A kind of preparation method of glutamic acid dipeptide

A technology of dipeptide and alanine, applied in biochemical equipment and methods, peptides, transferases, etc., can solve the problems of high production cost, complex synthesis process, unfavorable industrial scale-up, etc., achieve low cost, wide source of raw materials, easy The effect of large-scale industrial production

Active Publication Date: 2020-11-06
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, it has been reported that amino acid ester acyltransferase is used to catalyze L-alanine methyl ester hydrochloride and L-glutamine to generate glutamic acid dipeptide (201410663050.8; 201410670694.X; 201510064439.5), but due to the The synthesis process of ester hydrochloride is complicated, which leads to high production cost of this method, which is not conducive to industrial scale-up

Method used

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  • A kind of preparation method of glutamic acid dipeptide
  • A kind of preparation method of glutamic acid dipeptide
  • A kind of preparation method of glutamic acid dipeptide

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Experimental program
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Embodiment 1

[0023] The construction of embodiment 1 amino acid ligase strain

[0024] ①According to the nucleotide sequence of the amino acid ligase encoding gene ywfE of B. subtilis ATCC 15245 on Genbank, use the codon software commonly used in Escherichia coli to optimize its codon, and add the restriction site BamH I to the optimized sequence and Hind III (sequence shown in Sequence Listing 400) were sent to Jinweizhi Company for synthesis.

[0025] ② Use Takara restriction endonucleases BamH I and Hind III to double digest the target gene fragment and pET-His vector plasmid in step ① to obtain ywfE and pET-His linear fragments with the same cohesive ends.

[0026] ③ Use Takara T4 DNA ligase to connect the two gene fragments in step ② to obtain the recombinant expression vector pET-His-ywfE.

[0027] ④ Transform the recombinant expression vector in step ③ into E.coli BL21 (ACCC11171) to obtain the amino acid ligase-producing strain E.coli pET-His-ywfE.

Embodiment 2

[0028] The construction of embodiment 2 acetate kinase bacterial strains

[0029] ①A pair of gene amplification primers ( The upstream primer is the sequence shown in Sequence Listing 400 , the downstream primer is the sequence shown in Sequence Listing 400 ), and the ack gene fragment is obtained by amplification. The pair of primers respectively comprise enzyme cutting sites BamH I and EcoRI.

[0030] ② Use Takara restriction endonucleases BamH I and EcoRI to double digest step ① to obtain the target fragment and pET-His vector plasmid, and obtain ack and pET-His linear fragments with the same sticky ends.

[0031] ③ Use Takara T4 DNA ligase to connect the two gene fragments obtained in step ② to obtain the recombinant expression vector pET-His-ack.

[0032] ④ Transform the recombinant expression vector in step ③ into E.coli BL21 (ACCC11171) to obtain the acetate kinase-producing strain E.coli pET-His-ack.

Embodiment 3

[0033] The preparation of embodiment 3 amino acid ligase and acetate kinase

[0034] ① Inoculate the strains in (1) and (2) from the glycerol bacteria preservation tube with an inoculum of 0.1% (v / v) into 100 mL of LB liquid medium containing ampicillin (100 μg / mL), and culture at 37°C and 200 rpm After 12 hours, transfer to 400 mL LB liquid medium containing ampicillin (100 μg / mL) according to 1% (v / v) inoculum amount, and continue culturing at 37° C. and 200 rpm. Strain concentration OD 600nm When it reached 0.6, IPTG with a final concentration of 0.1 mmol / L was added, and the expression protein was induced and cultured at 28°C for 10 h.

[0035] ② After the cultivation, the fermentation broth was centrifuged at 4°C and 6000rpm to collect the bacterial cells. After washing three times with PBS buffer at pH 7.4, resuspend with PBS buffer at pH 7.4, sonicate for 20 min, and centrifuge at 4°C at 10,000 rpm to obtain the supernatant.

[0036] ③ Pass the supernatant through Ni...

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Abstract

The invention relates to a preparation method of glutamine dipeptide. Escherichia coli is used for expressing amino acid ligase from bacillus subtilis and acetokinase from clostridium acetobutylicum respectively, and a double-enzyme coupling system is formed through mixing after purification. The amino acid ligase is used for catalyzing L-alanine and L-glutamine to generate the glutamine dipeptide, meanwhile ADP is formed along with ATP dephosphorylation, and the acetokinase is used for catalyzing acetyl phosphate and the ADP to form ATP, so that cyclic regeneration of the ATP is realized. By using the double-enzyme coupling system, 32.5 mM of glutamine dipeptide can be obtained through reaction for 8 h under appropriate reaction conditions, and the molar conversion rate is 64.5%. The production method of the glutamine dipeptide provided by the invention has the advantages of low raw material cost, short enzymatic conversion time, simple and convenient operation, low production cost and the like, and has relatively high industrial application value.

Description

technical field [0001] The invention relates to the field of biotechnology production of nucleoside products, in particular to a preparation method of glucodipeptide. Background technique [0002] As an essential amino acid for human metabolism, L-glutamine is converted into glycosamine in the body. As a precursor for the synthesis of mucin, it can promote ulcer healing. It is used as a parenteral nutrition drug for severe infection, compound fractures, and trauma. , major surgery, large area burns, treatment and recovery of patients with chemical poisoning injuries, radiation injuries, and harmful substances; it can also be used as a brain function improving agent for improving brain function in children with mental retardation and mental disorders, alcoholism, and epilepsy. In addition, it can also be used as a therapeutic drug for immunodeficiency syndrome such as AIDS, critical illness or immunocompromised patients after bone marrow transplantation, so it is widely used ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C12N9/12C12N9/00C12N15/70
CPCC07K5/06026C12N9/1217C12N9/93C12N15/70C12P21/02C12Y207/02001C12Y603/02
Inventor 范晓光陈宁谢希贤洪翔朱新雅贾子樊徐庆阳张成林
Owner TIANJIN UNIV OF SCI & TECH
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