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Production method of glycine

A glycine and amino acid technology, applied in the biological field, can solve the problems of high toxicity of the substrate aminoacid, inactivation of microbial enzymes, etc.

Active Publication Date: 2022-02-18
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the 1990s, Japan published a patent (US5238827) that used aminocyanic acid as a substrate and catalyzed by microbial enzymes. Although the glycine production could reach 148g / L in 30 hours, the substrate aminocyanic acid was highly toxic and microbial enzymes were easy to produce. Inactivated by organic matter
De novo synthesis of glycine in the cell can solve this problem, but there is no enzyme involved in the synthesis pathway to catalyze the reaction from glyoxylate to glycine, which needs to be discovered, and it is also the key point to realize this technology
[0006] In summary, the de novo synthesis of glycine by biological methods is still in the blank

Method used

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  • Production method of glycine
  • Production method of glycine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Embodiment 1, the whole gene synthesis of glyoxylate ammonialase gene on the expression plasmid

[0107] Will be derived from Aeromonas veronii, Bacillus aquimaris, Bacillus cereus, Bacillus flexus, Bacillus licheniformis, Bacillus velezensis, Bacillus substilis, Geobacillus stearothermophilus, Halomonas boliviensis, Labrenzia aggregata, Lysinibacillus fusiformis, Pseudomonas aeruginosa, Paucisalibacillus globuius, smegmatis Mycobacterium smegmatis and Mycobacterium tuberculosis glyoxylate ammonialase gene ald (stop codon removed) were synthesized between NdeI and XhoI of pET30a(+) (GenScript) and verified by sequencing After being correct, the corresponding plasmids pET30a-Avald, pET30a-Ba ald, pET30a-Bc ald, pET30a-Bf ald, pET30a-Bl ald, pET30a-Bv ald, pET30a-Bs ald, pET30a-Gs ald, pET30a-Hb were obtained in sequence ald, pET30a-La ald, pET30a-Lf ald, pET30a-Pa ald, pET30a-Pg ald, pET30a-Ms ald and pET30a-Mt ald (Table 1). These plasmids were respectively transforme...

Embodiment 2

[0109] Embodiment 2, analysis of glyoxylate ammonialase enzyme activity

[0110] Glyoxylate ammonase expression bacterial strain Av, Ba, Bc, Bf, Bv, Bl, Bs, Gs, Hb, La, Lf, Pa, Pg, Ms, Mt of the different sources of glyoxylate ammonialase obtained in embodiment 1 carry out glyoxylate Enzyme activity analysis of ammonase (Ald).

[0111] The single clone of the inoculated strain was cultured in LB liquid medium (the final concentration of kanamycin was 50mg / L) at 37°C and 250rpm until OD550=0.1, and the inducer IPTG was added with a final concentration of 0.1mM, and the culture was continued until logarithmic Expect. Take 30ml culture solution in a 50ml centrifuge tube, centrifuge at 10,000rpm at 4°C for 5min, discard the supernatant, collect the bacteria, wash twice with 15ml 100mM Tris-HCl buffer, suspend the bacteria in 3ml 100mM Tris-HCl , sonicate in an ice bath (power: 25W; on: 1s; off: 3s) for 3-5min until clarification, centrifuge at 10,000rpm for 20min at 4°C, and col...

Embodiment 3

[0118] Embodiment 3, glyoxylate ammonialase (Ald) catalyzes and produces glycine in vitro

[0119] The glyoxylate ammoniaase pure enzymes obtained in Example 2 from different sources were catalyzed in vitro, and the yield of glycine synthesis was determined.

[0120] Glyoxylate ammoniation enzyme in vitro catalyzed glycine production reaction system is: 1.5ml centrifuge tube, add reaction buffer 990μl (40mM potassium phosphate buffer, 400mM ammonium sulfate, 0.2mM NADPH, 50mM sodium glyoxylate; pH7.0), Add 10 μl of 3U pure glyoxylate ammoniaase, react for 10 min, and perform amino acid analysis on an amino acid analyzer (Hitachi Automatic Amino Acid Analyzer L-8900) to detect the yield of glycine.

[0121] The ability to produce glycine in vitro can be achieved by in vitro catalysis of pure glyoxylate ammoniaase enzymes from different sources. Derived from Aeromonas veronii, B. aquimaris, B. cereus, B. flexus, B. licheniformis, B. velezensis, Bacillus subtilis B.substilis, B...

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Abstract

The invention discloses a production method of glycine. The invention provides a method for constructing an engineering strain capable of producing glycine, and the method comprises the following steps of: expressing glyoxylic acid ammoniation enzyme from mycobacterium smegmatis, paucisalibacillus globuius, mycobacterium tuberculosis, bacillus aquimaris, halomonas boliviensis, aeromonas veronii or labrenzia aggregata by recipient bacteria, thus obtaining a strain named as an engineering bacterium 1; the engineering bacterium 1 is an engineering strain capable of producing glycine. The invention opens up the precedent of synthesizing glycine by using a biological method.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a production method of glycine. Background technique [0002] Glycine (glycine) is the simplest structure in the amino acid series, and it is a non-essential amino acid for the human body. As an important fine chemical intermediate, it is widely used in the fields of pesticides, medicines, food and feed additives. Adding glycine to food can be used as a food preservative to extend its shelf life; in the processing of alcoholic beverages and animal and plant foods, it can be used as a flavoring agent and flavor enhancer. In terms of medicine, glycine can be used to synthesize a variety of drugs, such as the drug delapril hydrochloride for the treatment of hypertension, calcium carbonate preparations for inhibiting gastric ulcers, paracetamol glycinate, monoglycine acetylsalicylate calcium, reseramine injection, antipatric Kimson's drugs L-dopa, thiamphenicol, etc. Industria...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/06C12N9/10C12N9/88C12N15/53C12N15/54C12N15/60C12N15/31C12P13/04C12R1/19
CPCC12N9/0016C12N9/1025C12N9/88C07K14/245C12P13/04C12Y104/01001C12Y203/03009C12Y401/01064
Inventor 张学礼朱欣娜徐洪涛
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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