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Biosynthesis method of ademetionine

A technique for the synthesis of adenosylmethionine and its synthesis method, which is applied in the field of biosynthesis of adenosylmethionine by beneficial engineering bacteria, which can solve the problems of high price and achieve the effects of reducing the cost of SAM synthesis, easy separation and operation, and large-scale synthesis

Inactive Publication Date: 2014-08-20
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limited production technology level, the price of SAM is still relatively expensive at pres

Method used

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  • Biosynthesis method of ademetionine
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  • Biosynthesis method of ademetionine

Examples

Experimental program
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Example Embodiment

[0022] Example 1 Construction of engineering bacteria

[0023] The specific construction method of SAM engineering bacteria containing Saccharomyces cerevisiae SAM synthetase gene 2 (sam2) used in the present invention is as follows (preparation of Actinomucor elegans protoplasts, cloning of target genes, restriction enzyme digestion verification, recombinant plasmid construction and transformation, protoplasts The specific conditions and methods of regeneration and transformant screening are detailed in the paper "Cloning and Expression of the Key Enzyme Gene APT1 for ATP Synthesis in Actinomucor elegans" (Zhu Jiarong, Yang Shanyan, Chen Lifen, etc. Food and Fermentation Industry, 2012, 38(6) ,43-47.):

[0024] 1. Experimental materials

[0025] Actinomucor elegans ZGB1 has been disclosed in patent CN102154117A.

[0026] E.coli DH5α was purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.;

[0027] Saccharomyces cerevisiae 1964 (Saccharomyces cerevisiae1964) was purchased fro...

Example Embodiment

[0062] Example 2 SAM engineering bacteria culture collection and activation treatment

[0063] Liquid seed medium (g / L): glucose 20, peptone 10, yeast extract 5, NaCl5, solvent is water, pH 6.0-6.2; (slant medium plus agar 20).

[0064] Fermentation medium (g / L): glucose 40, yeast extract 3, corn steep liquor 10, NH 4 Cl3, MgSO 4 ·7H 2 O1, FeSO 4 ·7H 2 O0.2, K 2 HPO 4 ·3H 2 O3, the solvent is water, pH 6.0-6.5.

[0065] Inoculate the strains of SAM engineered bacteria in a slant medium (test tube or eggplant-shaped bottle), culture at 28-30°C for 3-4 days; wash the spores with an appropriate amount of liquid seed medium to obtain a spore suspension, and then use the spore suspension to Inoculate a 10% volume concentration inoculum into a 250mL Erlenmeyer flask containing the above-mentioned 100mL seed culture medium, 150-220r / min shaker, 28-30°C for 10-14h, to obtain a seed liquid; The inoculum is inoculated into a 500mL Erlenmeyer flask containing the above 300mL bacterial culture ...

Example Embodiment

[0067] Example 3 Transformation of SAM engineering bacteria into SAM

[0068] The activated bacteria are put into the following SAM reaction solution (g / L) in proportion: glucose 80, K 2 HPO 4 ·3H 2 O70, MgSO 4 ·7H 2 O4, adenine 3, L-methionine 4, active cell 80 (prepared by the method in Example 2), and the balance is water. The reaction was terminated at 33-37° C., 160r / min shaker for 10-14 hours, and liquid chromatography (the detection conditions and methods described in Example 1) determined that the yield of SAM in the reaction-terminated clear liquid was 1.0 g / L.

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Abstract

The invention discloses a biosynthesis method of ademetionine. The method comprises the following steps: subjecting ademetionine engineering bacteria to fermentation culture so as to obtain fermentation broth, filtering the fermentation broth, drying the wet bacteria for 2 to 3 hours at a temperature of 60 to 65 DEG C, then activating the dried bacteria, adding adenine, glucose, phosphate, sulfate, methionine and water into the obtained activated bacteria to form a reaction system, and carrying out conversion reactions at a temperature of 33 to 37 DEG C, when the conversion reactions finish, the reaction liquid is a mixed solution containing ademetionine. An SAM engineering bacterium is constructed, and can add methionine in a reaction liquid of an ATP synthesis reaction through adenine, so the steps of SAM synthesis enzyme culture, SAM synthesis enzyme extraction, and SAM synthesis enzyme addition during the reaction process are saved, and the production is more convenient. Moreover, the raw materials such as adenine can be bought from the market at a low price in a large amount. The biosynthesis method of ademetionine can efficiently synthesize SAM in a large scale.

Description

(1) Technical field [0001] The invention relates to a method for synthesizing adenosylmethionine, in particular to a method for biosynthesizing adenosylmethionine with beneficial engineering bacteria. (2) Background technology [0002] S-adenosylmethionine (S-adenosyl-L-methionine, SAM), also known as S-adenosylmethionine, mainly plays the role of transmethylation, transsulfuration and transaminopropyl in organisms, and participates in many important It has a variety of therapeutic effects, and has good curative effects on depression, arthritis, liver and gallbladder diseases, pancreatitis, etc. It has been prescribed in Europe for depression and arthritis. From 1999 when the US FDA approved it as a food nutritional supplement to the present, the efficacy of SAM has gradually become known to the public. Due to the limited level of production technology, the price of SAM is relatively expensive at present, so research and development of low-cost SAM production technology st...

Claims

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Application Information

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IPC IPC(8): C12P19/40C12R1/865C12R1/645
Inventor 朱家荣郑计瑞陈丽芬杨善岩
Owner ZHEJIANG UNIV OF TECH
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