阅读说明：本技术 一种利用酵母全细胞催化合成三磷酸腺苷的方法 (Method for catalytically synthesizing adenosine triphosphate by using yeast whole cell ) 是由 汤亦文 陈晓春 张磊 李梓阳 蔡家栋 于 2021-04-23 设计创作，主要内容包括：本发明公开了一种利用酵母全细胞催化合成三磷酸腺苷的方法,将酵母接种到含解偶联剂的培养基中,催化合成培养,即得。本发明通过添加解偶联剂进行催化合成,单批催化合成时间由7h缩短至5h,产量由20.5g/L提高至38.6g/mL。该方法可以显著的缩短催化合成工时提高催化合成产量,所用的催化合成促进剂添加量少,成本低,操作简单,效果明显,减少生产成本。(The invention discloses a method for catalytically synthesizing adenosine triphosphate by using yeast whole cells, which comprises the steps of inoculating yeast into a culture medium containing a uncoupler, and carrying out catalytic synthesis culture. According to the invention, the catalytic synthesis is carried out by adding the uncoupler, the single-batch catalytic synthesis time is shortened from 7h to 5h, and the yield is improved from 20.5g/L to 38.6 g/mL. The method can obviously shorten the working hour of catalytic synthesis and improve the yield of catalytic synthesis, and the used catalytic synthesis accelerator has the advantages of small addition amount, low cost, simple operation, obvious effect and production cost reduction.)

1. A method for catalytically synthesizing adenosine triphosphate by using yeast whole cells is characterized in that yeast is inoculated into a culture medium containing a uncoupler and subjected to catalytic synthesis culture to obtain the adenosine triphosphate.

2. The method of claim 1, wherein the decoupling agent is any one or more of 2, 4-dinitrophenol, carbonyl cyanide-m-chlorophenyl, carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone, valinomycin, ethanol, and salicylic acid.

3. The method of claim 1, wherein the concentration of the coupling agent is 0.1-20g/L of the culture medium.

4. The method of claim 1, wherein the culture medium further comprises the following components: 0.5-50g/L glucose, 5-50g/L adenosine, 1-40g/L potassium dihydrogen phosphate, 1-40g/L dipotassium hydrogen phosphate, 0.1-20g/L ammonium sulfate, 0.1-20g/L magnesium chloride and 0.1-20g/L sodium chloride.

5. The method of claim 1, wherein the solvent of the culture medium is water.

6. The method of claim 1, wherein the pH of the medium is 5.0 to 8.0.

7. The method of claim 1, wherein the yeast is Saccharomyces cerevisiae.

8. The method of claim 1, wherein the amount of yeast inoculated is 20 to 300g/L of medium.

9. The method of claim 1, wherein the catalytic synthesis is cultured at a temperature of 38-40 ℃.

10. The method of claim 1, wherein the catalytic synthesis is cultured for 4-10 hours.

Technical Field

The invention belongs to the field of microbial catalytic synthesis, and particularly relates to a method for synthesizing adenosine triphosphate by using yeast whole cell catalysis.

Background

Adenosine triphosphate is an important functional substance, and ATP plays a role of a metabolic activity center in various metabolic processes of a living body; is a direct supplier of energy required for various physiological and biochemical activities (such as muscle contraction, body movement, cardiac muscle pulsation, respiration, nerve conduction, synthesis of macromolecular substances by small molecules, etc.) in the body. The production and supplementation of CTP, GTP and UTP all depend on ATP. ATP is called "universal currency" for intracellular energy. ATP is used as the most important energy donor and clinically used for treating and assisting in treating heart, brain and vascular diseases and inflammation caused by a series of functions and metabolism.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of the prior art and provides a method for synthesizing adenosine triphosphate by using yeast whole cell catalysis.

In order to solve the technical problems, the invention discloses a method for catalytically synthesizing adenosine triphosphate by using yeast whole cells, which comprises the steps of inoculating yeast into a culture medium containing a decoupling agent, and carrying out catalytic synthesis culture.

Wherein the uncoupler is any one or a combination of several of 2, 4-Dinitrophenol (DNP), carbonyl cyanide-m-chlorphenyl (CCCP), carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP), valinomycin, ethanol and salicylic acid; preferably, the decoupling agent is any one or two combination of DNP, CCCP, and FCCP; further preferably, the decoupling agent is DNP, or a combination of DNP with either or both of CCCP and FCCP.

Wherein the concentration of the uncoupler is 0.1-20g/L of the culture medium; preferably, the concentration of the coupling agent is 1-10g/L of the culture medium; further preferably, the concentration of the coupling agent is 1-8g/L of the culture medium.

Wherein, the culture medium also comprises the following components: 0.5-50g/L glucose, 5-50g/L adenosine, 1-40g/L potassium dihydrogen phosphate, 1-40g/L dipotassium hydrogen phosphate, 0.1-20g/L ammonium sulfate, 0.1-20g/L magnesium chloride and 0.1-20g/L sodium chloride; preferably, the culture medium comprises the following components: 40g/L glucose, 40g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride and 5g/L sodium chloride.

Wherein the solvent of the culture medium is water.

Wherein the pH of the culture medium is 5.0-8.0.

Wherein the yeast is saccharomyces cerevisiae; wherein, the saccharomyces cerevisiae in the prior art can be applied to the invention; preferably, the yeast is the saccharomyces cerevisiae deposited by the company with the preservation number of CGMCC No.7522, which is disclosed in Chinese patent CN 201410057913.7: saccharomyces cerevisiae for producing ribonucleic acid by fermentation and application thereof are provided.

Wherein the inoculation amount of the yeast is 20-300g/L of culture medium; preferably, the inoculation amount of the yeast is 100-300g/L of culture medium; further preferably, the inoculation amount of the yeast is 200-300g/L of the culture medium; still more preferably, the yeast is inoculated in an amount of 285g/L of medium.

Wherein the temperature of the catalytic synthesis culture is 38-40 ℃.

Wherein the time of the catalytic synthesis culture is 4-10 h; preferably, the time of the catalytic synthesis culture is 7 h.

Has the advantages that: compared with the prior art, the invention has the following advantages:

according to the invention, the catalytic synthesis is carried out by adding the uncoupler, the single-batch catalytic synthesis time is shortened from 7h to 5h, and the yield is improved from 20.5g/L to 38.6 g/mL. The method can obviously shorten the working hour of catalytic synthesis and improve the yield of catalytic synthesis, and the used catalytic synthesis accelerator has the advantages of small addition amount, low cost, simple operation, obvious effect and production cost reduction.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

The yeast used in the following examples is Saccharomyces cerevisiae (CGMCC No. 7522).

Example 1.1

Preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 3.0g/L DNP, water as a solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 33g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Comparative example 1

Preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, water as solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 20.5g/L after catalytic synthesis is carried out for 7 hours in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 1.2

Preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 3.0g/L CCCP, water as solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 25g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 1.3

Preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 3.0g/L FCCP, water as solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 22g/L after catalytic synthesis is carried out for 5 hours in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 2.1

Preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 1.5g/L DNP and 1.5g/L CCCP solvent are water, and the pH value is 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 38g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 2.2

Preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 1.5g/L DNP, 1.5g/L FCCP solvent is water, and the pH value is 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 35g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 2.3

Preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 1.5g/L CCCP, 1.5g/L FCCP solvent is water, and the pH value is 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 23g/L after catalytic synthesis is carried out for 5 hours in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 3

Preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 1.0g/L DNP, 1.0g/L CCCP, 1.0g/L FCCP solvent is water, and the pH value is 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 34g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 4

Preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 2.0g/L DNP, 2.0g/L CCCP, 2.0g/L FCCP solvent is water, and the pH value is 6.8.

Chemical synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 37g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 5.1

Preparing a catalytic synthesis culture medium: 30g/L glucose, 30g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 1.5g/L DNP, 1.5g/L CCCP, water as solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 42g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 5.2

Preparing a catalytic synthesis culture medium: 40g/L glucose, 40g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 3g/L DNP, 1.5g/L CCCP, water as solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 50g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 6

Preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 2.0g/L DNP, 2.0g/L CCCP, water as solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 38g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 7:

preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 3.0g/L DNP, 1.0g/L CCCP, water as solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 37g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 8:

preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 1.0g/L valinomycin, water as solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 23g/L after catalytic synthesis is carried out for 5 hours in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 9:

preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 1.0g/L ethanol, water as solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 22g/L after catalytic synthesis is carried out for 5 hours in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 10:

preparing a catalytic synthesis culture medium: 20g/L of glucose, 20g/L of adenosine, 15g/L of monopotassium phosphate, 5g/L of dipotassium phosphate, 2g/L of ammonium sulfate, 2g/L of magnesium chloride, 5g/L of sodium chloride, 1.0g/L of salicylic acid, water as a solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 23g/L after catalytic synthesis is carried out for 5 hours in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

Example 11:

preparing a catalytic synthesis culture medium: 20g/L glucose, 20g/L adenosine, 15g/L potassium dihydrogen phosphate, 5g/L dipotassium hydrogen phosphate, 2g/L ammonium sulfate, 2g/L magnesium chloride, 5g/L sodium chloride, 1.0g/L DNP, water as a solvent and pH 6.8.

Catalytic synthesis: and 6L of culture medium is filled into a 10L stirring tank, 285g/L of saccharomyces cerevisiae is inoculated for catalytic synthesis culture, the yield is stabilized at 28g/L after 5 hours of catalytic synthesis in the catalytic synthesis tank at the temperature of 38 ℃, and the catalytic synthesis is stopped.

As can be seen from the results of the above examples, the catalytic synthesis of the invention by adding the uncoupler substance shortens the single-batch catalytic synthesis time from 7h to 5h, and improves the yield from 20.5g/L to 38 g/L. The method can obviously shorten the catalytic synthesis working hours and improve the catalytic synthesis yield, and has the advantages of small addition amount of the used accelerant, low cost, simple operation, obvious effect and production cost reduction.

The present invention provides a method and a concept for a method for synthesizing adenosine triphosphate by yeast whole cell catalysis, and a method and a way for implementing the technical scheme are many, the above description is only a preferred embodiment of the present invention, it should be noted that, for those skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.