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Method for improving SAM synthetase expression level

A technology for expression and synthesis of enzymes, which is applied in the field of biopharmaceuticals, can solve the problems of low enzyme activity, low content, and difficulty in separation and purification, and achieve the effect of increasing expression

Active Publication Date: 2010-10-27
BEIJING KAWIN TECH SHARE HLDG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Adenosylmethionine synthase, also known as adenosylmethionine synthase, exists widely in animals, plants and microorganisms. Later, some people isolated adenosylmethionine synthase from Escherichia coli and yeast and used it for the preparation of adenosylmethionine. However, adenosylmethionine synthetase has less content in animals, plants and microorganisms, and the enzyme activity is not high, and it is difficult to separate and purify. For example, 400g yeast stem cells can only be separated and purified to obtain 8U adenosylmethionine synthetase, and the specific activity is 0.05U / mg

Method used

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  • Method for improving SAM synthetase expression level
  • Method for improving SAM synthetase expression level
  • Method for improving SAM synthetase expression level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 Fermentation experiments without adding divalent metal ions and flocculants

[0043] 1. Preparation of fermentation broth:

[0044] Prepare seed medium, fermentation medium and feed medium respectively according to the following formulas:

[0045] (1) Seed medium (LB medium): peptone: yeast powder: sodium chloride = 2:1:2

[0046] (2) Fermentation basal medium (g / 100ml medium):

[0047] Glucose: 1.47 Peptone: 0.4

[0048] Yeast powder: 0.55 Potassium dihydrogen phosphate: 0.4

[0049] Dipotassium hydrogen phosphate: 0.65 Ammonium sulfate: 0.047

[0050] Ammonium chloride: 0.01 Ferrous sulfate: 0.002

[0051] Manganese sulfate: 0.0007

[0052] 200mg / ml Oxytetracycline: 0.0125 (V / V)

[0053] (3) Feed medium (g / 100ml medium):

[0054] Glucose: 1.12 Yeast powder: 0.55

[0055] Magnesium sulfate: 0.035

[0056] 2. Fermentation process:

[0057] (1) Insert the glycerol species containing the recombinant bacteria into the primary seed medium (LB medium)...

Embodiment 2

[0066] (7) Stop the fermentation after 8.5 hours, take a sample and measure the expression level of SAM synthetase in it by SDS-PAGE polyacrylamide electrophoresis, and determine that the expression level of the target enzyme accounts for 30.8% of the soluble protein of the bacteria. Embodiment 2 adds Mg 2+ Fermentation experiment with flocculant

[0067] 1. Preparation of fermentation broth:

[0068] Prepare seed medium, fermentation medium and feed medium respectively according to the following formulas:

[0069] (1) Seed medium (LB medium) (%): peptone: yeast powder: sodium chloride = 2:1:2

[0070] (2) Fermentation basal medium (g / 100ml medium):

[0071] Glucose: 1.47 Peptone: 0.4

[0072] Yeast powder: 0.55 Potassium dihydrogen phosphate: 0.4

[0073] Dipotassium hydrogen phosphate: 0.3 Disodium hydrogen phosphate: 0.35

[0074] Ammonium sulfate: 0.047 Magnesium sulfate: 0.05

[0075] Ammonium chloride: 0.01 Ferrous sulfate: 0.002

[0076] Calcium chloride: 0.002 ...

Embodiment 3

[0090] Embodiment 3 adds Zn 2+ Fermentation experiment with flocculant

[0091] Repeat the steps of Example 2, the difference is that in the fermentation basal medium, the added Zn 2+ The amount of flocculant added is 0.15g / 100ml medium, the amount of flocculant disodium hydrogen phosphate added is 0.2g / 100ml medium, the amount of flocculant calcium chloride added is 0.001g / 100ml medium, other conditions are the same .

[0092] Stop fermentation after 8.5 hours, stop fermentation after 8.5 hours, take a sample and measure the expression level of SAM synthetase wherein with SDS-PAGE polyacrylamide electrophoresis, measure the expression level of target enzyme to account for 36.7% of thalline soluble protein, relative embodiment 1 ( Without adding this divalent metal ion), the expression level of SAM synthetase increased by 5.9%; the turbidity after 8 hours was 12.2, which decreased by 6.5.

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Abstract

The invention provides a method for preparing an ademetionine synthetase in large scale. In the method, an SAM expression level of engineering bacteria in fermentation liquor is improved, and simultaneously the turbidity of the fermentation liquor can be reduced so as to bring more contribution to further extraction and preparation of an enzyme by selectively adding divalent metal ions and a flocculating agent.

Description

technical field [0001] The invention relates to a method for increasing the expression of SAM synthetase, which belongs to the technical field of biopharmaceuticals. Background technique [0002] China is a country with a large population, but also a "big hepatitis country". According to the national viral hepatitis epidemiological survey in 1992, about 120 million people in the country carry hepatitis B virus, and the infection rate of hepatitis C in the general population is 3.1%. There are currently 20 million chronic hepatitis B patients in my country, some of whom may transform into liver cirrhosis or even liver cancer. [0003] Adenosylmethionine (SAM) is an extremely important intermediate metabolite in the human body. It can generate the key antioxidant and detoxification substance in the body-cysteine ​​through the transsulfurization pathway, and regenerate another key antioxidant and detoxification substance-glutathione to relieve the oxidative stress state in th...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12R1/19
Inventor 熊国裕吉春周德胜
Owner BEIJING KAWIN TECH SHARE HLDG
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