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Method for simultaneously producing glutathione and S-adenosyl methionine at high yield

A technology of adenosylmethionine and glutathione, which is applied in the field of bioengineering and can solve the problem of low yield

Active Publication Date: 2010-03-24
浙江拜克生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, Liu Hui et al. have also reported using S. cerevisiae strain zju1 to simultaneously ferment glutathione and adenosylmethionine (H. Liu et al. Process Biochemistry 39 (2004), p1993-1997), but according to the literature It is reported that the adenosylmethionine and glutathione obtained by its fermentation are 45mg / g dry weight and 18mg / g dry weight respectively, that is, 1.35g / L and 0.54g / L, and the yield is relatively low. Therefore, it is necessary to Provides a new method to simultaneously high-yield glutathione and S-adenosylmethionine

Method used

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  • Method for simultaneously producing glutathione and S-adenosyl methionine at high yield
  • Method for simultaneously producing glutathione and S-adenosyl methionine at high yield
  • Method for simultaneously producing glutathione and S-adenosyl methionine at high yield

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1 1. Construction of strains co-expressing glutathione synthetase and SAM synthetase

[0044] In this example, plasmids for expressing glutathione synthetase and SAM synthetase for Pichia or Saccharomyces cerevisiae were first constructed, and then the above-mentioned plasmids that had been constructed were transformed into corresponding Pichia Yeast or Saccharomyces cerevisiae, so as to construct strains that can co-express glutathione synthetase and SAM synthetase in Pichia pastoris and Saccharomyces cerevisiae.

[0045] 1.1, GSH1, GSH2, SAM fragment amplification

[0046] In this example, primers for amplification were first designed, the genome as the amplification template was extracted, and then the fragments of GSH1, GSH2 and SAM synthetase 2 were respectively amplified by PCR, as follows:

[0047] According to the GSH1 and GSH2 sequences reported by Genebank (EF633694, EF633695), the following 4 primers were designed for cloning the GSH1 and GSH2 sequ...

Embodiment 2

[0138] Example 2 , HZ111 fermentation to produce glutathione and S-adenosylmethionine simultaneously or separately

[0139] Select a single colony of HZ111 that has grown for three days and inoculate it into 30ml of YPD medium, culture it at 30°C and 240rpm for 20h, then insert it into 320ml of YPD medium, cultivate it at 30°C and 240rpm for about 8h, and insert it into 3.15L of fermentation medium BMGY (7.5L fermenter), add a final concentration of 0.01% defoamer (polyoxypropylene polyoxyethylene glyceryl ether, i.e. foam enemy), and ferment, the temperature is controlled at 30 ° C, and glycerin is added during the fermentation process to meet the needs of cell growth Need, by adding ammonia water, the pH is controlled at 6.0. Adding a total amount of 30 grams of L-cysteine ​​and a total amount of 30 grams of L-methionine to 30 hours of fermentation continued to ferment, and the fermentation was carried out at 70 hours. The test results showed that glutathione in the fermen...

Embodiment 3

[0141] Example 3 , HZ203 fermentation to produce glutathione and S-adenosylmethionine simultaneously or separately

[0142] The formula of the medium used in the present embodiment is as follows (g / L):

[0143] Galactose 40, yeast extract 20, peptone 40, ammonium sulfate 2.0, urea 2.0, KH 2 PO 4 1.5, MgSO 4 ·7H 2 O0.5, ZnSO 4 ·7H 2 O 4.0×10 -3 , FeSO 4 ·7H 2 O 3.0×10 -3 , MnCl 2 4H 2 O 0.3×10 -3 , CuSO 4 ·5H 2 O0.5×10 -3 , CaCl 2 2H 2 O 1.0×10 -3 .

[0144] Pick a single colony of HZ203 that has grown for two days and inoculate it into 30ml of the above-mentioned medium, culture at 30°C and 240rpm for 20h, then insert 320ml of the above-mentioned medium, cultivate at 30°C and 240rpm for about 6h, and insert 3.15L of the above-mentioned fermentation culture In the base (7.5L fermenter), add 0.01% final concentration of antifoaming agent foam enemy, carry out fermentation, the temperature is controlled at 30 ℃, add galactose during the fermentation process, ...

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Abstract

The invention provides a recombined strain capable of simultaneously compressing glutathione synthetase system and adenosyl methionine synthetase and a method for simultaneously producing glutathione and S-adenosyl methionine at high yield. The recombined strain comprises: fragment capable of exogenously expressing gama-glutamyl cysteine synthetase or glutathione synthetase and fragment capable of exogenously expressing adenosyl methionine synthetase and the strain is capable of expressing glutathione synthetase system and / or adenosyl methionine synthetase, thereby fermenting glutathione and adenosyl methionine. The recombined strain is capable of simultaneously producing glutathione and adenosyl methionine by fermentation at high yield and independently producing glutathione or adenosyl methionine by fermentation.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for simultaneously high-yielding glutathione and S-adenosylmethionine. Background technique [0002] Glutathione, namely γ-L-glutamyl-L-cysteinyl-glycine, referred to as GSH. It is composed of precursor substances L-glutamic acid, L-cysteine ​​and glycine through γ-glutamylcysteine ​​synthetase (GSH I, GSH1) and glutathione synthase (GSH II, GSH2) Catalytic synthesis, the two constitute the glutathione synthetase system, of which γ-glutamylcysteine ​​synthetase is considered to be the rate-limiting enzyme in the GSH synthesis pathway. GSH can effectively remove free radicals in the human body, purify the environmental pollution in the human body, and improve human health. It has broad application prospects in clinical medicine, food industry and related biological research fields. Its industrial production mainly includes extraction method, chemical synthesis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P21/02C12P13/00C12R1/84C12R1/865
Inventor 杨晟杨俊杰范文超陶荣盛曹传增沈正权胡传峰程跃孟松
Owner 浙江拜克生物科技有限公司
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