Method for preparing immobilized adenosylmethionine synthetase and adenosylmethionine

A technology for adenosylmethionine and synthetase, which is applied in the field of preparation of immobilized adenosylmethionine synthetase, can solve the problems of various SAM extraction and purification processes, low conversion rate of L-methionine, and SAM solution composition Complexity and other problems, to achieve the effect of excellent continuous operation performance, simplify the subsequent separation process, and facilitate continuous production

Inactive Publication Date: 2011-03-16
江苏中丽新材料有限公司 +1
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Problems solved by technology

Appling et al constructed a chimeric gene consisting of methylenetetrahydrofolate reductase and adenosylmethionine synthetase, transformed Saccharomyces cerevisiae to express SAM, and increased the conversion rate of L-methionine to 40% (Appling ; Dean R.United States Patent: 7,033,815); Although the technology of fermentative production of SAM is constantly improving, there are several inherent and difficult problems in the fermentative process: (1). The conversion rate of substrate L-methionine Low; (2). The fermentation product is a mixture of (-) SAM and (+) SAM, while (-) SAM with biological activity only accounts for about 70%,

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Embodiment Construction

[0028] Now in conjunction with embodiment the present invention will be further described:

[0029] Materials and methods for the examples:

[0030] 1. Cells and reagents:

[0031] Escherichia coli K12, Escherichia coli JM109, and Escherichia coli expression plasmid pBR322 are all preserved in our laboratory, and can also be purchased; the plasmid DNA extraction kit and DNA gel recovery kit were purchased from Beijing Biotech Company. T4 DNA ligase, Taq DNA polymerase, various restriction enzymes, and Protein Marker were purchased from Takara Company. Phenyl-Sepharose Fast Flow prepacked column was purchased from GE Company, adenosylmethionine standard was purchased from Sigma Company, the amino resin carrier Seplite LX-1000HA used to immobilize enzymes was purchased from Xi’an Lanxiao Technology Co., Ltd., and the rest of the reagents All are domestic or imported analytically pure.

[0032] Escherichia coli engineering bacteria carry the gene encoding wild-type Escherichia...

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Abstract

The invention relates to a method for preparing immobilized adenosylmethionine synthetase and adenosylmethionine. The method has the following technical characteristics of: crosslinking amino resin carrier Seplite LX-1000HA and glutaraldehyde solution; and reacting the amino resin carrier crosslinked with glutaraldehyde with solution of recombinant adenosylmethionine synthetase to obtain the immobilized adenosylmethionine synthetase. The amino resin carrier has high protein fixation rate, the recovery rate of the enzyme activity of the immobilized adenosylmethionine synthetase is high, and the stability and continuous operation performance are good. When the immobilized adenosylmethionine synthetase is used for producing the adenosylmethionine, the reaction operation is simple, the production cycle is short, and products are easy to purify; the immobilized enzyme can be recycled, the production cost is greatly reduced, the production efficiency is improved, and a new way is provided for industrialized production of the adenosylmethionine.

Description

technical field [0001] The invention relates to a method for preparing immobilized adenosylmethionine synthetase, and a method for preparing adenosylmethionine by using the prepared immobilized adenosylmethionine synthetase, involving enzyme immobilization and the use of immobilized The technical field of enzyme-catalyzed synthesis of high-value compounds. Background technique [0002] S-adenosylmethionine (S-adenosylmethionine, SAM) is an important metabolic intermediate widely present in organisms, first discovered by Cantoni in 1952, with a relative molecular mass of 399. SAM is a pair of chiral substances, with two isomers: (R, S)-SAM and (S, S)-SAM, only (S, S)-SAM has biological activity. In organisms, SAM is synthesized by L-methionine (L-Met) and adenosine triphosphate (ATP) catalyzed by adenosylmethionine synthetase. Because SAM contains a high-energy sulfonium atom that can activate the nucleophilic attack reaction of adjacent carbon atoms, it has the effects of ...

Claims

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Application Information

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IPC IPC(8): C12N11/02C12P19/40
CPCY02P20/50
Inventor 牛卫宁钦传光徐洪杰曹珊珊尚晓娅徐春兰
Owner 江苏中丽新材料有限公司
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