Method for synthesizing acetyl glutathione by biological enzyme method
By using acyltransferase to catalyze the reaction of glutathione and vinyl acetate, and using the immobilized enzyme to perform continuous flow reaction in polyvinyl alcohol gel, the problems of harsh reaction conditions and low product purity in traditional chemical synthesis methods are solved. Efficient, environmentally friendly production of acetyl glutathione.
Patent Information
- Application Number
- CN202411817269.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-01-24
AI Technical Summary
When traditional chemical synthesis methods are used to produce acetyl glutathione, harsh reaction conditions are required, resulting in low purity of the product, unstable yield, and high environmental pollution and production costs.
An acyltransferase is used to catalyze the reaction between glutathione and vinyl acetate, and the immobilized enzyme is used to perform a continuous flow reaction in a polyvinyl alcohol gel to generate acetyl glutathione.
A high conversion rate and high selectivity have been achieved. The product obtain rate has reached more than 95%, fewer by -products, mild response conditions, suitable for industrial production, reducing the negative impact on the environment.
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, in particular to a method for synthesizing acetyl glutathione by using an enzyme catalysis method. Background Art
[0002] Glutathione is a tripeptide composed of glutamic acid, cysteine and glycine. It plays an important antioxidant role in the body, protecting cells from damage by free radicals and maintaining the normal function of the immune system. Acetyl glutathione, a derivative of glutathione, is formed by introducing an acetyl group into the glutathione molecule. Acetyl glutathione not only retains the antioxidant properties of glutathione, but also has better stability and bioavailability. Therefore, it has broad application prospects in the fields of medicine, food additives and cosmetics. For example, acetyl glutathione is often used in anti-aging products, skin care products, and health foods to help enhance immunity, detoxify and whiten the skin.
[0003] However, traditional chemical synthesis methods for producing acetyl glutathione usually involve harsh reaction conditions, such as high temperature, high pressure or the use of toxic chemical reagents. This method not only requires complex post-processing to remove by-products, but may also result in low product purity, unstable yield, environmental pollution, and increased production costs. Therefore, a safe and reliable method for producing acetyl glutathione is urgently needed. Summary of the invention
[0004] The present invention aims to overcome the above defects and provides a method for generating acetyl glutathione by using acyltransferase to catalyze the reaction of glutathione and vinyl acetate.
[0005] The method uses acyltransferase from Pseudomonas sp. as a catalyst, immobilizes it using polyvinyl alcohol gel, and then performs efficient catalysis in a continuous flow reactor to ultimately obtain the product acetyl glutathione.
[0006] The specific technical solutions of the present invention are as follows:
[0007] The invention provides a method for synthesizing acetyl glutathione by bioenzymatic method, which is characterized in that glutathione and vinyl acetate are synthesized into acetyl glutathione under the catalytic action of bioenzyme; wherein the bioenzyme is acyltransferase derived from Pseudomonas.
[0008] Furthermore, the present invention provides a method for synthesizing acetyl glutathione by bioenzymatic method, characterized in that the amino acid sequence of the bioenzyme is as shown in SEQ ID No.1.
[0009] Acyltransferase amino acid sequence SEQ ID No.1: MDRARLHVEHASEVIGMDERPSQALRGKPDASMRVALELVRCGRAKACVSAGNTGALMALSRYVLKTLPGIDRPAMVSPVPTATGSCQLLDLGANVDCSAEHLYQFAVMGSVAAEALGVVRPRVALLNVGTEEVKGNQQ VKLAASLLQQARGLNYTGFIEGDGLYRGEADVVVCDGFVGNILLKSSEGLASMISARIEALFRENIATRLVGGMALPLLRRLQTELTPAQHNGASFLGLQGIVVKSHGAAGVEGFQSAIRRALLEVRENLPQRLHGRLEDLLL.
[0010] Furthermore, the present invention provides a method for synthesizing acetyl glutathione by bioenzymatic method, characterized in that the bioenzyme is immobilized.
[0011] Furthermore, the present invention provides a method for synthesizing acetyl glutathione by bioenzymatic method, characterized in that: the bioenzyme is immobilized by using a gel matrix; wherein the gel matrix is selected from at least one of polyvinyl alcohol, sodium alginate, gelatin, polyurethane, polyacrylamide, and carbomer.
[0012] The mass ratio of enzyme to gel matrix is 15-5:1, which is determined according to the properties of different matrices.
[0013] In the above-mentioned bioenzyme immobilization method, the enzyme is embedded in a polyvinyl alcohol gel network. The pores of the gel allow small molecule substrates and products to pass through, but the enzyme molecules are confined within the gel. This method can not only protect the enzyme from the external environment, but also maintain the activity of the enzyme over a larger range.
[0014] Furthermore, the present invention provides a method for synthesizing acetyl glutathione by bioenzymatic method, characterized in that: the reaction is carried out in a continuous flow reactor.
[0015] Specifically, in the present method, the immobilized acyltransferase is filled into the reactor to construct a packed bed reactor, the substrate glutathione and vinyl acetate are mixed in a certain proportion, and the mixed solution is pumped into the packed bed by a peristaltic pump. After the catalytic reaction, the solution flowing out from the other side is acetyl glutathione.
[0016] Furthermore, the present invention provides a method for synthesizing acetyl glutathione by bioenzymatic method, characterized in that the reaction temperature is controlled within the range of 25°C to 40°C.
[0017] Furthermore, the present invention provides a method for synthesizing acetyl glutathione by bioenzymatic method, characterized in that the pH of the reaction system is controlled within the range of 6.5 to 8.0. Specifically, the pH of the system is controlled by using a buffer solution commonly used in the art, the most commonly used being 100 mM KPB (phosphate buffer), which can adjust the ratio so that the pH is 6.5, 7.0, 7.5 and 8.0 respectively.
[0018] Furthermore, the present invention provides a method for synthesizing acetyl glutathione by bioenzymatic method, characterized in that the molar ratio of vinyl acetate to glutathione is 1:1.0-2.0.
[0019] Furthermore, the present invention provides a method for synthesizing acetyl glutathione by a bioenzymatic method, characterized in that: liquid chromatography is used to monitor the product / reaction process; the specific monitoring method is: after high-speed centrifugation of the test liquid, the supernatant is taken, and liquid chromatography analysis is performed after filtering with a filter membrane; the chromatographic conditions are: mobile phase A is 50mM KH2PO4, mobile phase B is acetonitrile, the ratio of mobile phase A to B is 90-80:10-20, the flow rate is 1-2mL / min, the column temperature is 40-60°C, and the detection wavelength is 210nm.
[0020] Functions and effects of the present invention:
[0021] Compared with the existing technology, the bioenzymatic synthesis of acetyl glutathione has the advantages of mild reaction conditions, high product yield and environmental friendliness. Therefore, it is of great significance to develop a method for synthesizing acetyl glutathione using the bioenzymatic method, which can not only improve industrial production efficiency, but also reduce the negative impact on the environment, and has important industrial application value and social benefits. This new synthesis method provides a safer, more economical and environmentally friendly solution for the large-scale production of acetyl glutathione in the future.
[0022] Specifically:
[0023] (1) The method of the present invention has the characteristics of high conversion rate and high selectivity: by using immobilized enzyme to carry out continuous flow reaction and optimizing reaction conditions, the product yield of acetyl glutathione reaches more than 95% with less by-products.
[0024] (2) The method of the present invention is characterized by simple operation and reusability: the immobilized enzyme can be used for a long time in a continuous flow reactor, which simplifies the enzyme recovery step and reduces the production cost.
[0025] (3) The method of the present invention has the characteristic of mild reaction conditions: the enzymatic synthesis is carried out at room temperature, avoiding the high temperature and high pressure chemical synthesis environment, which is conducive to maintaining the structural integrity of glutathione.
[0026] (4) The method of the present invention has the characteristics of great industrial potential: the method is suitable for large-scale production, and the continuous flow reactor can realize industrial continuous production, thereby improving production efficiency. DETAILED DESCRIPTION
[0027] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.
[0028] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods in the art. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained through conventional commercial channels.
[0029] Example 1. Preparation of acyltransferase
[0030] The specific steps are as follows:
[0031] (1) Gene synthesis and cloning: The acyltransferase sequence from Pseudomonas sp. was fully synthesized, and plasmid pET-28a(+) was selected as the expression vector. After obtaining the recombinant plasmid, it was transformed into Escherichia coli BL21(DE3) competent cells to obtain a recombinant strain.
[0032] (2) Induction and expression of target protein: After successful transformation, a single colony was picked from the plate and inoculated into 4 mL LB liquid medium containing 50 μg / mL Kan, and cultured in a shaker at 37°C and 200 rpm for 12 h. Then, 1 mL of bacterial solution was inoculated into a shake flask containing 100 mL LB liquid medium (containing 50 μg / mL Kan) at a 1% inoculation rate, and cultured in a shaker at 37°C and 200 rpm for 3 h until OD 600 When the pH reached 0.6-0.8, IPTG was added to a final concentration of 0.2 mM for induction, and the shake flask was transferred to a shaker at 16°C and 200 rpm for 20 h. After 20 h of induction, the fermentation broth was collected and centrifuged at 10,000 rpm for 5 min in a floor centrifuge at 4°C to collect the bacteria.
[0033] (3) Cell disruption and enzyme extraction: The cells were disrupted by ultrasonic disruption, and the crude enzyme extract was obtained by centrifugation. The crude enzyme activity was found to be above 1000 U / mg.
[0034] Example 2. Preparation of immobilized acyltransferase
[0035] The bioenzyme is immobilized by using a gel matrix; wherein the gel matrix is selected from at least one of polyvinyl alcohol, sodium alginate, gelatin, polyurethane, polyacrylamide, and carbomer, and the amount thereof is 5-15:1 by mass ratio of enzyme to gel matrix. Considering the cost and ease of use, polyvinyl alcohol is finally used as the gel matrix in the optimal embodiment. The specific test method is as follows:
[0036] (1) Preparation of polyvinyl alcohol solution: Weigh the required polyvinyl alcohol according to a 20% aqueous solution, add it to deionized water, and then heat it to 80° C. to completely dissolve it, and then cool it to room temperature for use.
[0037] (2) Preparation of crude enzyme solution: According to the description in Example 1, the crude enzyme solution of acyltransferase required for the experiment was prepared for use.
[0038] (3) Preparation of immobilized acyltransferase: Slowly add the crude enzyme solution to the cooled polyvinyl alcohol solution (immobilization can be achieved in the range of 5-15:1, and the most preferred mass ratio of enzyme to polyvinyl alcohol is 10:1), and gently stir to ensure uniform dispersion. Then add 5% glutaraldehyde to the solution to promote the cross-linking reaction between PVA and enzyme, and gently stir to mix to ensure uniform distribution of the cross-linking agent. Then refrigerate the mixed solution in a refrigerator overnight to allow it to gel. Finally, remove the gel block from the reaction vessel, place it in deionized water, gently wash it to remove uncross-linked PVA and free cross-linking agent, and activate the immobilized enzyme in buffer. The activated immobilized enzyme is placed in a 4°C refrigerator for use.
[0039] The enzyme activity retention rate was used as the evaluation criterion, which was defined as: immobilized enzyme activity / free enzyme activity*100%.
[0040] Example 2.1. Comparison of gels with different ratios
[0041] The polyvinyl alcohol (PVA) concentrations were set at 5%, 7.5%, 10%, 12.5% and 15%.
[0042] At the above concentrations, the enzyme activity retention rates were 28%, 53%, 73%, 62% and 39%, respectively. Therefore, the final PVA concentration was selected as 10%.
[0043] Example 2.2. Comparison of different gel matrices
[0044] Polyvinyl alcohol, sodium alginate and gelatin were selected, and their concentrations were all 10%. The three results were 73%, 56% and 38% respectively based on the enzyme activity retention rate. Therefore, polyvinyl alcohol was finally selected as the immobilization carrier.
[0045] Example 3: Preparation of acetyl glutathione by continuous flow reaction
[0046] The immobilized enzyme in Example 2 was loaded into a reactor to obtain a packed bed reactor. 10 mM glutathione and 20 mM vinyl acetate were configured into a mixed solution, and the mixed solution was pumped into the reactor using a peristaltic pump. After catalysis by the immobilized enzyme, acetyl glutathione was obtained. After testing, the yield of acetyl glutathione was 83%. In order to further improve the product titer, the reaction pH, reaction temperature and reaction time were optimized.
[0047] Example 4, Optimization of reaction temperature
[0048] The reaction was carried out at 25°C, 30°C, 35°C and 40°C, and the conversion rate under each temperature condition was monitored. The product yields at the four temperatures were 78%, 89%, 94% and 66%, respectively. The results showed that at 30°C, the conversion rate was the highest, reaching 94%, and 30°C was selected as the reaction temperature for further study.
[0049] Example 5. Optimization of reaction pH
[0050] Before entering the reactor, glutathione and vinyl acetate are prepared into a solution in a certain proportion using buffered salt solutions of different pH values, and then enter the reactor. Specifically, 100mM KPB buffer (phosphate buffer) can be prepared under four pH conditions of pH 6.5, 7.0, 7.5 and 8.0, and the product yields are 55%, 76%, 92% and 88% respectively. The results show that the reaction is optimal at pH 7.5, with a product yield of 92%, and the subsequent reaction pH is selected to be 7.5.
[0051] Example 6, Optimization of reaction time
[0052] The reaction time was gradually extended from 30 min to 120 min, and the product yield at different time points was monitored. The results showed that at 90 min, the product yield reached a maximum of 96%, and the subsequent product concentration was stable. Therefore, the subsequent reaction time was set to 90 min.
[0053] Example 7: Optimization of substrate loading
[0054] The concentration gradient was set to 10mM, 20mM, 50mM and 100mM. The product yields at different substrate loadings were determined to be 99%, 98%, 96% and 96%, respectively. We found that as the substrate loading reached 100mM, the product yield remained above 96%, so we finally chose a substrate loading of 100mM.
[0055] It can be found that in the method of this embodiment, the substrate concentration was increased by 10 times, and the conversion rate hardly decreased, which has great prospects for industrial production.
[0056] Example 8: Experiment on repeated use of immobilized enzyme
[0057] Under the optimal reaction conditions (glutathione concentration of 100mM, vinyl acetate concentration of 200mM, pH 7.5, 30°C), multiple reaction cycles were carried out. After each reaction, the immobilized enzyme did not need to be re-fixed and was only rinsed with buffer. After 20 reaction cycles, the product yield of acetyl glutathione remained above 90%. Through the repeated use of the immobilized enzyme, the present invention has the characteristics of high efficiency, economy, convenience and environmental friendliness, and provides a reliable solution for industrial production.
[0058] It can be found that the present embodiment provides a novel method for synthesizing acetyl glutathione by enzymatic method, which combines the advantages of immobilized enzyme and continuous flow reactor, and realizes efficient and sustainable production of acetyl glutathione. In the present invention, the enzyme is fixed on a carrier by polyvinyl alcohol gel method to enhance its stability and reusability. A continuous flow reactor is used to carry out catalytic reaction, so that the substrate is continuously contacted with the immobilized enzyme in the reactor, thereby improving the reaction rate and substrate conversion rate.
[0059] In order to further improve the product yield, the present invention systematically studies the reaction temperature, reaction pH and substrate loading. This optimization process significantly improves the conversion rate of acetyl glutathione, and the product yield reaches more than 95% at a substrate loading of 100mM. In addition, the immobilized enzyme was repeatedly used. After 20 reaction cycles, the product yield of acetyl glutathione is still greater than 90%, showing that the present invention has the characteristics of high efficiency, economy, convenience and environmental friendliness.
[0060] In summary, the enzymatic method for synthesizing acetyl glutathione of the present invention has the significant advantages of being green, efficient, and sustainable, has broad prospects for industrial application, and provides a reliable and economical solution for the green production of acetyl glutathione.
Claims
1. A method for synthesizing acetyl glutathione by bioenzymatic method, characterized in that: Glutathione and vinyl acetate are synthesized into acetyl glutathione under the catalysis of biological enzymes; Wherein, the biological enzyme is an acyl transferase derived from Pseudomonas.
2. The method for synthesizing acetyl glutathione by bioenzymatic method as claimed in claim 1, characterized in that: The amino acid sequence of the biological enzyme is shown in SEQ ID No.
1.
3. The method for synthesizing acetyl glutathione by bioenzymatic method as claimed in claim 1, characterized in that: Immobilization of biological enzymes.
4. The method for synthesizing acetyl glutathione by bioenzymatic method as claimed in claim 1, characterized in that: Immobilize biological enzymes using gel matrix; Wherein, the gel matrix is selected from at least one of polyvinyl alcohol, sodium alginate, gelatin, polyurethane, polyacrylamide and carbomer.
5. The method for synthesizing acetyl glutathione by bioenzymatic method as claimed in claim 4, characterized in that: The mass ratio of the biological enzyme to the gel matrix is 5-15:
1.
6. The method for synthesizing acetyl glutathione by bioenzymatic method as claimed in claim 1, characterized in that: The reaction is carried out in a continuous flow reactor.
7. The method for synthesizing acetyl glutathione by bioenzymatic method as claimed in claim 1, characterized in that: The reaction temperature is controlled in the range of 25°C to 40°C.
8. The method for synthesizing acetyl glutathione by bioenzymatic method as claimed in claim 1, characterized in that: The pH of the reaction system was controlled in the range of 6.5 to 8.
0.
9. The method for synthesizing acetyl glutathione by bioenzymatic method as claimed in claim 1, characterized in that: The molar ratio of vinyl acetate to glutathione is 1:1.0-2.
0.
10. The method for synthesizing acetyl glutathione by bioenzymatic method as claimed in claim 1, characterized in that: Liquid chromatography was used to monitor the product / reaction process; The specific monitoring method is: take the liquid to be tested and centrifuge it at high speed, take the supernatant, filter it with a filter membrane, and then perform liquid chromatography analysis; The chromatographic conditions were as follows: mobile phase A was 50 mM KH2PO4, mobile phase B was acetonitrile, the ratio of mobile phase A to B was 90-80:10-20, the flow rate was 1-2 mL / min, the column temperature was 40-60°C, and the detection wavelength was 210 nm.