CN1760359A - Method for producing glutathione thorugh biologic engineering method - Google Patents
Method for producing glutathione thorugh biologic engineering method Download PDFInfo
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- CN1760359A CN1760359A CN 200410079363 CN200410079363A CN1760359A CN 1760359 A CN1760359 A CN 1760359A CN 200410079363 CN200410079363 CN 200410079363 CN 200410079363 A CN200410079363 A CN 200410079363A CN 1760359 A CN1760359 A CN 1760359A
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Abstract
A process for preparing glutathion by biologic engineering method features the enzymatic reaction between a screened microbial strain (CGMCC 1231) containing the glutathion synthetase I (GSH-I) and II (GSH-II), the substrate (L-glutamic acid, L-cysteine and glycine), glucose and magnesium sulfate. Its advantages are high output rate (45-58%) and high purity (98%).
Description
It is substrate that technical field the present invention relates to L-L-glutamic acid, L-halfcystine, glycine, use the CGMCC1231 microorganism strains, glutathion production by fermentation synthetic enzyme I (GSH-I) and glutathione synthetase II (GSH-II) carry out the method for a kind of producing glutathione thorugh biologic engineering method of biocatalytic reaction synthesizing glutathion.
Gsh is a kind of active kyrine with important biomolecule function.Reduced form (GSH) and two kinds of patterns of oxidized form (GSSG) are arranged, and what play a major role in vivo is reduced glutathion.Gsh, Glutathione is a kind of important biochemical drug and foodstuff additive, has many-sided important biological function.Be the coenzyme of body metabolism enzyme Triose phosphate dehydrogenase, lactoyl-glutathione lyase and triosephosphate dehydrogenase, participate in tricarboxylic acid cycle and carbohydrate metabolism in the body, make human body obtain high-energy, and can activate various enzymes.As intravital sulfydryl enzyme, cholinesterase etc.; thereby promote sugar, fat and protein metabolism; and provide glutamyl in the γ-Gu Anxianji circulation in vivo; keep the integrity of the eubolism and the cytolemma of cell; also can combine, have antioxygenation widely with toxicants such as electrophilic base, oxyradicals.After the quantity of gsh increases in the human body, helpful to anti-aging, cardiovascular, Digestive tract, poisoning, transmissible disease and immunizing power, cancer, respiratory system and metabolism.Gsh is used for the treatment of toxic hepatitis and infectious hepatitis clinically; Suppress the improvement of cataract and cornea and retinal diseases; The detoxifcation of organism and heavy metal; The protection of cancer radiation and chemotherapy; To recovery after renal failure, uremia, diabetic complication, DPN, the surgical operation, tumour and acquired immune deficiency syndrome (AIDS) also is effective drug combination.
Gsh still is a kind of important biological anti-oxidant.In food-processing industry, that gsh not only can play at aspects such as meat product, milk preparation, flour products, infant foods is anti-oxidant, strengthen amino acid, strengthen flavour of food products, improve vital role such as food quality.The what is more important gsh is the perfect additive of preparation functional foodstuff.In developed country, be considered to one of most promising protective foods of 21 century, be called as the longevity factor and the anti-ageing factor.
For many years, the main dependence on import of the demand of gsh.Especially in developed country, gsh as biologically active additives, is widely used in the manufacture field of food except that being used for field of medicaments, and realizes commercialization production.Along with the development of China's medical health career and food-processing industry, the imbalance between supply and demand meeting is very outstanding.Producing glutathione thorugh biologic engineering method has more practical significance.
Background technology gsh preparation method has following several:
1. extraction process: mainly be the separation and Extraction of from the animal vegetable tissue that contains gsh, carrying out gsh by extraction and sedimentary method.
2. chemical synthesis: the production cycle is long, complicated operation, and separation difficulty, the cost height, and exist problems such as environmental pollution.
3. fermentation method: domestic do not have an industrialization manufacturing enterprise.
4. enzyme process: the present invention adopts the CGMCC1231 microorganism strains, the synthetic gsh of producing of enzymatic reaction.Have that technology is simple, environmental friendliness, with short production cycle, reaction temperature and, counter investment is few, the product purity height.
Summary of the invention the present invention adopts the CGMCC1231 bacterial strain enzymatic reaction synthesizing glutathion of stereospecificity, does not require substrate is synthesized thoroughly, and the reaction times is short, and partly substrate can reuse through processing for synthetic.
The CGMCC1231 bacterial strain that the present invention is used.Present accessible level is: produce 1～3 day enzymic fermentation time, a transformation efficiency of enzymatic reaction is 25～40%, and the extract yield of product is 45～58%, the content of gsh 〉=98%.
The CGMCC1231 microorganism strains that contains glutathione synthetase I (GSH-I) and glutathione synthetase II (GSH-II) that the purpose of this invention is to provide a kind of high yield provides a kind of method of new producing glutathione thorugh biologic engineering method.Resulting enzymatic reaction synthetic gsh, through extract refining after, the content of gsh 〉=98%.
The present invention realizes by following scheme:
(1) method of the present invention adopts the parental plant that the contriver provides, and again by specific mutagenesis, screening, optimization, sift out mutant strain again, can high yield contains the microbial mutation bacterial strain of glutathione synthetase I (GSH-I) and glutathione synthetase II (GSH-II).This bacterial strain is kept at Zhong Guan-cun, BeiJing, China Chinese microorganism strain on October 11st, 2004 and preserves management committee common micro-organisms preservation center, and preserving number CGMCC1231 is as the bacterial strain in enzyme source.
(2) bacterial strain is through slant activation, fermentation culture, with its wet thallus as the enzyme source.
(3) with L-L-glutamic acid, L-halfcystine, glycine substrate as enzymatic reaction.
(4) the enzyme source of (2) is added the substrate of (3), and add glucose and sal epsom carries out the enzymatic reaction synthesizing glutathion, through solvent extraction, concentrate, decolouring, make gsh.
Bacterial classification and mutagenesis thereof:
From the microorganism strains of contriver laboratory preservation, select an Accharomyces cerevisiae (Saccharomyces cerevisiae) E478C bacterial strain.Again this bacterial strain is carried out ultraviolet ray, Co according to a conventional method
60Mutagenic treatment.Promptly bacterial strain is made bacteria suspension and places culture dish, under aseptic and agitation condition with ultraviolet lamp 30cm apart from irradiation 1～5 minute; Dense bacterium liquid Co
60Irradiation, irradiation dose 5～120,000 roentgens, the time is 10～30 minutes.The aseptic technique of postradiation bacterium liquid is inoculated in the sudden change plate culture medium and cultivates after 3～7 days, transfers and cultivates 2～5 days in the screening plate culture medium, and choosing colony is optimized, and sifts out mutant strain again.
Through mutagenic obtained mutant strain: bacterium colony be ore deposit candle look, big and thick, moistening sticky, the surface is more smooth, provoke easily, the edge rounding; Cell is oval, and its monoploid and amphiploid can both be bred in the budding mode, sometimes long holidays mycelia; Circular ascus and thecaspore; Anaerobism bottom fermentation carbohydrate can produce ethanol.And called after yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) E6322.3C32.28 bacterial strain, compare the morphological specificity unanimity with parental plant.Increased by 25% but produce the bacterium amount, but the processing condition operating restraint is wide, adaptability is strong, and enzymatic reaction is stable.This bacterial strain is kept at Zhong Guan-cun, BeiJing, China Chinese microorganism strain on October 11st, 2004 and preserves management committee common micro-organisms preservation center, preserving number CGMCC1231.
The cultivation of bacterium producing multi enzyme preparation and fermentation:
Slant culture: preparation contains glucose 0.5～10%, extractum carnis 0.5～5.0%, and peptone 0.5～5%, agar 1.5～2.0%, PH5～9, each components contents of substratum is percent weight in volume, and promptly g/100ml is together following.Sterilized 20～50 minutes for 110～121 ℃, the sterilization postcooling, bevel, the aseptic technique inoculation was cultivated 3～7 days for 20～40 ℃.
The product enzyme is cultivated: preparation contains glucose 0.1～10%, peptone 0.5～5%, extractum carnis 0.5～5%, vitamin H 0.01～0.05%, corn steep liquor 0.5～5%, PH5～9, sterilized 20～50 minutes for 110～121 ℃, the sterilization postcooling, the aseptic technique inoculation, inoculum size is 5～10%, 20～40 ℃, 200～230r/min cultivated 1～3 day.
The mensuration of gsh enzymatic reaction vigor: get the 100mL fermented liquid, separating thallus, add the aqueous solution contain 4% glucose and be mixed with the substrate 60mL of L-L-glutamic acid, L-halfcystine, each 0.02mol/L concentration of glycine, put 22～25 ℃ of shaking tables, 200rpm carried out enzymatic reaction 30 minutes, and extracting solution detects glutathione content with ALLOXAN reagent method.The definition of enzyme activity unit: under these conditions, the enzyme amount of per minute enzymatic reaction synthesizing glutathion 1 μ mol amount is defined as 1 enzyme activity unit (U).
The ALLOXAN reagent method of gsh is measured: use 0.24mol/L ALLOXAN reagent; 0.1mol/L glycine solution; 0.24mol/L, PH7.6 phosphoric acid buffer and gsh example reaction made the gsh derivatize in 20 minutes, this moment with ultraviolet spectrophotometer in 305nm place mensuration absorbance.
Enzymatic is synthetic:
Thalline after the cultivation is as the enzyme source, with L-L-glutamic acid, L-halfcystine and glycine is substrate, concentration of substrate respectively is 0.01～1mol/L, glucose concn is 2～15%, sal epsom 0.02～0.1%, wet thallus is 5～20%, and the enzymatic reaction temperature is 20～40 ℃, and the enzymatic reaction generated time is 3～24 hours.The extracting solution that contains gsh of gained is measured with ALLOXAN reagent method and is shown that enzymatic reaction synthetic major ingredient is a gsh with this understanding.
The extraction of gsh and purifying:
Extracting solution is synthesized in enzymatic reaction, major ingredient is a gsh, contain unconverted amino acid substrate, also contain some inorganic salt impurity and pigments, can remove unconverted amino acid substrate with the appropriate solvent extraction earlier, isolate the gsh that has transformed, extract with the appropriate solvent extraction again, and carry out desalination and decolouring is handled or recrystallization method is made with extra care, thereby the high purity of obtaining, high-quality gsh product.
Major advantage of the present invention is the CGMCC1231 bacterial strain that a kind of energy of seed selection high yield contains gamma-glutamylcysteine synthetase (GSH-I) and glutathione synthetase (GSH-II), and the method for the simple producing glutathione thorugh biologic engineering method of a kind of technology is provided.Resulting enzymatic synthetic product gsh, through extract refining after, the content of gsh 〉=98%.
Description of drawings process flow diagram of the present invention is seen Figure of description.
The embodiment the following examples elaborate to the present invention
Embodiment 1:
Slant culture: substratum is extractum carnis 10g, peptone 10g, glucose 5g, agar 20g, add water to 1000ml, PH6.5,121 ℃ of sterilizations 30 minutes, the sterilization postcooling, aseptic technique inserts bacterial classification CGMCC1231, cultivates 5 days for 25 ℃, as the slant activation seed.
The product enzyme is cultivated: substratum is 0.3% glucose, 1.0% peptone, 0.5% extractum carnis, vitamin H 0.02%, 0.5% corn steep liquor, PH6.5, liquid amount is the bottled liquid 50mL of the triangle of 250mL, sterilized 30 minutes for 121 ℃, the sterilization postcooling, aseptic technique inserts the inclined-plane seed, put 28 ℃ of shaking tables, 200～230r/min cultivated 1～3 day, the centrifugal wet thallus that obtains.
Embodiment 2:
Enzymatic reaction liquid is with containing 0.02mol/L glycine, 0.02mol/LL-L-glutamic acid, 0.02mol/LL-halfcystine, 4% glucose, sal epsom 0.1%.Press 100g/L in the reaction solution, add the wet thallus of pressing the preparation of embodiment 1 method, put shaking table for 22～25 ℃, 200～230r/min, sampling is on time measured enzymatic synthesizing glutathion content with ALLOXAN reagent method, and the enzymatic reaction generated time is 3～24 hours.Repeat so repeatedly 3 times, obtain that enzymatic is synthetic to the results are shown in Table 2, table 2 is the result show, after enzymatic reaction in 3～72 hours was synthetic, the average conversion that obtains was 30.5%.After extraction, obtain crude product glutathione content 〉=90%.
Table 2 is the result of enzymatic conversion in batches repeatedly
| Transform number of times | 1 | 2 | 3 | Mean value |
| Transformation efficiency % | 30.3 | 28.7 | 32.5 | 30.5 |
| Enzyme U/g (dry mycelium) alive | 0.89 | 0.88 | 0.92 | 0.90 |
Embodiment 3:
Press the method for embodiment 2, preparation enzymatic reaction liquid, the method that adds by embodiment 1 prepares wet thallus, pressing the concentrated solution of 100mL glutathione content 0.09mol/L of the method enzymatic building-up reactions gained of embodiment 2, is 1: 2～4 alcohol extraction with volume ratio, the separating and extracting phase, ethanol is reclaimed in evaporation, obtain gsh crude product 1.77g, detect glutathione content 〉=90% with ALLOXAN reagent method, extraction yield is 58%.Crude product restrains with extraction using alcohol, decolouring, freezing and crystallizing, vacuum-drying, the pure product 1.36 that obtain white crystalline powder gsh, and the purified yield is 85%, glutathione content 〉=98%.
Embodiment 4:
Press embodiment 1 method, ferment tank with 15L produces enzyme, press the method for embodiment 2, preparation enzymatic reaction liquid, put the 15L jar and carry out enzymatic reaction, carry out product by embodiment 3 methods and extract, the result is: enzyme work reaches the 1.02U/g dry mycelium, the enzymatic reaction transformation efficiency is 35%, crude product glutathione content 〉=90%., extraction yield is 56%.
Claims (4)
1. microbial mutation bacterial strain that contains glutathione synthetase I (GSH-I) and glutathione synthetase II (GSH-II), it is the CGMCC1231 bacterial strain.
2. the method for a producing glutathione thorugh biologic engineering method comprises:
(1) with claims 1 described bacterial strain, to carry out routine and cultivate, fermentation obtains wet thallus as the enzyme source;
(2) with L-L-glutamic acid, L-halfcystine, glycine substrate as enzymatic reaction;
(3) wet thallus of (1) is joined the substrate enzymatic reaction synthesizing glutathion of (2).
3. according to claims 2 described methods, it is characterized in that: the concentration of substrate in the step (3) respectively is 0.01～1mol/L, glucose 2～15%, sal epsom 0.02～0.1%, adding wet thallus is 5～20%, the temperature of enzymatic reaction is 20～40 ℃, and the enzymatic reaction generated time is 3～24 hours.
4. according to claims 2 described methods, it is characterized in that the substratum that produces the enzymic fermentation cultivation is: glucose 0.1～10%, peptone 0.5～5.0%, vitamin H 0.01～0.05%, corn steep liquor 0.5～5.0%, extractum carnis 0.5～5%, PH5～9,20～40 ℃ of temperature were cultivated 1～3 day.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319244B (en) * | 2008-07-11 | 2011-05-04 | 重庆大学 | Method of preparing glutathione freeze-dried powder |
| CN101255454B (en) * | 2008-03-14 | 2011-07-20 | 华东理工大学 | Method for biosynthesis of glutathione by using yeast |
| CN105581344A (en) * | 2015-12-16 | 2016-05-18 | 开平牵牛生化制药有限公司 | Probiotic product containing reduced glutathione and preparation method of probiotic product |
| JP2022552488A (en) * | 2019-10-29 | 2022-12-16 | シージェイ チェイルジェダン コーポレーション | Glutathione-producing yeast strain and method for producing glutathione using the same |
| WO2023198006A1 (en) * | 2022-04-12 | 2023-10-19 | 元素驱动(杭州)生物科技有限公司 | Method for preparing s-lactoylglutathione |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MY128920A (en) * | 2000-05-25 | 2007-02-28 | Ajinomoto Kk | METHOD FOR PRODUCING y-GLUTAMYLCYSTEINE |
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2004
- 2004-10-17 CN CNB2004100793635A patent/CN1313597C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101255454B (en) * | 2008-03-14 | 2011-07-20 | 华东理工大学 | Method for biosynthesis of glutathione by using yeast |
| CN101319244B (en) * | 2008-07-11 | 2011-05-04 | 重庆大学 | Method of preparing glutathione freeze-dried powder |
| CN105581344A (en) * | 2015-12-16 | 2016-05-18 | 开平牵牛生化制药有限公司 | Probiotic product containing reduced glutathione and preparation method of probiotic product |
| JP2022552488A (en) * | 2019-10-29 | 2022-12-16 | シージェイ チェイルジェダン コーポレーション | Glutathione-producing yeast strain and method for producing glutathione using the same |
| JP7377353B2 (en) | 2019-10-29 | 2023-11-09 | シージェイ チェイルジェダン コーポレーション | Yeast strain that produces glutathione and method for producing glutathione using the same |
| WO2023198006A1 (en) * | 2022-04-12 | 2023-10-19 | 元素驱动(杭州)生物科技有限公司 | Method for preparing s-lactoylglutathione |
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