阅读说明：本技术 一种生物工程法生产谷胱甘肽的方法 (Method for producing glutathione by bioengineering method ) 是由 许对兴 于 2019-11-20 设计创作，主要内容包括：本发明公开了一种生物工程法生产谷胱甘肽的方法,该生物工程法生产谷胱甘肽的方法如下：将GshI酶和GshII酶加入培养皿中进行产酶发酵的培养,将获得的突变酶株进行培养和发酵,将得到的酶菌作为反应的酶源,将酶源加入反应罐中,并加入L-谷氨酸、L-半肤氨酸及甘氨酸作为底物,进行酶促反应,反应得到谷胱甘肽的含有体,进行树脂吸附的工作,然后进行水洗,再进行酸解反应,将得到的谷胱甘肽通过干燥处理制备出成品的谷胱甘肽。通过对材料的质量以严格的比例进行,对ph值、温度也加以控制,使得得到的谷胱甘肽纯度可以达到96%以上,通过结晶干燥机进行快速的脱水干燥,得到高纯度的成品谷胱甘肽,提高了谷胱甘肽的产量以及缩短了周期时间。(The invention discloses a method for producing glutathione by a bioengineering method, which comprises the following steps: adding GshI enzyme and GshII enzyme into a culture dish for culture of enzyme production fermentation, culturing and fermenting the obtained mutant enzyme strain, taking the obtained enzyme strain as an enzyme source for reaction, adding the enzyme source into a reaction tank, adding L-glutamic acid, L-cysteine and glycine as substrates, carrying out enzymatic reaction to obtain a glutathione-containing body, carrying out resin adsorption, then washing with water, carrying out acidolysis reaction, and drying the obtained glutathione to prepare the finished product of glutathione. The quality of the material is strictly proportioned, the ph value and the temperature are also controlled, so that the purity of the obtained glutathione can reach over 96 percent, and the high-purity finished product glutathione is obtained by performing rapid dehydration and drying through a crystallization dryer, thereby improving the yield of the glutathione and shortening the cycle time.)

1. A method for producing glutathione by a bioengineering method is characterized by comprising the following steps: the method for producing glutathione by the bioengineering method comprises the following steps:

step one, adding GshI enzyme and GshII enzyme into a culture dish to regulate the ph value and the temperature, and carrying out enzyme production fermentation culture to obtain a mutant enzyme strain;

secondly, culturing and fermenting the obtained mutant enzyme strain to obtain a mould, and taking the enzyme strain as an enzyme source for reaction;

adding an enzyme source obtained by culture into a reaction tank, adding L-glutamic acid, L-cysteine and glycine as substrates, adding glucose and magnesium sulfate into the substrates, and carrying out enzymatic reaction in the reaction tank to obtain a glutathione-containing body;

fourthly, performing resin adsorption on the obtained glutathione-containing body, and washing the adsorbed glutathione-containing body with water;

fifthly, adding the glutathione-containing body after washing into acidolysis equipment for acidolysis reaction to obtain glutathione;

and sixthly, drying the obtained glutathione by a crystallization dryer to prepare the finished glutathione.

2. The method for producing glutathione by bioengineering according to claim 1, wherein: the culture dish contains 0.5-8% of glucose, 0.3-6% of peptone, 0.02-0.06% of biotin, 0.3-6% of corn steep liquor and 0.3-6% of beef extract, the pH value is adjusted to be about 6-10, the temperature needs to be controlled to be 25-45 ℃, and the culture time is 1-3 days, so that the mutant enzyme strain can be obtained.

3. The method for producing glutathione by a bioengineering method according to claim 1, wherein the method comprises the following steps: the culture and fermentation of the mutant enzyme strain are divided into two steps, the culture of the mutant enzyme strain is to prepare a culture medium containing 1 to 10 percent of glucose, 0.3 to 0.6 percent of beef extract, 0.3 to 6 percent of peptone and 1.6 to 2.2 percent of agar, the pH value is adjusted to be about 6 to 10, the contents of all components in the culture medium are weight volume percent, namely g/100ml, then the culture medium is sterilized and sterilized at 105 to 115 ℃ for 30 to 60min, the culture medium is cooled after the sterilization and is made into a slope, the inoculation is carried out in a sterile environment, the culture is carried out at 25 to 40 ℃ for 2 to 5 days,

then carrying out enzyme production culture, preparing 2-8% of glucose, 0.3-6% of peptone, 0.3-0.6% of beef extract, 0.02-0.06% of biotin and 0.3-0.6% of corn steep liquor, adjusting the pH value to about 6-10, sterilizing at 105-115 ℃ for 30-60 min, cooling after sterilization, inoculating in a sterile environment, and culturing at 25-40 ℃ for 2 days.

4. The method for producing glutathione by a bioengineering method according to claim 1, wherein the method comprises the following steps: the reaction tank is a container for chemical reaction, has the advantages of rapid heating, high temperature resistance, corrosion resistance, sanitation and no environmental pollution, and can automatically heat and control the temperature.

5. The method for producing glutathione by a bioengineering method according to claim 1, wherein the method comprises the following steps: the concentration of each content of the substrate in the reaction tank is 0.3-1mol/L, the glucose content is about 5% -15%, the magnesium sulfate is 0.03% -0.09%, the added enzyme source is 8% -22%, the temperature of the reaction tank is controlled to be 25 ℃ -45 ℃, the reaction time is about 5-18 hours,

a glutathione-containing product was obtained.

6. The method for producing glutathione by a bioengineering method according to claim 1, wherein the method comprises the following steps: the adsorption of the glutathione-containing body is carried out by using adsorption resin, and the adsorption resin refers to a class of high molecular polymers and can be used for removing organic matters in wastewater, decoloring sugar liquor, separating and refining natural products and biochemical products and the like.

7. The method for producing glutathione by a bioengineering method according to claim 1, wherein the method comprises the following steps: the acidolysis equipment is an air bubbling tank type reactor, nitric acid is used as a solvent, and acidolysis work is carried out on a glutathione-containing body, so that one of reaction products is an acid gas, and the other reaction product is glutathione solid-phase crystal.

8. The method for producing glutathione by a bioengineering method according to claim 1, wherein the method comprises the following steps: the drying process of the crystallization dryer is that the dried material is contacted with the inner wall of the container heated by the jacket for heat transfer and temperature rise, and the water is gasified and discharged outside after reaching the gasification point in vacuum, so that the material is quickly dehydrated and dried.

Technical Field

The invention relates to the technical field of glutathione preparation, in particular to a method for producing glutathione by a bioengineering method.

Background

Glutathione (GSH) is a non-protein sulfhydryl compound in cells, is a tripeptide with biological activity formed by condensing L-glutamic acid, L-cysteine and glycine, has free sulfhydryl and redox capability, is the most abundant antioxidant in cells, can protect DNA and protein from being damaged by ROS, is also involved in cell signal transduction, and is an essential substance for maintaining normal physiological activities of cells; meanwhile, the glutathione can also enhance the immunity of the organism and play a role in resisting viruses. In the food industry, the glutathione-containing dairy products and the glutathione-containing meat can improve the fresh-keeping capacity of the dairy products and the meat, prevent browning and keep color and nutrition. Glutathione is also used in the cosmetic industry, and can improve skin luster and relieve the phenomenon of increased skin wrinkles, so the demand of glutathione in the fields of domestic and foreign medical markets, foods and cosmetics is steadily increasing.

In the existing production process, the current main preparation methods of glutathione include: solvent extraction, chemical synthesis, biological fermentation and enzymatic methods. Glutathione is extracted from cereal germs, because glutathione has low yield, high cost, serious pollution of organic solvents, low purity and large consumption of grains, glutathione is synthesized by a chemical synthesis method, because active products are difficult to separate and need chemical separation, the product purity is low and difficult to popularize, glutathione production basically adopts a fermentation method, and the principle is to clone genes encoding glutathione synthetase systems into escherichia coli or yeast and use microorganisms for fermentation to produce glutathione. 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.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a method for producing glutathione by a biological engineering method.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for producing glutathione by a bioengineering method comprises the following steps:

step one, adding GshI enzyme and GshII enzyme into a culture dish to regulate the ph value and the temperature, and carrying out enzyme production fermentation culture to obtain a mutant enzyme strain;

secondly, culturing and fermenting the obtained mutant enzyme strain to obtain a mould, and taking the enzyme strain as an enzyme source for reaction;

adding an enzyme source obtained by culture into a reaction tank, adding L-glutamic acid, L-cysteine and glycine as substrates, adding glucose and magnesium sulfate into the substrates, and carrying out enzymatic reaction in the reaction tank to obtain a glutathione-containing body;

fourthly, performing resin adsorption on the obtained glutathione-containing body, and washing the adsorbed glutathione-containing body with water;

fifthly, adding the glutathione-containing body after washing into acidolysis equipment for acidolysis reaction to obtain glutathione;

and sixthly, drying the obtained glutathione by a crystallization dryer to prepare the finished glutathione.

Preferably, the culture dish contains 0.5-8% of glucose, 0.3-6% of peptone, 0.02-0.06% of biotin, 0.3-6% of corn steep liquor and 0.3-6% of beef extract, the pH value is adjusted to be about 6-10, the temperature needs to be controlled to be 25-45 ℃, and the culture time is 1-3 days, so that the mutant enzyme strain can be obtained.

Preferably, the culture and fermentation of the mutant enzyme strain are divided into two steps, the culture of the mutant enzyme strain is to prepare a culture medium containing 1 to 10 percent of glucose, 0.3 to 0.6 percent of beef extract, 0.3 to 6 percent of peptone and 1.6 to 2.2 percent of agar, the pH value is adjusted to be about 6 to 10, the contents of all components in the culture medium are weight volume percent, namely g/100ml, then the culture medium is sterilized, the sterilization is carried out at the temperature of between 105 and 115 ℃ for 30 to 60min, the culture medium is cooled after the sterilization, a slope is formed, the inoculation is carried out in the sterile environment, and the culture is carried out at the temperature of between 25 and 40 ℃ for 2 to 5 days.

Then carrying out enzyme production culture, preparing 2-8% of glucose, 0.3-6% of peptone, 0.3-0.6% of beef extract, 0.02-0.06% of biotin and 0.3-0.6% of corn steep liquor, adjusting the pH value to about 6-10, sterilizing at 105-115 ℃ for 30-60 min, cooling after sterilization, inoculating in a sterile environment, and culturing at 25-40 ℃ for 2 days.

Preferably, the reaction tank is a container for chemical reaction, has the advantages of rapid heating, high temperature resistance, corrosion resistance, sanitation and no environmental pollution, and can automatically heat and control the temperature.

Preferably, the respective concentration of the substrate in the reaction tank is 0.3 to 1mol/L, the glucose content is about 5 to 15%, the magnesium sulfate is 0.03 to 0.09%, the enzyme source is added in 8 to 22%, and the reaction tank is used to control the temperature to 25 ℃ to 45 ℃ for about 5 to 18 hours. A glutathione-containing product was obtained.

Preferably, the glutathione-containing body is adsorbed by using an adsorbent resin, which is a polymer and can be used for removing organic substances from wastewater, decoloring sugar solution, separating and refining natural products and biochemical products, and the like.

Preferably, the acidolysis equipment is an air bubbling tank reactor, and acidolysis is performed on the glutathione-containing body by using nitric acid as a solvent, so that one of reaction products is an acid gas, and the other is glutathione solid-phase crystals.

Preferably, the drying process of the crystallization dryer is that the dried material is contacted with the inner wall of the container heated by the jacket for heat transfer and temperature rise, and water is gasified and discharged out in vacuum after reaching the gasification point, so that the material is quickly dehydrated and dried.

The invention provides a method for producing glutathione by a bioengineering method, which comprises the steps of culturing and fermenting GshI enzyme and GshII enzyme to obtain mutant enzyme strains, fermenting and culturing the enzyme strains to obtain mould, performing a series of reaction culture processes, controlling the quality of materials used in the processes according to a strict proportion, controlling the pH value and the temperature, enabling the purity of the obtained glutathione to reach more than 96 percent, and performing rapid dehydration and drying by a crystallization dryer to obtain high-purity glutathione finished products, thereby improving the yield of the glutathione and shortening the cycle time.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.