to consume meso in a reaction system. The application generates high-concentration (S)-nornicotine from meso by using immobilized enzyme catalysis, is a process route for obtaining high-concentration and high-purity chiral (S)-nornicotine and has low cost, and has high industrial application value and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological chemical industry, in particular to an enzyme catalysis method for generating high-purity (S)-nornicotine from mesembrine. BACKGROUND

[0002] Alkaloids are an important class of chemical components in tobacco plants, and nicotine accounts for more than 95% of the total alkaloids in tobacco. Nicotine, also known as nicotine (chemical name: 1-methyl-2-(3-pyridyl) pyrrolidine, CAS number: 54-11-5), has a chiral center and exists as chiral enantiomers, namely (S)-nicotine and (R)-nicotine. (S)-nicotine and its derivatives are effective drugs for treating Parkinson's syndrome, Alzheimer's disease, schizophrenia, epilepsy and depression. In agriculture, nicotine is used as an insecticide and herbicide, which is a highly effective and low-toxicity broad-spectrum pesticide. In addition, (S)-nicotine can be used as a raw material for health cigarettes, smoking cessation paste, and external medicine for treating joint pain and muscle spasms. Common administration methods include chewable tablets, creams, transdermal patches, tablets, nasal sprays, and electronic cigarettes. In recent years, the electronic cigarette industry has developed rapidly, and the market demand is very large. (S)-nicotine, as an important active ingredient of electronic cigarettes, has also seen a surge in market demand.

[0003] Currently, (S)-nicotine on the market is mainly extracted and purified from plants such as tobacco, which is affected by factors such as raw materials, climate, and cycle. In addition, (S)-nicotine extracted and purified from plants such as tobacco often contains many impurities that are unhealthy and potentially carcinogenic to the human system. In contrast, racemic nicotine, i.e., (R,S)-nicotine, obtained by artificial synthesis from chemical raw materials, does not contain the impurities present in naturally purified (S)-nicotine. It has the irreplaceable advantages of high purity, low cost, large yield, and wide application. However, the reagents used are expensive, the steps are complicated, and the cycle is long. Low-temperature reactions are often used, and each step of separation and purification is complex and difficult to handle. At the same time, the prepared (R,S)-nicotine also needs to be further separated and the non-target product (R)-nicotine needs to be destroyed to obtain high-purity (S)-nicotine, so the cost is very high, large-scale production is difficult, and the product price is high.

[0004] The chemical synthesis methods for preparing (S)-nicotine reported so far include chemical resolution, asymmetric hydrogenation, chiral auxiliary reagent method, etc. For example, Chinese patent CN107406411A discloses a synthesis method of (R,S)-nicotine, which is prepared by using nicotinic acid ester and N-vinyl-2-pyrrolidone as starting materials, and then through base-catalyzed Claisen condensation, acidic decarboxylation and ring closure to obtain intermediate mesamine (CAS No.: 532-12-7), and then mesamine is subjected to palladium-carbon hydrogenation reduction and Eschweiler-Clarke methylation to obtain racemic mixture (R,S)-nicotine. This process is the mainstream process for artificial synthesis of nicotine at present, but it has a major drawback that the prepared product (R,S)-nicotine is a mixture of (S)-nicotine and (R)-nicotine two chiral enantiomers, which is essentially different from natural (S)-nicotine, and has a large difference in physicochemical properties, drug efficacy and experience of electronic cigarette. (S)-nicotine can also be obtained by synthesis method, and the existing technology for synthesizing (S)-nicotine usually prepares racemic mixture of (R)-nicotine and (S)-nicotine and then separates to obtain. For example, patent CN111511726A reports the synthesis of racemic nicotine, and chiral separation is carried out by using L-DBTA to obtain (S)-nicotine; but this method needs to separate the mixture, and the process is complex, the reaction conditions are harsh, and the yield of the obtained product is low. Therefore, it is of high industrial application value and economic benefit to develop a process route for obtaining high-purity chiral (S)-nicotine.

[0005] Currently, there is also a method of using enzymes as biological catalysts to catalytically reduce mesmene in a biological enzyme system to obtain an intermediate (S)-nornicotine (CAS No.: 494-97-3) with high optical purity, and finally undergo aminomethylation reaction to obtain (S)-nicotine. For example, patent CN112409327A discloses a preparation method of high optical purity nicotine. The method first prepares mesmene from nicotinic acid ester and vinyl pyrrolidone, catalytically reduces mesmene in a biological enzyme system to obtain an intermediate (S)-nornicotine with high optical purity, and finally undergoes aminomethylation reaction to obtain (S)-nicotine. However, this method has the problems of using complex and multiple enzymes, and the enzymes cannot be reused after use. Patent CN113272289A discloses reducing mesmene to (S)-nornicotine using imine reductase, and methylating (S)-nornicotine to form (S)-nicotine. However, this method also has the problems of needing to prepare two enzymes and the enzymes cannot be reused. Patent WO2020098978 A1 discloses using imine reductase to reduce 3-(1-pyrrolin-2-yl)pyridine, using glucose dehydrogenase / glucose as a coenzyme regeneration system to catalyze the reaction of (S)-nicotine. When the concentration is 400 mM, the conversion rate is 99.6% in 24 h, and the ee value is 99.8%. The optimal imine reductase has a conversion rate of only 52.4% in 24 h and an ee value of 99.6% when the substrate concentration is 1 M. It is not resistant to high-concentration substrate 3-(1-pyrrolin-2-yl)pyridine, which is not conducive to scale-up production and has high production cost.

[0006] Therefore, it is of high industrial application value and economic benefit to develop a process route for obtaining high-concentration and high-purity chiral (S)-nornicotine at a low cost. SUMMARY

[0007] The technical problem to be solved by the present application is to provide an enzyme catalysis method for generating high-purity (S)-nornicotine from mesmene, which generates high-concentration (S)-nornicotine from mesmene through immobilized enzyme catalysis.

[0008] To solve the above technical problems, the present application provides an enzyme catalysis method for generating high-purity (S)-nornicotine from mesmene, which includes the following steps: (1) using mesmene as raw material, and generating racemic (R,S)-nornicotine through immobilized enzyme catalysis;

[0009] (2) using (R)-nornicotine in the racemic (R,S)-nornicotine generated in step (1) as raw material, and generating mesmene from (R)-nornicotine through immobilized enzyme catalysis; and continuously recycling the above steps to consume mesmene in the reaction system and obtain high-purity (S)-nornicotine.

[0010] Specifically, the immobilized enzyme is high (R) selective monoamine oxidase, imine reductase, glucose dehydrogenase, catalase and resin covalently bound.

[0011] Specifically, the resin is an epoxy resin, and the model of the epoxy resin is resin LXTE-600, LXTE-601, LXTE-602, LXTE-603, LXTE-604, LXTE-605, LXTE-606, LXTE-607, LXTE-608, LXTE-609 of Xi'an Lanxiao, and resin ES-1, ES-107, ES-108, ES-103B of Tianjin Nankai Heng.

[0012] Specifically, the imine reductase is selected from the following group:

[0013] (1) the amino acid sequence shown in SEQ ID NO. 1; and

[0014] (2) an amino acid sequence obtained by substitution, deletion, modification, insertion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO. 1 within the range of maintaining the enzyme activity.

[0015] Specifically, the high (R) selective monoamine oxidase is selected from the following group:

[0016] (3) the amino acid sequence shown in SEQ ID NO. 2; and

[0017] (4) an amino acid sequence obtained by substitution, deletion, modification, insertion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO. 2 within the range of maintaining the enzyme activity.

[0018] Specifically, the imine reductase is selected from the following group:

[0019] (a) the nucleotide sequence shown in SEQ ID NO. 3;

[0020] (b) a polynucleotide complementary to the sequence defined in (a); or

[0021] (c) any polynucleotide or complementary sequence having at least 70%, or 90% or more sequence identity to the sequence defined in (a).

[0022] Specifically, the high (R) selective monoamine oxidase is selected from the following group:

[0023] (d) the nucleotide sequence shown in SEQ ID NO. 4;

[0024] (e) a polynucleotide complementary to the sequence defined in (d); or

[0025] (f) any polynucleotide or complementary sequence having at least 70%, or 90% or more sequence identity to the sequences defined in (d).

[0026] Specifically, the preparation method of the immobilized enzyme is that 500-2000 ml of the enzyme solution of the high (R) selective monoamine oxidase, 200-500 ml of the enzyme solution of the imine reductase, 100-300 ml of the enzyme solution of the glucose dehydrogenase, and 100-300 ml of the enzyme solution of the catalase are added with 140-600 g of K2HPO4·3H2O and 14-60 g of KH2PO4, stirred and dissolved, then 50-300 g of epoxy resin is added, stirred at 25℃ and 180 rpm for 24 h, filtered, washed with deionized water for three times, then washed with 500 ml of PBS buffer (200 mM, pH 8.0, containing 0.5 M NaCl) for two times, and filtered to obtain the immobilized enzyme.

[0027] Specifically, the enzyme activity of the immobilized enzyme is 200-400 U / g.

[0028] Specifically, the mass molar ratio (M / mol) of the immobilized enzyme to the mesmene is (20-200): 1.

[0029] Specifically, the reaction temperature of the reaction system is 15-40℃.

[0030] Specifically, the reaction time of the reaction system is 16-48 hours.

[0031] Specifically, the reaction system further comprises maintaining pH 7.0-7.2 by using PBS buffer (200 mM, pH 7.0) and alkali.

[0032] The present application provides a process route for obtaining high-concentration, high-purity chiral (S)-nornicotine with low cost by catalyzing the conversion of mesmene to high-concentration (S)-nornicotine through immobilized enzyme, which has high industrial application value and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the present application, the following briefly introduces the drawings needed to be used in the present application. Obviously, the drawings described in the following only constitute some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0034] Figure 1 is the route map of enzyme catalysis of mesmene to produce high-purity (S)-nornicotine;

[0035] Figure 2SDS-PAGE map of imine reductase E1, high (R) selective monoamine oxidase E2, catalase E3, glucose dehydrogenase E4. DETAILED DESCRIPTION

[0036] The technical solutions in the present application will be described clearly and completely below in combination with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0037] Example 1.

[0038] In the present application, the imine reductase E1 is derived from Ureibacillus thermosphaericus, the amino acid sequence is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 3; the high (R) selective monoamine oxidase E2 is derived from Erythrobacteraceae bacterium, the amino acid sequence is shown as SEQ ID NO. 2, and the nucleotide sequence is shown as SEQ ID NO. 4; the catalase E3 is derived from Bacillus pumilus (Gene ID: WP_016837596.1); and the glucose dehydrogenase E4 is derived from Exiguobacterium sibiricum (Gene ID: ACB59697.1). The genes of the above four enzymes are all obtained by gene synthesis, and each gene is connected to the vector pET28a. The strain is subjected to plate culture, and finally single colonies are picked for step-by-step liquid culture. First, 3 ml of LB culture solution containing 50 μM kanamycin is inoculated (37°C, 220 rpm) overnight, the inoculation amount is 1%, and then 100 ml of LB culture solution containing 50 μM kanamycin is inoculated, and cultured for 4-8 h. Finally, it is transferred into a 10 L fermenter for culture; when the cell OD reaches about 25, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added, and then the protein expression is induced at 30°C for 18 h. Finally, the cells are collected by high-speed centrifugation (6000 rpm, 30 min), and 600-1000 g of wet bacteria are obtained, which are stored at -20°C for use. 10 g of wet bacteria is suspended in 100 ml of PBS buffer (50 mM, pH 8.0) and mixed uniformly, the cells are broken by high-pressure homogenization, centrifuged at 12000 rpm for 20 min, and the supernatant is filtered through a 0.22 μm membrane to obtain the above four enzyme solutions.

[0039] Take 40ul of each of the filtered E1, E2, E3 and E4 enzyme solution, add 10ul of 5* loading dye, treat at 100 degrees Celsius for 10 minutes, centrifuge at 12000 rpm for 30 seconds, take 10ul for SDS-PAGE to verify the expression of the protein and the size, the results are shown in Figure 2 E1 is about 30kDa, E2 is about 49kDa, E3 and E4 are about 57kDa and 29kDa respectively, which is consistent with the expectation.

[0040] Example 2.

[0041] Mix the high (R) selective monoamine oxidase enzyme solution (1000ml, enzyme activity 69.4U / mL) obtained in Example 1, imine reductase enzyme solution (300ml, enzyme activity 73.4U / mL), glucose dehydrogenase enzyme solution (200ml, enzyme activity 81.4U / mL), and catalase enzyme solution (150ml, enzyme activity 82.7U / mL), then add 250g of K2HPO4·3H2O and 25g of KH2PO4, stir to dissolve, add 100g of epoxy resin carrier, stir at 25 degrees Celsius and 180rpm for 24 hours, suction filter, wash with deionized water three times, then wash with 500ml of PBS buffer (200mM, pH 8.0, containing 0.5M NaCl) twice, suction filter, and obtain the immobilized complex enzyme. Select different types of resins to immobilize and screen for the resin with the highest immobilized enzyme activity.

[0042] The immobilized enzyme activity detection method is as follows:

[0043] Use mesmene as the substrate to detect the activity of the immobilized enzyme. The enzyme activity detection method is as follows: 2.5mmol (0.37g) of mesmene and 3mmol (0.54g) of glucose are dissolved in PBS buffer (200mM, pH 7.0) to make up to 50mL, 1.0g of immobilized enzyme is added to the reaction system, oxygen is passed, and the reaction is carried out at 30 degrees Celsius and 200rpm for 20 minutes. After standing for 30 seconds, 2ml of supernatant is taken and centrifuged at 12000rpm for 3 minutes. HPLC detection is performed, and the concentration of the product (S)-norsinemine in the reaction system is calculated according to the standard curve of the standard (S)-norsinemine concentration, and the activity of the immobilized enzyme is calculated.

[0044] The immobilized enzyme activity of different types of resins is detected, and the detection results are shown in Table 1 below:

[0045] Table 1. Immobilized enzyme activity of different types of resins

[0046] Resin model Immobilized enzyme activity (U / g) LXTE-600 179 LXTE-601 312 LXTE-602 336 LXTE-603 328 LXTE-604 349 LXTE-605 315 LXTE-606 324 LXTE-607 281 LXTE-608 264 LXTE-609 312 ES-1 168 ES-103B 289 ES-107 327 ES-108 358

[0047] Note: Resins LXTE-600, LXTE-601, LXTE-602, LXTE-603, LXTE-604, LXTE-605, LXTE-606, LXTE-607, LXTE-608, LXTE-609 are from Xi'an Lanxiao Science and Technology New Material Co., Ltd.; Resins ES-1, ES-107, ES-108, ES-103B are from Tianjin Nankai Hecheng Technology Co., Ltd.

[0048] Example 3.

[0049] In this example, the immobilized enzyme (amine oxidase, imine reductase, glucose dehydrogenase and catalase) with higher enzyme activity prepared in Example 2 (ES-108 resin for preparing immobilized enzyme) was used to catalyze the generation of (S)-nor nicotine from myosmine.

[0050] In 500 ml of PBS buffer (200 mM, pH 7.0), 0.25 mol (36.5 g) of myosmine and 0.3 mol (54 g) of glucose were added and stirred uniformly, 0.01 mol (6.8 g) of sodium salt of nicotinamide adenine dinucleotide was added and stirred uniformly, and then 30 g of immobilized enzyme was added. The reaction temperature was 30°C, the stirring speed was 200 rpm, and the pH was adjusted to about 7.0 using 2M NaOH during the reaction. After 30 hours of reaction, the conversion rate and the ee value of the product were detected by HPLC, 50 ml of PBS buffer (100 mM, pH 7.0) was added to wash the immobilized enzyme, and the immobilized enzyme was collected by suction filtration. The washing liquid was collected, and the same operation was repeated twice. The collected washing liquid and the previously collected reaction liquid were mixed for subsequent treatment, and the recovered immobilized enzyme was used for the next batch of reaction. The same operation was repeated for 20 batches of immobilized enzyme.

[0051] The results are shown in Table 2 below. The conversion rate of 20 batches of repeated experiments of immobilized enzyme under the condition of 0.5 mol / L substrate concentration was greater than 98.2%, and the optical purity (ee value) of (S)-nor nicotine was greater than 99.6%. The immobilized enzyme can be reused after the reaction is completed.

[0052] Table 2. Conversion rate of 20 batches of repeated experiments of immobilized enzyme under the condition of 0.5 mol / L substrate concentration

[0053] Batch Reaction time / h Conversion rate / % ee value / % 1 30 99.4 99.8 2 30 99.2 99.8 3 30 99.1 99.8 4 30 99.1 99.8 5 30 98.8 99.8 6 30 98.9 99.8 7 30 99.2 99.7 8 30 98.7 99.7 9 30 98.8 99.8 10 30 98.6 99.7 11 30 98.6 99.7 12 30 98.5 99.8 13 30 98.7 99.7 14 30 99.1 99.7 15 30 98.6 99.8 16 30 98.1 99.7 17 30 98.3 99.7 18 30 98.3 99.7 19 30 98.4 99.6 20 30 98.2 99.6

[0054] Example 4.

[0055] In this example, the immobilized enzyme (amine oxidase, imine reductase, glucose dehydrogenase and catalase) with higher enzyme activity prepared in Example 2 (ES-108 resin for preparing immobilized enzyme) was used to catalyze the generation of (S)-nor nicotine from myosmine.

[0056] In 500 ml PBS buffer (200 mM, pH 7.0) was added 0.5 mol (73 g) of myosmine and 0.6 (108 g) mol of glucose, stirred uniformly, 0.02 mol (13 g) of sodium salt of nicotinamide adenine dinucleotide, stirred uniformly, and then 30 g of immobilized enzyme was added. The reaction temperature was 30°C, the stirring speed was 200 rpm, and the pH was adjusted to about 7.0 using 2M NaOH during the reaction. After 30 h of reaction, the conversion rate and ee value of the product were detected by HPLC. The reaction solution was collected by suction filtration, 50 ml of PBS buffer (100 mM, pH 7.0) was added, the immobilized enzyme was washed by stirring, and the washing solution was collected by suction filtration. The same operation was repeated twice, and the collected washing solution and the previously collected reaction solution were mixed for subsequent post-treatment. The recovered immobilized enzyme was used for the next batch of reaction. The same operation was repeated for 20 batches of immobilized enzyme.

[0057] The results are shown in Table 3 below. The conversion rate of 20 batches of repeated experiments of the immobilized enzyme under the condition of 1 mol / L substrate concentration was greater than 87.9%, and the optical purity (ee) of (S)-nornicotine was greater than 99.2%.

[0058] Table 3. Conversion rate of 20 batches of repeated experiments of the immobilized enzyme under the condition of 1 mol / L substrate concentration

[0059] Batch Reaction time / h Conversion rate / % ee value / % 1 30 98.2 99.6 20 30 87.9 99.2

[0060] Example 5.

[0061] In this example, the immobilized enzyme (amine oxidase, imine reductase, glucose dehydrogenase, and hydrogen peroxidase, ES-108 resin) with higher enzyme activity prepared in Example 2 was used to catalyze the generation of (S)-nornicotine from myosmine.

[0062] In 500 ml PBS buffer (200 mM, pH 7.0) was added 0.5 mol (73 g) of myosmine and 0.6 (108 g) mol of glucose, stirred uniformly, 0.02 mol (13 g) of sodium salt of nicotinamide adenine dinucleotide, stirred uniformly, and then 30 g of immobilized enzyme was added. The reaction temperature was 30°C, the stirring speed was 200 rpm, and the pH was adjusted to about 7.0 using 2M NaOH during the reaction. After 30 h of reaction, the conversion rate and ee value of the product were detected by HPLC. The reaction solution was collected by suction filtration, 50 ml of PBS buffer (100 mM, pH 7.0) was added, the immobilized enzyme was washed by stirring, and the washing solution was collected by suction filtration. The same operation was repeated twice, and the collected washing solution and the previously collected reaction solution were mixed for subsequent post-treatment. The recovered immobilized enzyme was used for the next batch of reaction. The same operation was repeated for 20 batches of immobilized enzyme.

[0063] The results are shown in Table 4. The conversion rate of the 20 batches of repeated experiments of the immobilized enzyme under the condition of 2 mol / L substrate concentration is greater than 63.3%, and the optical purity (ee) of (S)-nor nicotine is greater than 98.5%.

[0064] Table 4. Conversion rate of the 20 batches of repeated experiments of the immobilized enzyme under the condition of 2 mol / L substrate concentration

[0065] Batch Reaction time / h Conversion rate / % ee value / % 1 30 88.7 99.4 20 30 63.3 98.5

[0066] The above description is merely preferred embodiments of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An enzyme-catalyzed process for the production of high purity (S)-nornicotine from myosmine, characterized by, It comprises the following steps: (1) taking mesmene as raw material, using immobilized enzyme to catalyze the generation of racemic (R, S)-norsosaline; (2) taking (R)-norsosaline in the racemic (R, S)-norsosaline generated in step (1) as raw material, using immobilized enzyme to catalyze (R)-norsosaline to generate mesmene; Through the continuous circulation of the above steps, the mesmene in the reaction system is consumed to obtain high-purity (S)-norsosaline; The immobilized enzyme is high (R) selective monoamine oxidase, imine reductase, glucose dehydrogenase, catalase and resin covalently combined; The amino acid sequence of the imine reductase is shown in SEQ ID NO.

1. The amino acid sequence of the high (R) selective monoamine oxidase is shown in SEQ ID NO.

2.

2. The enzyme-catalyzed process according to claim 1, characterized in that, The resin is an epoxy resin, and the model of the epoxy resin is any one of resin LXTE-600, LXTE-601, LXTE-602, LXTE-603, LXTE-604, LXTE-605, LXTE-606, LXTE-607, LXTE-608, LXTE-609 of Xi'an Lanxiao and resin ES-1, ES-107, ES-108, ES-103B of Tianjin Nankai Hengcheng.

3. The enzyme-catalyzed process according to claim 1, characterized in that, The nucleotide sequence of the imine reductase is shown in SEQ ID NO.

3.

4. The enzyme-catalyzed process of claim 1, wherein, The nucleotide sequence of the high (R) selective monoamine oxidase is shown in SEQ ID NO.

4.

5. The enzyme-catalyzed process according to claim 1 or 3 or 4, characterized in that, The preparation method of the immobilized enzyme is that 500-2000 ml of enzyme solution of the high (R) selective monoamine oxidase, 200-500 ml of enzyme solution of the imine reductase, 100-300 ml of enzyme solution of the glucose dehydrogenase, 100-300 ml of enzyme solution of the catalase, 140-600 g of K2HPO4·3H2O and 14-60 g of KH2PO4 are added, stirred and dissolved, then 50-300 g of the epoxy resin is added, stirred at 25°C and 180 rpm for 24 h, filtered, washed with deionized water for three times, then washed with 500 ml of PBS buffer solution with a concentration of 200 mM, pH 8.0 and containing 0.5 M NaCl for two times, and the immobilized enzyme is obtained by filtering.

6. The enzyme-catalyzed process according to claim 5, characterized in that, The enzyme activity of the enzyme solution of the immobilized enzyme, the high (R) selective monoamine oxidase, the imine reductase, the glucose dehydrogenase and the catalase is 50-100 U / mL.

7. The enzyme-catalyzed process of claim 5, wherein, The enzyme activity of the immobilized enzyme is 200-400 U / g.

8. The enzyme-catalyzed process of claim 1, wherein, The mass molar ratio (M / mol) of the immobilized enzyme to the mesmene is (20-200):

1.

9. The enzyme-catalyzed process of claim 1, wherein, The reaction temperature of the reaction system is 15-40°C.

10. The enzyme-catalyzed process of claim 1, wherein, The reaction time of the reaction system is 16-48 hours.

11. The enzyme-catalyzed process of claim 1, wherein, The reaction system further comprises using PBS buffer solution with a concentration of 200 mM and pH 7.0 to maintain the reaction pH at 6.8-7.2.

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