CN1800410A

CN1800410A - Glutathion production method

Info

Publication number: CN1800410A
Application number: CN 200510023119
Authority: CN
Inventors: 朱文洲
Original assignee: SHANGHAI SINO-BIOTECH Co Ltd
Current assignee: SHANGHAI SINO-BIOTECH Co Ltd
Priority date: 2005-01-05
Filing date: 2005-01-05
Publication date: 2006-07-12

Abstract

The invention relates to a method for producing glutathione, which comprises the following steps: doing large scale culture and separating purify technology to two types of expressing recombining gene's gene project bacillus coli with glutathione enzyme A and enzyme B to obtain enzyme A and enzyme B and doing solid to them by suit materials (1); mixing the two solid enzymes with the ratio EA: EB=1:10-10:1 into the GSH synthesis reactor, which is used in the synthesis of glutathione (2); forming the total producing system into a large fluid cycle type reaction system because GSH synthesis reactor and ATP re-generating reactor are fluid cycle(3); the aminothiopropionic acid availability ratio is above 40%; the glutathione recovery ratio s above 50%; the main synthesis reacting stage can hold the density of glutathione above 0.8 g/L.

Description

The production method of gsh

Technical field

The present invention relates to a kind of production method of gsh.

Background technology

Gsh (GSH) extensively is present in animals and plants and the microorganism cells, and it and SOD are human body two big restoring systems also, has to participate in the amino acid whose important physiological function such as reduced state of striding the film transportation, keeping cell.GSH as the sulfydryl buffer reagent, keeps oxyphorase and the proteic cysteine residues of other red corpuscle and is in and goes back ortho states in red corpuscle.GSH is widely used at field of food: GSH content is lower usually in the foodstuffs industry flour products, adds GSH therein and can strengthen its nutritive value greatly; Join in the milk-product and can effectively prevent enzymatic and non-enzymatic browning, be equivalent to vitamins C, play stabilization; Add GSH and can suppress the nucleic acid decomposition in fish and bird food-processing, the nutrient fortified food local flavor also extends the shelf life greatly; In the processing of fruit and vegetable based food, add GSH, can effectively prevent brown stain, keep its original tempting color and luster, local flavor and nutrition.In addition, it also can be used as the reductive agent of Sparkling wine.

Medicine GSH purposes clinically also has many aspects:

The amino acid that participates in vital tissues such as red corpuscle, kidney by the gamma-glutamyl circulation absorbs, and guarantees the nutritional requirement of these tissues;

GSH participates in drug metabolism, medicine in liver by containing the mixed-function oxidase system of cytochrome P-450, after reactions such as oxidation, hydroxylation and the sulfydryl of GSH be combined into water-soluble cpds and excrete;

With the active intermediate be combined into non-carcinogenic thing of potential carcinogen (precarcinogen) in liver;

GSH is that this enzyme of substrate of glutathione peroxidase can be removed deleterious hydrogen peroxide in the body, can remove intravital superoxide ion and other free radical with SOD, prevents primary cellular defect.

Present current research shows, GSH is fine to propylene, the poisoning of fluorochemical, carbon monoxide, organic solvent etc. all has detoxification; For the leukopenia that radiation exposure, radiopharmaceuticals or tumour medicine cause, myeloid tissue inflammation and oral mucositis that radioactivity causes all have mitigation;

GSH protects liver, suppresses the formation of fatty liver, alleviates the symptom of toxic and infectious hepatitis; Tissue adherence there is provide protection, can prevents haemolysis, reduce the loss of methemoglobin; Correct the imbalance of vagusstoff, cholinesterase, eliminate the allergic symptom that causes thus; Has mitigation for anoxenia, uncomfortable disease nauseating and that hepatic diseases causes;

Prevent skin pigment deposition and melanic formation, reduce its oxidation, improve skin gloss, delay senility; Suppress the development of cataract, cornea and retinal diseases, prevent to form muddiness behind the retina transplantation; Sexual function improving; At American market, except being used for the disease treatment as medicine, gsh also is used for protective foods (food supplements) in a large number, brings into play it and beautifies skin, delays senility health care functions such as protection liver.Because U.S.'s health food market is very huge, and Acyclovir can not bought the gsh healthcare products, so very big at the health food market gsh consumption of the U.S. and other developed country.

Recently find that GSH may have the function that suppresses acquired immune deficiency syndrome (AIDS).In addition, gsh adds in the food can play function of stabilizer, plays simultaneously and strengthens amino acid whose effect; Gsh transmits amino acid whose biological function in vivo and helps lend some impetus to growing of infant; The pregnant woman lacks protein, mainly is in fact to lack gsh; Therefore aspect functional health-care food, gsh also has very big potential application market.

The preparation of gsh has chemical synthesis, extraction method, microbe fermentation method, enzyme process etc.

Solvent extration is a raw material with the animal vegetable tissue that is rich in gsh, by adding appropriate solvent or in conjunction with processing such as amylase, proteolytic enzyme, separation and purification forms again.Domestic extraction process produced in small quantities gsh early has successfully precedent in China, but production technique is backward, and small scale yields poorly, and is of poor quality, and popular supercritical extraction technique is not used widely as yet at present.

Fermentation method comprises yeast mutagenic treatment method, green alga cultivation extraction method and immobilization cereuisiae fermentum continuous production method etc., and it is the most common wherein to produce gsh with the yeast mutation bacterial strain of mutagenic treatment acquisition homoglutathion content.Green alga cultivation extraction method is comparatively easy, and production cost is also lower, but is subjected to the influence of regions resources bigger.Immobilization cereuisiae fermentum continuous production method is produced gsh, because its substratum formation raw material is more expensive, production cost is higher, still is not suitable for carrying out industrial mass production.

The present gsh of chemical synthesis chemistry synthesis and production process is ripe, but have that cost height, reactions steps are many, long reaction time, complicated operation, light requirement learn problems such as fractionation and contaminate environment.Its used basic raw material is L-glutamic acid, halfcystine and glycine.

Enzyme process utilizes biological intravital glutathione synthetase, is substrate with L-L-glutamic acid, L-half Guang acid and glycine, and adds a small amount of Triphosaden and get final product synthesizing glutathion.This method is the most promising at present gsh production method.How tame unit is arranged at present in research both at home and abroad.The technology that obtains engineering bacteria after utilizing genetically engineered to clone out two key enzymes of gsh synthetic be mature on the whole at present [Shen Lixin etc. biotechnology journal .200117 (4): 452-455; Li Huazhong etc. Wuxi light industry college journal .1999,18 (4): 1-5; Li Yin etc. microorganism journal .2001,41 (2): 191-197].The bright prospect of utilizing recombinant bacterial strain to produce gsh is generally acknowledged by everybody.

The biosynthesizing of microorganism cells glutathion inside is subjected to the control of two enzymes, simultaneously these two enzymes are subjected to the feedback inhibition of gsh again, and born of the same parents' glutathion inside level is roughly suitable can produce the concentration (about 2.5mmol/L) of the gsh of 50% feedback inhibition effect and cell and be in stationary phase the time.Therefore on to gsh synthetic efficiency, the activity of two enzymes plays crucial effects.

It is all good that Li Huazhong etc. [microorganism journal .2001,41 (1): 16-24] have obtained a strain gsh composite reactive, plasmid stability and mitotic stability, and can reusable recombination bacillus coli E.coli II.This bacterial strain can accumulate the gsh (GSH) about 4g/L through after the O for toluene outside born of the same parents.Improving the L-aminoglutaric acid concentration in the building-up reactions system can promote GSH synthetic.But the L-semicystinol concentration can suppress the synthetic of GSH after being increased to 20mmol/L.

In addition, in building-up process, the abundant supply of ATP is to realize gsh high-performance bio synthetic prerequisite.Shimosaka[Agric Biol Chem.1988,52:2753-2762] will carry glycolysis-key gene (pfkA and tpi) the heterozygosis plasmid clone in intestinal bacteria, significantly improved the activity of the synthetic ATP of cell.Can make up the regeneration system rapidly of energy factors or cofactor, directly have influence on the economy of the multistep enzyme reaction of cell catalysis.In the biosynthesizing of gsh, the transformation efficiency of substrate depends on the supply of ATP in the system, so the regeneration of ATP and ATP keeping in reaction system is the key that improves gsh synthesis system operational efficiency.

.[Wuxi light industry college journal .1999 such as Li Huazhong, 18 (4): 1-5] utilize the mix suspending cultured method, having made up a gsh biosynthetic enzyme by recombination bacillus coli is the ATP regenerating system of forming with the glycolytic pathway of bread yeast, has verified that this system is used for the biosynthetic feasibility of gsh.

.[microorganism journal .2001 such as Li Yin, 41 (2): 191-197] utilize glutathione synthetase system and the ATP biosynthetic enzyme among the bread yeast WSH-J7 among the recombination bacillus coli II-1 to be, having made up one is coupling ATP regenerating system between the kind of the energy with glucose.Almost can not consumption of glucose through the yeast cell that permeability is handled.In reaction system, add 1mmol/L AMP and 0.05mmol/L NADH, can start zymic glycolysis approach.Improve the glucose concn in the coupling system, can promote synthesizing of GSH.

Lin Jinping [structure and the building-up process research of the bacterial strain of the biosynthetic research of gsh-homoglutathion composite reactive] has made up the biosynthetic Fourier Series expansion technique of GSH by the enzymatic ATP regenerating system in the glycolytic pathway of introducing S.cerevisiaeWSH-J702.

.[Sanop Misaengmul Hakhoechi such as Koh, 1998,26 (3): 213-220] the ATP regenerating system method of producing as gsh.Gsh is synthetic by the continuous action of g-glutamyl cysteine synthetase and glutathione synthetase.Its synthetic enzyme then results from recombination bacillus coli.

But, in these researchs, all exist in the gsh building-up process its feedback inhibition and be the insoluble problem of research at present.

Summary of the invention

The technical issues that need to address of the present invention have provided a kind of production method of gsh, are intended to solve above-mentioned defective.

In order to solve the problems of the technologies described above, the present invention realizes by following steps:

The gene engineering colibacillus of two kinds of express recombinant genes that will have glutathione synthetase A and enzyme B obtains enzyme A, enzyme B through large scale culturing and extensive separating and purifying technology, and they are material immobilized with what be fit to respectively;

Two immobilized enzyme are pressed EA: EB=1: 10～10: after 1 the mixed, the GSH synthesis reactor of packing into is used for the biosynthesizing of gsh;

GSH synthesis reactor and ATP regeneration reactor are respectively liquid stream round-robin, and whole production system forms a big liquid again and flows circulating reactive system.

Compared with prior art, the invention has the beneficial effects as follows: the synthesizing glutathion combined coefficient is greatly improved: the utilization ratio of halfcystine is more than 40%; The rate of recovery of gsh is more than 50; It is more than the 0.8g/L in the concentration of synthesis reactor outlet that the main building-up reactions stage can be kept gsh; But have continuous production, running cost and raw materials cost are lower, and processing ease and productive rate height, product refine and be refining relatively easy, the yield advantages of higher.

Embodiment

Below in conjunction with embodiment the present invention is described in further detail:

The present invention realizes by following steps:

Gene order according to glutathione synthetase A and enzyme B, design two amplimers respectively, by utilizing the RT-PCR method from intestinal bacteria, to amplify glutathione synthetase A and two gene fragments of enzyme B, and clone respectively on prokaryotic expression carrier, the exactness of sequence verification gene order has obtained structure behind its plasmid transformation escherichia coli expression strain the bacterial classification that efficiently expresses afterwards.

The gene engineering colibacillus of two kinds of express recombinant genes that will have glutathione synthetase A and enzyme B is through large scale culturing and extensive separating and purifying technology, obtain enzyme A, enzyme B, and it is they are material immobilized with what be fit to respectively: as to use substratum such as the LB substratum of the improvement of forming with yeast extract, peptone, sodium-chlor, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, ammonium chloride etc. or 2YT, after cultivating in 3～5 hours, induce by IPTG, continue to cultivate centrifugal results thalline 3.5～4.5 hours; The thalline of results is behind the broken bacterium of ultrasonic or high-pressure homogenization, and centrifugal results supernatant adds the ammonium sulfate of 35%～45% saturation ratio, centrifugal results supernatant; Further handle centrifugal results precipitation with the ammonium sulfate of 60%～75% saturation ratio; Precipitation is dissolved with the Tris-HCl damping fluid of pH5.8～pH7.8 phosphate buffered saline buffer or pH8.0～pH9.5, after dialysis or G25 desalination, through sepharose CM fast flow or sepharose DEAE fast flow column chromatography, can obtain the synthetic enzyme EA and the EB of purifying; Two synthetic enzyme are cut into the fritter of 2mm * 2mm then respectively with glutaraldehyde cross-linking behind the gelatin embedding or use the carrageenin embedding, and-20 degree preservations are in order to being filled into the usefulness of synthesis reactor;

GSH synthesis reactor and ATP regeneration reactor are respectively liquid stream round-robin, and whole production system forms a big liquid again and flows circulating reactive system;

GSH is synthetic, adopts coupling GSH ion-exchange adsorption column, and enzymatic is closed the GSH that reaction is produced, and is adsorbed onto on the pillar, reduces and returns enzymatic synthesis reactor GSH concentration in a looping fashion, thereby remove product to the synthetic restraining effect; Two or more GSH adsorption columns are used alternatingly and regenerate.The process of wash-out also is the regenerative process of pillar, thereby guarantees that it is successive reaction that enzymatic synthesizes, and can improve the efficient of reactor like this; In whole enzymatic synthetic reaction process, L-glutamic acid, halfcystine, glycine add (Feed-batch) with stream mode with certain flow rate to the reactor continuous feeding.Gsh synthesis reactor self forms the circulation of liquid stream;

Gsh is after ion exchange column is adsorbed, all the other are because of providing energy to enter into the ATP regeneration reactor from ADP, the AMP etc. that the ATP degraded forms to the biosynthesizing reaction, by the multi-enzyme system of fixed yeast, regeneration becomes ATP, enters into the gsh synthesis reactor then; ATP regeneration reactor self forms the circulation of a liquid stream; Participate in ATP regenerated glucose and be added to the ATP regeneration reactor with certain flow rate stream;

Regenerated ATP returns the GSH synthesis reactor with the L-glutamic acid that does not work, halfcystine, glycine in the acidifying phosphorylation.

By directly synthetic enzyme being passed through purifying, utilize enzyme immobilization technology to be fixed again; And utilize GSH synthesis reactor and ATP regeneration reactor to form a big liquid and flow circulating reactive system in whole production system.

The utilization ratio of halfcystine is more than 40%;

The rate of recovery of gsh is more than 50%;

It is more than the 0.8g/L in the concentration of synthesis reactor outlet that the main building-up reactions stage can be kept gsh.

Claims

1. the production method of a gsh, realize by following steps:

The gene engineering colibacillus of two kinds of express recombinant genes that will have glutathione synthetase A and enzyme B obtains enzyme A, enzyme B through large scale culturing and extensive separating and purifying technology, and with them respectively with material immobilized (1) that is fit to;

Two immobilized enzyme are pressed EA: EB=1: 10～10: after 1 the mixed, the GSH synthesis reactor of packing into is used for the biosynthesizing (2) of gsh;

GSH synthesis reactor and ATP regeneration reactor are respectively liquid stream round-robin, and whole production system forms a big liquid again and flows circulating reactive system (3).

2. the production method of gsh according to claim 1, wherein in the step (1): according to the gene order of glutathione synthetase A and enzyme B, design two amplimers respectively, by utilizing the RT-PCR method from intestinal bacteria, to amplify glutathione synthetase A and two gene fragments of enzyme B, and clone respectively on prokaryotic expression carrier, the exactness of sequence verification gene order has obtained structure behind its plasmid transformation escherichia coli expression strain the bacterial classification that efficiently expresses afterwards; Substratum such as the LB substratum of the improvement that application is formed with yeast extract, peptone, sodium-chlor, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, ammonium chloride etc. or 2YT, after cultivating in 3～5 hours, induce by IPTG, continue to cultivate centrifugal results thalline 3.5～4.5 hours; The thalline of results is behind the broken bacterium of ultrasonic or high-pressure homogenization, and centrifugal results supernatant adds the ammonium sulfate of 35%～45% saturation ratio, centrifugal results supernatant; Further handle centrifugal results precipitation with the ammonium sulfate of 60%～75% saturation ratio; Precipitation is dissolved with the Tris-HCl damping fluid of pH5.8～pH7.8 phosphate buffered saline buffer or pH8.0～pH9.5, after dialysis or G25 desalination, through sepharose CM fast flow or sepharose DEAE fast flow column chromatography, can obtain the synthetic enzyme EA and the EB of purifying; Two synthetic enzyme are cut into the fritter of 2mm * 2mm then respectively with glutaraldehyde cross-linking behind the gelatin embedding or use the carrageenin embedding, and-20 degree preservations are in order to being filled into the usefulness of synthesis reactor;

In the step (3): GSH is synthetic, adopts coupling GSH ion-exchange adsorption column, and enzymatic is closed the GSH that reaction is produced, and is adsorbed onto on the pillar, reduces and returns enzymatic synthesis reactor GSH concentration in a looping fashion; Two or more GSH adsorption columns are used alternatingly and regenerate; In whole enzymatic synthetic reaction process, to the reactor continuous feeding, gsh synthesis reactor self forms the circulation of liquid stream to the mode that L-glutamic acid, halfcystine, glycine add with stream with certain flow rate; Gsh is after ion exchange column is adsorbed, all the other are because of providing energy to enter into the ATP regeneration reactor from ADP, the AMP etc. that the ATP degraded forms to the biosynthesizing reaction, by the multi-enzyme system of fixed yeast, regeneration becomes ATP, enters into the gsh synthesis reactor then; ATP regeneration reactor self forms the circulation of a liquid stream; Participate in ATP regenerated glucose and be added to the ATP regeneration reactor with certain flow rate stream; Regenerated ATP returns the GSH synthesis reactor with the L-glutamic acid that does not work, halfcystine, glycine in the acidifying phosphorylation.