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

A technology of adenosylmethionine and glutathione, which can be applied in the field of bioengineering and can solve problems such as low yield

Active Publication Date: 2013-02-06
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 Biochemistry39 (2004), p1993-1997), but according to the report , the adenosylmethionine and glutathione obtained by 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 low. Therefore, it is necessary to provide A Novel Approach to Simultaneously High Yield of 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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Embodiment 1

[0044] Example 1 , Construction of a strain co-expressing glutathione synthetase and SAM synthetase

[0045] In this example, the plasmids for expressing glutathione synthase and SAM synthetase for Pichia pastoris or Saccharomyces cerevisiae were constructed respectively, and then the constructed plasmids were transformed into corresponding Pichia In yeast or Saccharomyces cerevisiae, a strain capable of co-expressing glutathione synthetase and SAM synthetase can be constructed in Pichia pastoris and Saccharomyces cerevisiae.

[0046] 1.1, GSH1, GSH2, SAM fragment amplification

[0047] In this embodiment, the primers for amplification are first designed, the genome as the amplification template is extracted, and then fragments of GSH1, GSH2 and SAM synthetase 2 are obtained by PCR respectively, as follows:

[0048] According to the GSH1 and GSH2 sequences reported by Genebank (EF633694, EF633695), the following 4 primers were designed to clone the GSH1 and GSH2 sequences:

[0049] GS...

Embodiment 2

[0139] Example 2 , HZ111 fermentation simultaneously or separately to produce glutathione and S-adenosylmethionine

[0140] Select a single colony of HZ111 grown for three days to inoculate 30ml of YPD medium, culture at 30℃, 240rpm for 20h, then insert 320ml of YPD medium, culture at 30℃, 240rpm for about 8h, insert 3.15L of fermentation medium BMGY (7.5L fermenter), add a final concentration of 0.01% defoamer (polyoxypropylene polyoxyethylene glyceryl ether, that is, foam enemy), carry out fermentation, control the temperature at 30 ℃, and add glycerin during the fermentation process to meet cell growth If necessary, the pH is controlled at 6.0 by adding ammonia. When the fermentation reaches 30h, add a total of 30g of L-cysteine ​​and a total of 30g of L-methionine, continue the fermentation, and perform a test at 70h. The test results show that the glutathion is in the fermentation broth. The content of glycosyl is 3.0g / L, and the content of S-adenosylmethionine is 3.5g / L. ...

Embodiment 3

[0142] Example 3 , HZ203 fermentation simultaneously or separately to produce glutathione and S-adenosylmethionine

[0143] The formula of the medium used in this example is as follows (g / L):

[0144] 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 O3.0×10 -3 , MnCl 2 ·4H 2 O0.3×10 -3 , CuSO 4 ·5H 2 O0.5×10 -3 , CaCl 2 ·2H 2 O1.0×10 -3 .

[0145] Pick a single colony of HZ203 that has grown for two days and inoculate it into 30ml of the above medium, culture at 30℃ and 240rpm for 20h, then insert 320ml of the above medium, 30℃, 240rpm for about 6h, insert 3.15L of the above fermentation culture In the base (7.5L fermenter), add the final concentration of 0.01% defoamer foam enemy, and carry out fermentation. The temperature is controlled at 30℃. During the fermentation process, galactose is added to meet the needs of cell growth. Ammonia is added and pH is controlled. In 6.0. Ad...

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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 Patents(China)
IPC IPC(8): C12N1/19C12P21/02C12P13/00C12R1/865C12R1/84
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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