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Beta-N-acetylglucosaminidase 159 as well as cloning expression and application thereof

A technology of glucosidase and acetylaminosidase, which is applied to β-N-acetylglucosaminidase 159 and its cloning, expression and application fields, can solve the problems of difficult reaction process control, high cost of three-waste treatment, serious environmental pollution, etc.

Inactive Publication Date: 2021-05-14
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The method of preparing N-acetylglucosamine (GlcNAc) with chitin as a substrate can be mainly divided into chemical method and enzymatic method. At present, chemical methods are mostly used in industry. This method is difficult to control the reaction process and is accompanied by by-products. produced, and the biological activity of the product is poor
Moreover, it pollutes the environment seriously, and the cost of treating the three wastes is high.

Method used

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  • Beta-N-acetylglucosaminidase 159 as well as cloning expression and application thereof
  • Beta-N-acetylglucosaminidase 159 as well as cloning expression and application thereof
  • Beta-N-acetylglucosaminidase 159 as well as cloning expression and application thereof

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Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1 strain source

[0042] Bacterial strains used in the present invention Chitinibacter sp.GC72 It is separated from the pond silt in Nanjing. It is now preserved in the China Center for Type Culture Collection (Accession No.: CCTCC M 2014113)

Embodiment 2

[0044]Cloning of β-N-acetylglucosaminidase 159 comprises the following steps:

[0045] Step 1, the gene of β-N-acetylglucosaminidase, its nucleotide sequence is as shown in SEN ID NO: 1;

[0046] Step 2, design PCR amplification primers

[0047] 159-F-Primer: 5'- ATTCTAGCT ATGAACAAGCCAGCAGGTG-3';

[0048] 159-R-Primer: 5'- ATTCCGCTC CGCCAGCAAAGTACGCCTGAC-3', the full length of the gene was amplified;

[0049] Step 3, the reaction conditions for the above PCR amplification are: pre-denaturation at 95°C for 5 min; 30 cycles at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 40 sec; and finally extension at 72°C for 10 min;

[0050] Step 4, using restriction endonucleases NdeI and XhoI to double-enzyme-cut the PCR amplification product, and reclaim the digested product;

[0051] Step 5, double-digest the vector pET-28a(+) with restriction enzymes NdeI and XhoI, and recover the vector backbone (about 5400 bp);

[0052] Step 6, ligate the digested product of step 4 and the ...

Embodiment 3

[0054] The clone expression of β-N-acetylglucosaminidase 159 gene comprises the following steps:

[0055] In the first step, the recombinant plasmid pET-28a(+)-159 was introduced into Escherichia coli BL21 (DE3), and the Escherichia coli containing the recombinant plasmid pET-28a(+)-159 was obtained, which was named recombinant bacteria;

[0056] In the second step, select the single clone of the recombinant bacteria (expressing pET-28a(+)-159 containing His tag) and connect it to a shaking shaker containing 100 μg / mL kanamycin in 5 mL LB medium at 37°C and 200rmp Overnight culture in medium;

[0057] The third step is to inoculate the above-mentioned bacterial solution into 100 mL LB liquid medium containing 100 μg / mL kanamycin at a volume ratio of 1:100, and culture it at 37°C with shaking at 200 rpm / min until OD 600 Reach 0.6-0.8;

[0058] In the fourth step, add IPTG (inducer) to a concentration of 1mmol / L, shake and culture at 20°C and 200rmp for 20h;

[0059] The fift...

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Abstract

The invention discloses a beta-N-acetylglucosaminidase 159 as well as cloning expression and application of the beta-N-acetylglucosaminidase 159. The gene of the beta-N-acetylglucosaminidase 159 is constructed on a plasmid pET-28a (+) through a genetic engineering means and is transferred into E. coli BL21 (DE3) for expression; the molecular weight of the recombinase is 92 kDa; and according to a result of comparative analysis, the beta-N-acetylglucosaminidase 159 belongs to a glycoside hydrolase GH20 family. Research on enzymatic properties of the recombinase shows that the specific activity of a pure enzyme is 16.67 U / mg, an optimum temperature is 40 DEG C, an optimum pH value is 9.0, the enzyme activity can be maintained to be higher than 80% under the condition that the pH value is higher than 7, and the beta-N-acetylglucosaminidase 159 shows relatively high pH stability and synergistic effect when the recombinase is in synergism with other chitinase; and thus, a theoretical basis is provided for efficient preparation of N-acetylglucosamine.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a β-N-acetylglucosaminidase 159 and its clone expression and application. Background technique [0002] Chitin, also known as chitin and chitin, is a polymer linked by N-acetylglucosamine through β-1,4-glycosidic bonds. It is the polysaccharide with the highest content on the earth except cellulose. It is widely distributed and mainly exists in shrimp and crab shells, insect exoskeletons and fungal cell walls. Its hydrolyzed product N-acetylglucosamine can protect cartilage tissue and bone joints. Chitinase can prevent and treat fungal diseases of plants, and is widely used in food, medicine, agriculture, cosmetics and other industries. [0003] The methods for preparing N-acetylglucosamine (GlcNAc) using chitin as a substrate can be mainly divided into chemical methods and enzymatic methods. At present, chemical methods are mostly used in industry. This method is d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/26C12P19/14C12R1/19
CPCC12N9/2434C12N15/70C12P19/26C12P19/14C12Y302/01052
Inventor 陈可泉陈燕张阿磊陈雪曼周宁杨鹏帆王莹莹魏国光欧阳平凯
Owner NANJING UNIV OF TECH
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