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 bri

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

Example Embodiment

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

[0033] After activation on solid LB (1% peptone, 0.5% yeast powder and 1% sodium chloride, 0.4% agar, pH 7.0) plates, pick out a single colony on the enzyme production medium (1% peptone, 0.5% Yeast powder, 1% sodium chloride (NaCl), 3% acetoglucosamine, 0.5% phenol red, 0.4% agar, pH 7.0) on a plate. After incubating at 37°C for 24 hours, pick out a larger yellow transparent circle Strains. After comparative tests, the applicant's State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, screened a strain of Enterobacter sakazakii isolated from Xinjiang soil and identified by 16sDNA in 2014. The applicant named the isolated strain as Enterobacter sakazakii 0360, Cronobacter sakazakii 0360, and was sent to Wuhan, China. The collection number of the Chinese Type Culture Collection of Wuhan University, China, on April 1, 2016, was CCTCC N...

Example Embodiment

[0041] Example 2 Cloning of deacetylase gene

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

[0043] (1) After culturing the Enterobacter sakazakii strain selected in Example 1 on a LB liquid medium 37°C shaker for 12 hours, take 1.5 ml of the bacterial solution in a sterile 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) to suspend the bacteria , 37℃ water bath for one hour, so that lysozyme can fully exert its effect;

[0045] (3) Add 2 times the total volume of the above-mentioned DNA lysis solution (20mmol / L EDTA, 100mmol / L Tris-Cl (pH=8.0), 2% SDS), invert several times to mix, and water bath at 65℃ for 10min;

[0046] (4) Put in the refrigerator at -4℃ for 10min (DNA renaturation)

[0047] (5) Add 150μL of 5M NaCl, mix upside down, place at 4℃...

Example Embodiment

[0081] Example 3 Expression and Purification of Deacetylase

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

[0083] The recombinant plasmid pGEX-6p1-CsnagA was electro-transformed into E. coli BL21 (DE3) for expression and purification. Refer to Example 2 for the transformation steps. The obtained transformants were inoculated into 100mL liquid LB medium supplemented with 100μg / mL ampicillin, cultured overnight at 37°C, and transferred to 1L LB liquid medium containing 100μg / ml ampicillin at a rate of 1%, 37°C Cultivate 2-3hrs to make OD 600 To reach 0.6-0.7, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.2mM, and incubate for 14 hours on a shaker at 18°C ​​and 180 revolutions / min. Collect bacteria by centrifugation at low temperature (4℃) and high speed (8000r / min), then centrifuge at 8000r / min for 8min, then use appropriate amount of ddH 2 Resuspend the bacteria in O, centrifuge at 8000r / min for 8min, and then use phosphate buffer, ...

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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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