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Deacetylase capable of producing high-yield glucosamine and coding gene thereof

A technology of glucosamine and deacetylase, which is applied in the field of high-yield glucosamine deacetylase and its coding gene, can solve environmental problems, long production cycle of microbial fermentation method, high equipment requirements and other problems

Inactive Publication Date: 2017-08-08
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The material of the former method mainly comes from the exoskeleton of shellfish such as shrimp and crab. The acid hydrolysis method has many defects. For example, some patients who are allergic to shrimp and crab products cause allergic reactions. The glucosamine product from the hydrolysis of shrimp and crab shells of aquatic products has Fishy smell, in addition, the large use of concentrated hydrochloric acid will bring serious environmental problems, and the requirements for related equipment are relatively high, which will gradually be restricted by relevant national policies; compared with traditional methods, the production of GlcN by microbial fermentation has less environmental pollution , no allergenic factors (Chen Xin et al. 2012), but the microbial fermentation method has a long production cycle and low production efficiency

Method used

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  • Deacetylase capable of producing high-yield glucosamine and coding gene thereof
  • Deacetylase capable of producing high-yield glucosamine and coding gene thereof
  • Deacetylase capable of producing high-yield glucosamine and coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Screening and 16sDNA identification of the natural strain Enterobacter sakazakii producing deacetylase

[0033] After activation on a solid LB (1% peptone, 0.5% yeast powder and 1% sodium chloride, 0.4% agar, pH 7.0) plate, pick a single colony on the enzyme-producing medium (1% peptone, 0.5% Yeast powder, 1% sodium chloride (NaCl), 3% acetylglucosamine, 0.5% phenol red, 0.4% agar, pH 7.0) plate, after 24 hours of cultivation at 37 ° C, pick and produce a large yellow transparent circle strains. After comparative tests, the State Key Laboratory of Agricultural Microbiology of Huazhong Agricultural University, where the applicant works, screened a strain of Enterobacter sakazakii isolated from Xinjiang soil and identified as Enterobacter sakazakii by 16sDNA in 2014. The applicant named the isolated strain Enterobacter sakazakii 0360, Cronobacter sakazakii 0360, and it was sent to China. Wuhan. Wuhan University, China Type Culture Collection Center for preserva...

Embodiment 2

[0041] Cloning of embodiment 2 deacetylase gene

[0042] 1. Extract the chromosomal DNA of Enterobacter sakazakii:

[0043] (1) After the Enterobacter sakazakii strain screened in Example 1 was cultured on a LB liquid medium at 37° C. on a shaker for 12 hours, take 1.5 ml of bacterial liquid in a sterilized Ep tube and centrifuge on a centrifuge at 12,000 rpm 1 minute, discard the supernatant, and collect the bacteria;

[0044] (2) Wash the bacteria twice with TE buffer (50mmol / LTris-HCl, pH8.0; 10mmol / L EDTA, pH8.0), then add 50μl 100μg / ml lysozyme (purchased from sigma company) to suspend the bacteria , 37 ℃ water bath for one hour, so that the lysozyme can fully play its role;

[0045] (3) Add 2 times the total volume of DNA lysate (20mmol / L EDTA, 100mmol / L Tris-Cl (pH=8.0), 2% SDS), mix by inverting several times, and bathe in water at 65°C for 10 minutes;

[0046] (4) Place in -4°C refrigerator for 10 minutes (DNA renaturation)

[0047] (5) Add 150 μL of 5M NaCl, mix ...

Embodiment 3

[0081] Example 3 Deacetylase expression and purification

[0082] 1. Induced expression of recombinant deacetylase in Escherichia coli:

[0083] The recombinant plasmid pGEX-6p1-CsnagA was electrotransformed into E.coli BL21(DE3) for expression and purification, and the transformation steps refer to Example 2. Inoculate the obtained transformants in 100 mL liquid LB medium with 100 μg / mL ampicillin, culture overnight at 37°C, transfer to 1L LB liquid medium with 100 μg / ml ampicillin at 37°C Cultivate for 2-3hrs to make OD 600 When it reaches 0.6-0.7, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM, and incubate at 18° C. on a shaker at 180 rpm for 14 hours. Centrifuge at low temperature (4°C) and high speed (8000r / min) to collect the bacterial cells, then centrifuge at 8000r / min for 8min, and then use an appropriate amount of ddH 2 O resuspended bacterium, centrifuged at 8000r / min for 8min, then phosphate buffered saline, namely PBS (prepar...

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Abstract

The invention belongs to the technical field of microorganism gene engineering, and particularly relates to deacetylase capable of producing high-yield glucosamine and a coding gene thereof. The gene CsnagA is separated from the natural strain cronobacter sakazakii 0360. Genomic DNA is separated from cronobacter sakazakii with the preservation number of CCTCC NO:M 2016162. The nucleotide sequence of the gene is shown in SEQ ID NO:1. The sequence of protein coded by the gene is shown in SEQ ID NO:2. By means of genetic recombination, the gene is transferred into escherichia coli to construct genetically engineered bacteria, so as to obtain an efficient glucosamine fermentation production capacity, and glucosamine can be produced with the enzymatic biosynthesis method. Compared with the prior art, the deacetylase has the advantages that the production efficiency is improved, and the production cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and in particular relates to a high-yield glucosamine deacetylase and its coding gene. The gene is isolated from a natural strain Cronobacter sakazakii. Background technique [0002] Glucosamine (GlcN, the chemical name is 2-amino-2-deoxy-D-glucose, its structural formula is shown in Figure 8 ), the only alkaline monosaccharide in nature, widely exists in polysaccharides and conjugated polysaccharides from animals and microorganisms. In the human body, it mainly exists in articular cartilage, connective tissue, and cell membrane, providing protection and lubrication for human articular cartilage, and guaranteeing the normal operation of joint physiological activities. D-glucosamine not only has the functions of treating arthritis and gastric ulcer, inhibiting the growth of cancer cells, reducing inflammation, and stimulating the synthesis of proteoglycans, but also activat...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12P19/26C12N1/20C12N1/21C12R1/01C12R1/19
CPCC12N9/80C12P19/26C12Y305/01C12N1/205C12R2001/01
Inventor 刘子铎王倩
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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