CN106086126B - Method for synthesizing glutathione by enzyme catalysis - Google Patents
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Abstract
The invention discloses a method for synthesizing glutathione by enzyme catalysis, which comprises the following steps: s1, mixing the gamma-glutamylcysteine synthetase liquid and the glutathione synthetase liquid to obtain a mixed liquid A, and then mixing the mixed liquid A and the acetokinase liquid to obtain a mixed liquid B; s2, adding an immobilized carrier into the mixed solution B, stirring and immobilizing, and filtering to obtain an immobilized enzyme; s3, preparing glutamic acid, L-cysteine, glycine, magnesium sulfate, ATP and acetyl phosphate into a reaction solution, adding immobilized enzyme, and stirring for reaction; s4, after the reaction is finished, filtering the reaction solution, and extracting and refining the filtrate into glutathione. The method has high synthesis efficiency: the product content of the reaction solution reaches more than 10g/L, and the substrate conversion rate reaches more than 90 percent; an ATP regeneration coupling system is constructed by introducing acetate kinase, so that the dosage of ATP is greatly reduced, and the production cost is obviously reduced; the immobilized enzyme can be repeatedly used and has good stability.
Description
Technical Field
the invention relates to a method for preparing glutathione, in particular to a method for synthesizing glutathione by enzyme catalysis.
Background
glutathione (GSH) is a biologically active tripeptide compound containing gamma-2 glutamyl and sulfhydryl groups, which is obtained by condensing L-glutamic acid, L-cysteine and glycine, and plays a direct or indirect role in important biological phenomena such as protein and DNA synthesis, amino acid transport and cell protection. It is mainly distributed in animal, plant and microbial cell, is one of the most important non-protein sulfhydryl compounds in organism, is widely used for treating liver diseases, tumor, oxygen poisoning, aging and endocrine diseases, and has become one of the medically important drugs with the functions of regulating human body immunity and assisting anti-cancer. The oxidation resistance of GSH causes the application of GSH in the food industry to be concerned by people, and the GSH plays an increasingly better role in the aspects of food storage and preservation, product flavor improvement and product nutritive value improvement.
At present, the production methods of glutathione mainly include extraction methods, chemical synthesis methods, fermentation methods and enzymatic methods. GSH is extracted from cereal germs, and is rarely used due to low yield, high cost, serious pollution of organic solvents, low purity and consumption of a large amount of grains. The chemical synthesis method has the problems of high cost, multiple reaction steps, long reaction time, complex operation, difficult separation of active products, need of chemical resolution, low product purity, environmental pollution and the like, and is difficult to popularize. At present, GSH production at home and abroad basically adopts a fermentation method, and the principle is to clone genes of a GSH synthetase system into escherichia coli or yeast and produce the GSH by microbial fermentation. The yeast fermentation method has a mature process, but has long production period and low yield, and the downstream process treatment is complicated due to excessive byproducts.
Compared with the previous methods, the enzyme method has the advantages of strong catalytic specificity, mild reaction conditions, high conversion efficiency and the like. The enzymatic production of glutathione is carried out by catalyzing with key enzyme gamma-glutamylcysteine synthetase and glutathione synthetase for synthesizing glutathione, adding L-glutamic acid, L-cysteine, glycine and other substrates, and adding small amount of adenosine triphosphate to synthesize glutathione.
At present, the problems faced by the microbial enzyme method for producing glutathione mainly include mass transfer obstruction caused by cell walls or biological membranes and a continuous and effective ATP energy supply system. The synthesis of GSH is accompanied with the consumption of ATP (2 MATP is consumed for synthesizing 1M GSH), the cost of GSH synthesized by an enzyme method is obviously increased by directly adding ATP due to the high price of ATP, and the activity of GSH synthetase is also strongly inhibited by the existence of high-concentration ATP and a metabolite ADP thereof, so the GSH produced by the enzyme method at present adopts ATP regeneration, mainly adopts a yeast cell regeneration mode. However, the use of yeast introduces impurities such as pigments into the reaction system, which makes further purification difficult, and the regeneration of ATP by using an enzyme system has been the subject of recent research.
Immobilized enzymes are a technique for immobilizing enzymes on a carrier. Generally, the immobilized enzyme itself is soluble in water, and the enzyme is physically or chemically bound to or entrapped in a water-insoluble macromolecular carrier, so that the fluidity of the enzyme is lowered. The enzyme after being fixed at present has increased stability, is easy to separate from a reaction system, is convenient to transport and store, but has reduced activity.
for the reasons, the bottleneck of producing GSH by an enzyme method is urgently needed to be broken through, and a corresponding method for synthesizing glutathione by enzyme catalysis is developed.
disclosure of Invention
the object of the present invention is to provide a method for enzymatically synthesizing glutathione, thereby overcoming the above-mentioned drawbacks of the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for synthesizing glutathione by enzyme catalysis comprises the following steps:
S1, mixing the gamma-glutamylcysteine synthetase liquid and the glutathione synthetase liquid to obtain a mixed liquid A, and then mixing the mixed liquid A and the acetokinase liquid to obtain a mixed liquid B;
S2, adding an immobilized carrier into the mixed solution B, stirring and immobilizing, and filtering to obtain an immobilized enzyme;
S3, preparing glutamic acid, L-cysteine, glycine, magnesium sulfate, ATP and acetyl phosphate into a reaction solution, adding immobilized enzyme, and stirring for reaction;
S4, after the reaction is finished, filtering the reaction solution, and extracting and refining the filtrate into glutathione.
further, the mixed solution A is prepared by mixing gamma ~ glutamylcysteine synthetase solution and glutathione synthetase solution according to the ratio of 1: 0.3 ~ 1 of the enzyme protein amount.
further, the mixed solution B is obtained by mixing the mixed solution A and an acetate kinase solution according to the ratio of 1: 0.5 ~ 2 of the amount of the enzyme protein.
further, the immobilized carrier is a chitosan-silica gel compound.
further, the immobilized carrier is prepared by the following method that 1 part by weight of chitosan is weighed and added into 100 ~ 200 parts by weight of 0.5 ~ 2wt% acetic acid solution, stirred and dissolved, then 5 ~ 20 parts by weight of silica gel particles with the particle size of 0.1 ~ 0.5mm are added, stirred for 8 ~ 15h, after filtration, 80 ~ 120 parts by weight of 0.5 ~ 1mol/L sodium hydroxide solution is added for soaking for 1 ~ 3h, the chitosan and the silica gel particles are uniformly combined together, filtration is carried out, purified water is used for cleaning to be neutral, 80 ~ 120 parts by weight of 1 ~ 3wt% glutaraldehyde solution is added, stirring is carried out for 2 ~ 5h, cross ~ linking reaction and chitosan group activation are carried out, and after filtration, purified water is used for cleaning for 2 ~ 3 times, so that the chitosan ~ silica gel compound is obtained.
furthermore, the dosage of the immobilized carrier is that 1g of immobilized carrier is added into every 20 ~ 50mg of protein.
further, the stirring and immobilizing conditions in the step S2 are that the temperature is 2 ~ 10 ℃ and the time is 1 ~ 5 h.
furthermore, the formula of the reaction liquid comprises 20 ~ 60mmol/L, L ~ cysteine 20 ~ 60mmol/L of glutamic acid, 20 ~ 60mmol/L of glycine, 5 ~ 10mmol/L, ATP5 ~ 10mmol/L of magnesium sulfate and 40 ~ 100mmol/L of acetyl phosphate, and the dosage of the immobilized enzyme is 10 ~ 50 g/L.
further, the stirring reaction conditions in the step S3 are that the pH of the reaction solution is adjusted to 5.0 ~ 8.0 by sodium hydroxide, the reaction temperature is 25 ~ 35 ℃, and the reaction time is 2 ~ 6 h.
The invention designs a process method for synthesizing glutathione by immobilized enzyme catalysis, which is characterized in that three enzymes of gamma-glutamylcysteine synthetase, glutathione synthetase and acetate kinase are mixed and immobilized on the same carrier, and the obtained immobilized enzyme, amino acid, ATP and other substrates are put into a reactor to synthesize glutathione in one step.
The invention has the following beneficial effects:
1. the synthesis efficiency is high, the synthesis time is greatly reduced to 2 ~ 6h, the product content of the reaction solution reaches more than 10g/L under the optimized condition, and the substrate conversion rate (calculated by L ~ cysteine) reaches more than 90%.
2. The cost is reduced: the immobilized enzyme contains 3 enzyme proteins, wherein the function of synthesizing glutathione is realized by gamma-glutamylcysteine synthetase liquid and glutathione synthetase, the function of regenerating and utilizing ATP by acetate kinase is realized, and an ATP regeneration coupling system is constructed by introducing acetate kinase, so that the dosage of ATP is greatly reduced, and the production cost is obviously reduced.
3. The process is simplified: compared with a fermentation method and the existing enzyme catalysis synthesis method, the method adopts the immobilized enzyme to catalyze and synthesize the glutathione in one step, the synthesized reaction liquid does not have residual thalli, enzyme protein, host bacteria DNA or protein and the like after simple solid-liquid separation, and a high-quality refined product can be obtained only through simple extraction steps.
4. Environmental protection and energy conservation: the synthesis condition is mild, the reaction time is short, the energy-saving and consumption-reducing effects are obvious, three wastes generated in the process are very little, and the workshop and the surrounding environment are not influenced.
5. the immobilized enzyme can be repeatedly used, the stability is good, and after the immobilized enzyme is continuously used for 50 batches, the enzyme activity loss of the immobilized enzyme is less than 10 percent.
6. The immobilized carrier is a self-made chitosan-silica gel composite carrier, has low price and better enzyme immobilization effect.
Detailed Description
The invention will be further illustrated with reference to specific examples:
the gamma-glutamylcysteine synthetase, glutathione synthetase and acetate kinase used in the following examples are fermented by genetically engineered bacteria to express the corresponding enzyme proteins, and then the high-purity enzyme proteins are obtained after extraction and purification by a nickel column.
example 1
synthesizing glutathione according to the following steps:
1. Mixing gamma-glutamylcysteine synthetase solution and glutathione synthetase solution according to the ratio of enzyme protein amount of 1: 1 to obtain a mixed solution A, and mixing the mixed solution A with an acetate kinase solution according to the ratio of 1: 1 to obtain a mixed solution B;
2. adding an immobilized carrier into the mixed solution B, stirring and immobilizing for 3 hours at the temperature of 5 ℃ and filtering to obtain an immobilized enzyme, wherein the using amount of the immobilized carrier is that 1g of the immobilized carrier is added into every 40mg of protein, and the immobilized carrier is prepared by the following method that 1 part by weight of chitosan is added into 150 parts by weight of 1wt% acetic acid solution, stirring and dissolving, then 10 parts by weight of silica gel particles with the particle size of 0.1 ~ 0.5mm are added, stirring is carried out for 12 hours, after filtering, 0.5mol/L of 100 parts by weight of sodium hydroxide solution is added for soaking for 2 hours, so that the chitosan and the silica gel particles are uniformly combined together, filtering is carried out by using purified water to wash to neutrality, 100 parts by weight of 2wt% glutaraldehyde solution is added, cross ~ linking reaction and activation of chitosan group are carried out, after filtering, purified water is used for washing for 2 ~ 3 times to obtain a chitosan ~ silica gel compound, the mechanical strength of the chitosan ~ silica gel compound is greatly improved after cross ~ linking, the compound is used as the immobilized carrier, and the purified enzyme protein is added, and the stable;
3. preparing 20mmol/L, L-cysteine 20mmol/L, glycine 20mmol/L, magnesium sulfate 5mmol/L, ATP5mmol/L, and acetyl phosphate 40mmol/L into reaction liquid, adding 10g/L immobilized enzyme, stirring for reaction: adjusting the pH value of the reaction solution to about 6.0 by using sodium hydroxide, controlling the reaction temperature to about 30 ℃ and keeping the reaction time for 5 hours; the concentration of the glutathione product is 5.6g/L, the substrate conversion rate is 91 percent, and the conversion rate of the synthesis reaction is higher under the condition of low substrate concentration;
4. after the reaction is finished, the reaction solution is filtered, and the filtrate is extracted and refined into glutathione.
Example 2
synthesizing glutathione according to the following steps:
1. mixing gamma-glutamylcysteine synthetase solution and glutathione synthetase solution according to the ratio of enzyme protein amount of 1: 1 to obtain a mixed solution A, and mixing the mixed solution A with an acetate kinase solution according to the ratio of 1: 1 to obtain a mixed solution B;
2. adding an immobilized carrier into the mixed solution B, stirring and immobilizing for 3 hours at the temperature of 5 ℃, and filtering to obtain an immobilized enzyme, wherein the dosage of the immobilized carrier is that 1g of the immobilized carrier is added into every 40mg of protein, and the immobilized carrier is prepared by the following method that 1 part by weight of chitosan is weighed and added into 200 parts by weight of 0.5wt% acetic acid solution, stirred and dissolved, then 20 parts by weight of silica gel particles with the particle size of 0.1 ~ 0.5mm are added, stirred for 8 hours, 120 parts by weight of 0.5mol/L sodium hydroxide solution is added after filtering and soaked for 3 hours, so that the chitosan and the silica gel particles are uniformly combined together, filtering is carried out, purified water is used for cleaning to be neutral, 3 parts by weight of 3wt% glutaraldehyde solution is added, stirring is carried out for 5 hours, crosslinking reaction and chitosan group activation are carried out, and purified water is used for cleaning for 2 ~ 3 times after filtering, so as to obtain a chitosan;
3. preparing 60mmol/L, L-cysteine 60mmol/L glutamic acid, 60mmol/L glycine, 10mmol/L, ATP10mmol/L magnesium sulfate and 100mmol/L acetyl phosphate into a reaction solution, adding 50g/L immobilized enzyme, and stirring for reaction: adjusting the pH of the reaction solution to about 6.0 by using sodium hydroxide, wherein the reaction temperature is about 30 ℃, and the reaction time is 6 hours; the concentration of glutathione in the product is 15.0g/L, the substrate conversion rate (calculated by L-cysteine, the same below) is 81 percent, and after the substrate concentration is greatly increased, although the concentration of glutathione is increased, the conversion rate of the synthesis reaction is greatly reduced;
4. after the reaction is finished, the reaction solution is filtered, and the filtrate is extracted and refined into glutathione.
Example 3
synthesizing glutathione according to the following steps:
1. Mixing gamma-glutamylcysteine synthetase solution and glutathione synthetase solution according to the ratio of enzyme protein amount of 1: 1 to obtain a mixed solution A, and mixing the mixed solution A with an acetate kinase solution according to the ratio of 1: 1 to obtain a mixed solution B;
2. adding an immobilized carrier into the mixed solution B, stirring and immobilizing for 3 hours at the temperature of 5 ℃, and filtering to obtain an immobilized enzyme, wherein the dosage of the immobilized carrier is that 1g of the immobilized carrier is added into every 40mg of protein, and the immobilized carrier is prepared by the following method that 1 part by weight of chitosan is weighed and added into 100 parts by weight of 2wt% acetic acid solution, stirred and dissolved, then 5 parts by weight of silica gel particles with the particle size of 0.1 ~ 0.5mm are added, stirred for 15 hours, 80 parts by weight of 0.8mol/L sodium hydroxide solution is added after filtering and soaked for 1 hour, so that the chitosan and the silica gel particles are uniformly combined together, filtering is carried out, purified water is used for cleaning to be neutral, 1 part by weight of 1wt% glutaraldehyde solution is added, stirring is carried out for 2 hours, a crosslinking reaction and a chitosan group are activated, and the chitosan group is cleaned for 2 ~ 3 times by the purified water after filtering, so as to;
3. preparing 40mmol/L glutamic acid/L, L-cysteine, 60mmol/L glycine, 10mmol/L, ATP10mmol/L magnesium sulfate and 80mmol/L acetyl phosphate into a reaction solution, adding 50g/L immobilized enzyme, and stirring for reaction: adjusting the pH value of the reaction solution to about 6.0 by using sodium hydroxide, controlling the reaction temperature to about 30 ℃ and keeping the reaction time for 5 hours; the concentration of glutathione of the product is 11.5g/L, the substrate conversion rate is 94%, the balance between the production efficiency and the economic benefit is comprehensively considered, the synthesis formula is properly adjusted, the feeding concentration of L-cysteine with higher cost is reduced, and the yield and the conversion rate of the synthesis reaction can reach satisfactory effects;
4. After the reaction is finished, the reaction solution is filtered, and the filtrate is extracted and refined into glutathione.
example 4
Synthesizing glutathione according to the following steps:
1. mixing gamma-glutamylcysteine synthetase solution and glutathione synthetase solution according to the ratio of enzyme protein amount of 1: 0.3 to obtain a mixed solution A, and mixing the mixed solution A with an acetate kinase solution according to the ratio of 1: mixing at a ratio of 0.5 to obtain a mixed solution B;
2. adding an immobilized carrier into the mixed solution B, stirring and immobilizing for 5 hours at the temperature of 2 ℃, and filtering to obtain an immobilized enzyme, wherein the dosage of the immobilized carrier is that 1g of the immobilized carrier is added into every 20mg of protein, and the immobilized carrier is prepared by weighing 1 part by weight of chitosan, adding 180 parts by weight of 1.5wt% acetic acid solution, stirring and dissolving, adding 15 parts by weight of silica gel particles with the particle size of 0.1 ~ 0.5mm, stirring for 10 hours, adding 90 parts by weight of 0.8mol/L sodium hydroxide solution after filtering, soaking for 2 hours to uniformly combine the chitosan and the silica gel particles, filtering and washing with purified water to neutrality, adding 100 parts by weight of 2wt% glutaraldehyde solution, stirring for 3 hours, carrying out crosslinking reaction and activating chitosan group, and washing with purified water for 2 ~ 3 times after filtering to obtain a chitosan ~ silica gel compound;
3. Preparing a reaction solution from 40mmol/L, L-cysteine 30mmol/L, glycine 50mmol/L, magnesium sulfate 8mmol/L, ATP9mmol/L and acetyl phosphate 80mmol/L, adding 40g/L immobilized enzyme, and stirring for reaction: adjusting the pH value of the reaction solution to about 8.0 by using sodium hydroxide, wherein the reaction temperature is about 35 ℃, and the reaction time is 2 hours;
4. After the reaction is finished, the reaction solution is filtered, and the filtrate is extracted and refined into glutathione.
example 5
Synthesizing glutathione according to the following steps:
1. mixing gamma-glutamylcysteine synthetase solution and glutathione synthetase solution according to the ratio of enzyme protein amount of 1: 3 to obtain a mixed solution A, and mixing the mixed solution A with the acetate kinase solution according to the ratio of 1: 2 to obtain a mixed solution B;
2. adding an immobilized carrier into the mixed solution B, stirring and immobilizing for 2 hours at the temperature of 10 ℃, and filtering to obtain an immobilized enzyme, wherein the dosage of the immobilized carrier is that 1g of the immobilized carrier is added into every 50mg of protein, and the immobilized carrier is prepared by the following method that 1 part by weight of chitosan is weighed and added into 130 parts by weight of 1wt% acetic acid solution, stirred and dissolved, then 12 parts by weight of silica gel particles with the particle size of 0.1 ~ 0.5mm are added, stirred for 11 hours, after filtering, 0.7mol/L of 100 parts by weight of sodium hydroxide solution is added for soaking for 2 hours, so that the chitosan and the silica gel particles are uniformly combined together, filtering is carried out, purified water is used for cleaning to be neutral, 2 parts by weight of 2wt% glutaraldehyde solution is added, stirring is carried out for 4 hours, cross ~ linking reaction and chitosan group activation are carried out, after filtering, purified water is used for cleaning for 2 ~ 3 times to obtain a;
3. preparing 40mmol/L glutamic acid/L, L-cysteine, 30mmol/L glycine, 6mmol/L, ATP7mmol/L magnesium sulfate and 50mmol/L acetyl phosphate into a reaction solution, adding 30g/L immobilized enzyme, and stirring for reaction: adjusting the pH value of the reaction solution to about 5.0 by using sodium hydroxide, wherein the reaction temperature is about 25 ℃, and the reaction time is 6 hours;
4. After the reaction is finished, the reaction solution is filtered, and the filtrate is extracted and refined into glutathione.
Example 6
and (3) testing the stability of the immobilized enzyme:
the procedure of example 1 was followed with the difference that: glutamic acid 25mmol/L, L-cysteine 20mmol/L, glycine 25mmol/L, magnesium sulfate 5mmol/L, ATP5mmol/L, acetyl phosphate 40 mmol/L; the dosage of the immobilized enzyme is 15g/L, the reaction time is 4 hours, and 50 batches of reaction are continuously carried out. Results show results for the first batch: the concentration of glutathione in the reaction solution is 5.7g/L, and the substrate conversion rate is 93 percent; batch 50 results: the concentration of reaction liquid glutathione is 5.2g/L, the substrate conversion rate is 85%, the immobilized enzyme has good stability, the enzyme activity loss of the immobilized enzyme is less than 10% after continuous use of 50 batches, and the effect is obvious.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (1)
1. the method for synthesizing glutathione by enzyme catalysis is characterized by comprising the following steps:
S1, mixing the gamma-glutamylcysteine synthetase liquid and the glutathione synthetase liquid to obtain a mixed liquid A, and then mixing the mixed liquid A and the acetokinase liquid to obtain a mixed liquid B;
s2, adding an immobilized carrier into the mixed solution B, stirring and immobilizing, and filtering to obtain an immobilized enzyme;
s3, preparing glutamic acid, L-cysteine, glycine, magnesium sulfate, ATP and acetyl phosphate into a reaction solution, adding immobilized enzyme, and stirring for reaction;
s4, after the reaction is finished, filtering the reaction solution, and extracting and refining the filtrate into glutathione; the mixed solution A is prepared from gamma-glutamylcysteine synthetase solution and glutathione synthetase solution according to the ratio of 1: 1 in proportion;
the mixed solution B is prepared by mixing the mixed solution A with acetic acid kinase solution according to the ratio of the enzyme protein amount to 1: 1 in proportion;
The immobilized carrier is a chitosan-silica gel compound;
the immobilization carrier is prepared according to the following method: weighing 1 part by weight of chitosan, adding the chitosan into 150 parts by weight of 1wt% acetic acid solution, stirring for dissolving, adding 10 parts by weight of silica gel particles with the particle size of 0.1-0.5 mm, stirring for 12 hours, filtering, adding 100 parts by weight of 0.5mol/L sodium hydroxide solution, soaking for 2 hours to uniformly combine the chitosan and the silica gel particles, filtering, cleaning with purified water to be neutral, adding 100 parts by weight of 2wt% glutaraldehyde solution, stirring for 4 hours to perform a crosslinking reaction and activate chitosan groups, and cleaning with purified water for 2-3 times after filtering to obtain a chitosan-silica gel compound;
the dosage of the immobilized carrier is as follows: adding 1g of immobilized carrier into every 40mg of protein;
The conditions for stirring and immobilizing in step S2 are: the temperature is 5 ℃, and the time is 3 h;
The formula of the reaction solution is as follows: glutamic acid 25mmol/L, L-cysteine 20mmol/L, glycine 25mmol/L, magnesium sulfate 5mmol/L, ATP5mmol/L, acetyl phosphate 40 mmol/L; the dosage of the immobilized enzyme is 15 g/L;
The conditions of the stirring reaction in the step S3 are as follows: adjusting the pH value of the reaction solution to 6 by using sodium hydroxide, and reacting at the temperature of 30 ℃ for 4 hours.
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| CN111979206B (en) * | 2019-05-24 | 2021-08-17 | 深圳瑞德林生物技术有限公司 | Immobilized fusion enzyme and method for preparing glutathione by using same |
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