S-adenosyl-L-methionine synzyme mutant and preparation method using same

A technology for adenosylmethionine and methionine, which is applied in the field of S-adenosylmethionine synthetase mutants and its preparation, can solve the problems of low product concentration, inability to scale up applications, and product Inhibition of large problems, to achieve the effect of low enzyme activity decline, high specific activity, and small product inhibition

Active Publication Date: 2020-06-05
HUNAN FLAG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, it has been found in industrial production applications that up to now, both the wild-type SAM synthetase and the reported mutant modified enzymes generally have low enzyme specific activity, high product inhibition, poor stability, and low product concentration. , high production costs and other defects, resulting in the inability to scale applications, so it is urgent to develop a SAM synthetase mutant with higher catalytic activity, stronger substrate tolerance, and less product inhibition, so as to be applied to the SAM industry on a large scale Production

Method used

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  • S-adenosyl-L-methionine synzyme mutant and preparation method using same
  • S-adenosyl-L-methionine synzyme mutant and preparation method using same
  • S-adenosyl-L-methionine synzyme mutant and preparation method using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of prokaryotic expression strain of S-adenosylmethionine synthetase MATI derived from rat

[0052] Download the amino acid sequence of S-adenosylmethionine synthase MATI derived from rat liver in GenBank (SEQ ID NO.1 in this article, corresponding to GenBank accession number: NP_036992.2), and provide it to Beijing Qingke Biotechnology Co., Ltd. Total gene synthesis of encoding nucleic acid (using E. coli preferred codons). The C-terminal of the synthetic gene has a His tag, and it is constructed into the prokaryotic expression vector pET30a(+). The prokaryotic expression vector restriction site: Nde I at the 5' end, Xho I at the 3' end. Pass the constructed plasmid pET30a(+)-MATI through CaCl 2 Transformed into Escherichia coli expression strain BL21(DE3) by heat shock transformation method, spread on LB solid medium plate containing 50 μg / ml Kanamycin, and cultivate overnight at 37°C, the colony grown on the plate is S-adenosylmethylthio Amin...

Embodiment 2

[0054] Example 2: Purification and immobilization of rat-derived S-adenosylmethionine synthetase (MATI)

[0055] Using the His tag carried in the MATI recombinant protein, the supernatant obtained in Example 1 was treated with activated IDA resin (purchased from Anolon (Beijing) Biotechnology Co., Ltd., specific model: His.Bind Resin, Ni-charged). Carry out protein purification, the specific method is as follows: 4 ℃, 10000r / min, centrifuge the fermentation broth for 10min, discard the supernatant, collect the bacterial cells, the bacterial cells are repeatedly washed twice with phosphate buffer (pH 8.0, 0.1mol / L), centrifuged Afterwards, the cells were concentrated 5 times and resuspended in 20 ml of phosphate buffer (pH 8.0, 0.1 mol / L). The above-mentioned treated bacterial liquid was placed in ice water for ultrasonic crushing until clarification, the ultrasonic crushing conditions were: working for 2s, interval of 5s, ultrasonic power 500W. The crushed lysate was centrifu...

Embodiment 3

[0059] Example 3: Construction of MATI prokaryotic expression strain E.coli BL21(DE3) / pET30a(+)-MATI error-prone mutation library

[0060] Use the pET30a(+)-MATI recombinant plasmid as a PCR template, and conventional T7F / R as a universal primer (primer sequence: T7F: 5'-TAATACGACTCACTATAGGG-3'T7R: GCTAGTTATTGCTCAGCGG) for error-prone PCR amplification of the MATI gene, and adjust the PCR Mg in the amplification reaction system 2+ , Mn 2+ , dCTP and dTTP oligonucleotide concentrations, so that the base mismatch rate of the mutant library is only 2 / 1000, that is, it is guaranteed that only 1 to 2 amino acids are mutated in a mutant.

[0061] Error-prone PCR reaction system:

[0062]

[0063] Error-prone PCR reaction conditions: pre-denaturation at 95°C for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 1 minute, and extension at 72°C for 1.5 minutes, a total of 25 cycles; finally, extension at 72°C for 10 minutes.

[0064] Sampling 2 μL of the ...

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Abstract

The invention belongs to the technical field of enzyme engineering, and relates to a S-adenosyl-L-methionine synzyme mutant and a preparation method using the same. The mutant is mutated into a plurality of amino acid sites in wild S-adenosyl-L-methionine synzyme of which the amino acid sequence is shown as SEQID NO.1, and the mutated amino acid sites comprise C9R and K224R. According to the S-adenosyl-L-methionine synzyme mutant and the preparation method using the same, compared with the wild S-adenosyl-L-methionine synzyme, the mutant has higher specific activity and has higher product yield and lower enzyme activity reduction when catalyzing is performed for preparing the S-adenosyl-L-methionine synzyme.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and relates to an S-adenosylmethionine synthetase mutant and a preparation method using the same. Background technique [0002] S-adenosyl-L-methionine (SAM) is an important intermediate product in the metabolic process of organisms. It exists in all living cells and has the functions of transmethylation, transsulfurization and transaminopropylation. . SAM is a pair of chiral substances, there are two isomers: (R, S)-SAM and (S, S)-SAM, only (S, S)-SAM has biological activity. Clinically, SAM has extremely high medicinal value. It is mainly used to treat intrahepatic cholestasis, and can also be used to treat viral hepatitis and alcoholic liver disease, improve liver function, and relieve depression symptoms in patients with depression (with antidepressant effect), etc. . SAM can also be used in combination with L-dopa to treat Parkinson's disease, which can improve the curative eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P13/12
CPCC12N9/1085C12P13/12C12Y205/01006
Inventor 周晶辉许岗赵士敏黄斌刘亚刘洋
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD
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