Preparation method of glutamine dipeptide

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

Active Publication Date: 2017-06-13
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
View PDF10 Cites 14 Cited by
  • 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; 20

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method of glutamine dipeptide
  • Preparation method of glutamine dipeptide
  • Preparation method of glutamine dipeptide

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0023] Example 1 Construction of 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 codon software commonly used in E. coli to optimize the codon, and add the restriction site BamH I to the optimized sequence And Hind III (Sequence Listing 400 The sequence shown) was sent to Jinweizhi Company for synthesis.

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

[0026] ③Use Takara T4 DNA ligase to ligate 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.

Example Embodiment

[0028] Example 2 Construction of Acetate Kinase Strain

[0029] ①Using PCR technology with the genome of Clostridium acetobutylicum ATCC 4259 as a template, according to the nucleotide sequence of the acetate kinase encoding gene ack (sequence list 400 (Shown sequence) design a pair of gene amplification primers (upstream primer is 400 The sequence shown, the downstream primer is Sequence Listing 400 Shown sequence), amplify to obtain ack gene fragment. The pair of primers respectively contain restriction sites BamH I and EcoRI.

[0030] ②Using Takara restriction enzymes BamH I and EcoRI double digestion 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] ③Use Takara T4 DNA ligase to ligate the two two gene fragments obtained in step ② to obtain the recombinant expression vector pET-His-ack.

[0032] ④ Transform the recombinant expression vector of step ③ into E. coli BL21 (ACCC11171) to ob...

Example Embodiment

[0033] Example 3 Preparation of amino acid ligase and acetate kinase

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

[0035] ②After the cultivation, centrifuge the fermentation broth at 4°C and 6000rpm to collect the bacteria. After washing three times with pH 7.4 PBS buffer, resuspend in pH 7.4 PBS buffer, sonicate for 20 min, centrifuge at 4°C, 10000 rpm to obtain the supernatant.

[0036] ③Pass the supernatant through Ni at a flow rate of 1 mL / min + -After NTA resin, eluted with 50 mM imida...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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 UNIVERSITY OF SCIENCE AND TECHNOLOGY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products