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Glucosamine synthetase, engineering bacteria and application thereof

A technology of glucosamine and synthetase, applied in the direction of genetic engineering, application, enzymes, etc., can solve the problems of restricting the production of glucosamine, and achieve the effect of increasing fermentation yield and improving production efficiency

Active Publication Date: 2018-09-28
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In addition, the expression level and enzyme activity of the glucosamine synthase gmS gene have an important impact on the fermentation yield of glucosamine. At present, the glmS genes constructed by genetic engineering strains are all derived from Escherichia coli. During the fermentation process of E. coli engineering bacteria, the glmS gene Enzyme activity is subject to feedback inhibition by GlcN, which restricts the increase in the yield of glucosamine produced by fermentation

Method used

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  • Glucosamine synthetase, engineering bacteria and application thereof
  • Glucosamine synthetase, engineering bacteria and application thereof
  • Glucosamine synthetase, engineering bacteria and application thereof

Examples

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Effect test

Embodiment 1

[0033] Example 1 PCR amplification of the glucosamine synthase gene derived from Bacillus subtilis

[0034] The glucosamine synthase gene in this example is derived from Bacillus subtilis subsp. subsp.

[0035] The specific content is as follows:

[0036] Take the Bacillus subtilis stored on the slant of the test tube for plate activation, and then inoculate an appropriate amount of activated Bacillus subtilis seeds into LB liquid medium, and culture at 37°C, 200rpm for 12h. Take 10 mL of the culture solution, centrifuge at 10000r / min for 5min, collect the bacteria, and use the bacterial genome extraction kit to extract the total genome. The extraction steps refer to the bacterial genome DNA extraction kit (DP302- 02) Instructions.

[0037] Retrieve the nucleotide sequences of the glucosamine synthase genes belonging to different strains from GenBank, and design a pair of primers ES-glmS-F and ES-glmS-R for the conserved sequences. The sequences of the primers are as follows: ...

Embodiment 2

[0046] Example 2 Construction of glucosamine gene recombinant expression vector derived from Bacillus subtilis

[0047] Using the plasmid pUCm-ES-glmS as a template, use the primers BamH I-glmS-F / Sal I-glmS-R to amplify the amino group of Bacillus subtilis subsp. Full-length sequence of the glucose synthase gene. The primer sequences used are as follows:

[0048] BamH I-glmS-F: 5'-CGGGATCCATGTGTGGAATCGTAGGTTATA-3'

[0049] Sal I-glmS-R: 5'-ACGCGTCGACTTACTCCACAGTAACACTTTTC-3'

[0050] The PCR amplified product was double digested with BamH I and Sal I, and the target fragment was recovered, and the prokaryotic expression vector pET28a(+) (purchased from Novagen, Germany, catalog number 69864-3) was simultaneously treated with BamH I and Sal I. Carry out double enzyme digestion, recover the larger fragment in the vector, connect the recovered target gene fragment and the vector fragment, and transform the ligation product into E. After screening, positive clones were picked,...

Embodiment 3

[0057] The construction of the recombinant expression vector of glucosamine synthase gene of embodiment 3 Escherichia coli source and other microbial sources

[0058] Using the genome of Escherichia coli DH5α (purchased from Invitrogen, catalog number 18263-012) as a template, using primers BamH I-E-glmS-F / Sal I-E-glmS-R primers, amplified to obtain both sides with BamHI and Sal The full-length sequence of the glucosamine synthase gene derived from Escherichia coli at the I restriction site. The primer sequences used are as follows:

[0059] BamH I-E-glmS-F: 5'-CGGGATCCATGTGTGGAATTGTTGGCGCG-3'

[0060] Sal I-E-glmS-R: 5'-ACGCGTCGACTTACTCAACCGTAACCGATTTTG-3'

[0061] The PCR amplified product was digested with BamH I and Sal I, and the target fragment was recovered. At the same time, the constructed recombinant plasmid pET28a-ES-glmS-GAN1 was digested with BamH I and Sal I, and the larger one was recovered. fragments, the recovered target gene fragments were connected to the...

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Abstract

The invention discloses glucosamine synthetase, engineering bacteria and an application thereof. A glucosamine synthetase gene BS-glmS in high activity is obtained from a strain of bacillus subtilis by cloning, and the BS-glmS gene is used for constructing a recombinant expression vector for converting escherichia coli BL21 (DE3). The capacity of an obtained genetic engineering bacterial strain isremarkably improved in an aspect of glucosamine fermentation production. While the constructed genetic engineering bacterial strain is fermented for 16 h, the total fermentation yield of glucosamineand N-acetylglucosamine can reach 26.51 g / L. Compared with the genetic engineering bacterial strain obtained by a glmS gene from the escherichia coli, the fermentation yield is improved by 18.2%.

Description

technical field [0001] The invention relates to the technical field of microbial genetic engineering, in particular to a glucosamine synthase, engineering bacteria and applications thereof. Background technique [0002] Glucosamine (GlcN) is an important hexosamine sugar, which is formed by replacing a hydroxyl group of glucose with an amino group. It exists in all organisms, and in the human body it is hyaluronic acid, chondroitin sulfate and keratin sulfate. It is the precursor of the disaccharide unit of glucosaminoglycans such as glycosaminoglycans, and is the main component of various glycoproteins and proteoglycans in the body. [0003] Glucosamine is widely used in the fields of health food and medicine. It can specifically act on articular cartilage and effectively treat rheumatoid arthritis; it can inhibit the growth of various tumor cells, induce apoptosis, and play an anti-tumor effect; activate natural The function of immune cells such as killing cells regulates...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/26C12R1/19
CPCC12N9/1096C12N15/70C12P19/26C12Y206/01016
Inventor 程汝滨葛宇清方昀朱玲燕陈梦
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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