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Method for preparing S-adenosylmethionine by enzyme method capable of eliminating key impurities

A technology for adenosylmethionine and enzymatic preparation, which is applied in the field of enzymatic preparation of S-adenosylmethionine, can solve the problems of increasing the cost of purification, difficulty in product separation, residual SAM decarboxylase, etc., and achieves separation Purification cost reduction effect

Pending Publication Date: 2022-04-05
WUXI FORTUNE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even the enzyme solution after multi-step purification may still have SAM decarboxylase residue, which leads to the unavoidable production of impurity 5'-adenosylmethionine in the process of enzymatic synthesis of SAM
Since the chemical structure of the impurity 5'-adenosylmethionine is very similar to the target product SAM, subsequent product separation becomes difficult, which greatly increases the cost of purification

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Knockout of the speD gene in Escherichia coli BL21 (DE3)

[0026] 1. Obtaining DNA fragments for knockout

[0027] First, the plasmid pKD3 (addgene Plasmid #45604) was used as a template, and the chloramphenicol gene was amplified by PCR using primers with the upstream and downstream sequences of the gene speD to be knocked out at the 5' end respectively. The primer sequences are: Upstream:

[0028] 5'-TTGAAAAAACTGAAACTGCATGGCTTTAATAATCTGACCAAAAGTCTGAGGTGTAGGCTGGAGCTGCTTC-3'

[0029] Downstream:

[0030] 5'-TTAAACAGCTGGCATATTGCGCCCGTAATAAATCTCGCGCATTTCTTTTCCCATATGAATATCTCCTTA-3'. After the amplified product is recovered and purified, it is sent for sequencing to verify the correctness of the sequence.

[0031] 2. Preparation of Escherichia coli electroporation competent cells

[0032] Transform the pKD46 plasmid into Escherichia coli BL21(DE3) by the calcium chloride method, inoculate the transformants in 5 mL of LB liquid medium containing 50 μg / mL ampi...

Embodiment 2

[0037] Example 2: Construction of speD gene-deleted bacteria expressing SAM synthetase gene

[0038] Firstly, using the E. coli BL21 (DE3) genome as a template, the metK gene was amplified using the following primers.

[0039] Upstream primers:

[0040] 5'-GGAATTCCATATGATGGCAAAACACCTTTTTAC-3', downstream primer: 5'-CCCAAGCTTTTACTTCAGACCGGCAGC-3'. After purification, the amplified product was ligated to Nde I and Hind III sites of plasmid pET-22b by restriction endonuclease digestion. The obtained recombinant plasmid was transformed into the Escherichia coli BL21(DE3) gene-deleted bacteria obtained in Example 1 by the calcium chloride transformation method, and the LB plate containing 100 μg / mL ampicillin was used for screening. The plasmids of the positive clones were extracted and submitted for sequencing. According to the sequencing results, it was confirmed that the speD gene-deficient strain expressing the SAM synthetase gene was successfully constructed.

Embodiment 3

[0041] Embodiment 3: the expression of SAM synthetase gene and the extraction of SAM synthetase

[0042]Inoculate the recombinant Escherichia coli finally obtained in Example 2 into LB liquid medium containing 100 μg / mL ampicillin, cultivate at 37°C until the OD600 reaches between 0.6-0.8, and then add isopropyl-β at a final concentration of 1 mM -D-methylthiogalactopyranoside (IPTG), continue culturing at 25°C for 12-16h. After the cultivation is complete, take 100mL of the bacterial solution and centrifuge at 8000r / min for 5min, discard the supernatant, resuspend in 10mL of deionized water, perform ultrasonication under low temperature conditions, and centrifuge at 8000r / min for 10min after ultrasonication, the supernatant is the crude enzyme liquid.

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Abstract

The invention discloses a method for preparing S-adenosylmethionine by an enzyme method capable of eliminating key impurities. The method comprises the following steps: step 1, constructing gene-deleted escherichia coli: knocking out a speD gene in escherichia coli BL21 (DE3) to obtain gene-deleted escherichia coli which does not generate S-adenosylmethionine decarboxylase; and 2, transforming the escherichia coli by using a recombinant plasmid containing a target gene: expressing the S-adenosylmethionine synthetase gene metK by using a gene deletion bacterium, culturing and inducing by using an inducer, and directly applying a crude enzyme liquid extracted after expression to enzyme reaction to produce S-adenosylmethionine. The method has the effect of greatly reducing the subsequent SAM purification cost.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to an enzymatic method for preparing S-adenosylmethionine which can eliminate key impurities. Background technique [0002] S-adenosyl-L-methionine (SAM, hereinafter referred to as SAM) is an important active intermediate that widely exists in organisms and participates in various biochemical reactions including transmethylation. As a biologically active substance, it has significant therapeutic effects on diseases such as liver injury and depression, so it has been widely used in clinical practice. In recent years, studies have also shown that it has health effects such as delaying aging, and has certain development potential in health food. [0003] At present, the main production method of SAM is the fermentation method, mainly using strains such as Saccharomyces cerevisiae, Bacillus amyloliquefaciens, and Pichia pastoris. However, the fermentation method has disadva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/40C12N15/70C12R1/19
Inventor 付静游云龙金丹甜史佳栋张谨成
Owner WUXI FORTUNE PHARMA
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