Beta-N-acetylglucosaminidase of Paenibacillus barungensis, encode gene thereof and application of beta-N-acetylglucosaminidase

A technology of glucosidase and acetylamino, which is applied in the field of Paenibacillus barengerzia β-N-acetylglucosaminidase and its coding gene and application, can solve environmental pollution and other problems, achieve good thermal stability, High specific enzyme activity and excellent enzymatic properties

Active Publication Date: 2019-01-11
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditionally, N-acetyl-D-glucosamine is produced by acid hydrolysis of chitin, and the large amount of acidic waste generated will cause serious environmental pollution (Patil N.S. and Jadhav J.P. Enzymatic production of N-acetyl-D-glucosamine by solid state fermentation of chitinase by Penicillium ochrochloron MTCC 517 using agricultural residues. International Biodeterioration & Biodegradation, 2014, 91:9-17)

Method used

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  • Beta-N-acetylglucosaminidase of Paenibacillus barungensis, encode gene thereof and application of beta-N-acetylglucosaminidase
  • Beta-N-acetylglucosaminidase of Paenibacillus barungensis, encode gene thereof and application of beta-N-acetylglucosaminidase
  • Beta-N-acetylglucosaminidase of Paenibacillus barungensis, encode gene thereof and application of beta-N-acetylglucosaminidase

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Experimental program
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Effect test

Embodiment 1

[0056] Embodiment 1, the acquisition of β-N-acetylglucosaminidase and its coding gene

[0057] Extensive sequence analysis and functional verification of Paenibacillus barengoltzii, from Paenibacillus barengoltzii CAU904 (References: Zhang B., Liu Y., Yang H.Y., et al., Biochemical properties and application of a novel β-1,3-1,4-glucanase from Paenibacillus barengoltzii.Food Chemistry, 2017,234:68-75. The public can obtain from China Agricultural University) to obtain a β-N-acetylglucosaminidase The coding gene is 1038bp in full length, as shown in sequence 2 of the sequence listing. The DNA molecule shown in Sequence 2 of the Sequence Listing encodes the β-N-acetylglucosaminidase shown in Sequence 1 (composed of 345 amino acids, without signal peptide, named PbNag39).

Embodiment 2

[0058] Embodiment 2, the construction of engineering bacteria expressing β-N-acetylglucosaminidase

[0059] 1. Use the sequence 2 of the sequence listing to replace the fragment between the EcoRI and NotI restriction sites of the SUMO-pET28a vector (Novagen, Denmark) from the 1-1038 position of the 5' end to obtain the recombinant expression vector SUMO-pET28a-PbNag39( verified by sequencing).

[0060] 2. Introduce the recombinant expression vector SUMO-pET28a-PbNag39 obtained in step 1 into Escherichia coli BL21 (Bomad Gene Technology Co., Ltd.) to obtain recombinant bacteria.

[0061] 3. Introduce the SUMO-pET28a vector into Escherichia coli BL21 (Bomad Gene Technology Co., Ltd.) to obtain a recombinant bacterium converted to an empty vector.

Embodiment 3

[0062] Embodiment 3, preparation of β-N-acetylglucosaminidase and detection of enzymatic properties thereof

[0063] One, the preparation of β-N-acetylglucosaminidase

[0064] 1. Inoculate the recombinant bacterium prepared in Example 2 into LB liquid medium containing 50 μg / mL kanamycin, culture it with shaking at 37°C and 200 rpm to OD 600nm Reach between 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) in the culture system, the concentration of IPTG in the culture system is 1mmol / L, 30 ℃, 200rpm induce and cultivate overnight, then Centrifuge the culture system at 11510g, collect the bacterial precipitate, resuspend in 20mM acetic acid-sodium acetate (pH 5.5) buffer solution, and then ultrasonically break (250W, 20min). .

[0065] 2. Take the crude enzyme solution obtained in step 1, and use the agarose Ni Sepharose affinity column (GE Healthcare, product number: 17-5268-01) to purify the recombinant protein. The specific steps are as follows:

[0066]Equilibrate...

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Abstract

The invention discloses a Beta-N-acetylglucosaminidase of Paenibacillus barungensis, an encode gene thereof and an application of beta-N-acetylglucosaminidase. The provided Beta-N-acetylglucosaminidase PbNag39 is excellent in the enzymatic property, and has a specific activity against chitosan is 28.3 U/mg, and the final products of hydrolysis of chitosan oligosaccharides are N-Acetyl glucosamine.The Beta-N-Acetylglucosaminidase and chitinase can hydrolyze chitin powder synergistically and obtain N-Acetyl glucosamine. The Beta-N-acetylglucosaminidase PbNag39 has the characteristics of high specific activity, good thermal stability and excellent hydrolysis characteristics. It can stabilize and play a catalytic role in a wide range of pH, and has important application value in chitin conversion.

Description

technical field [0001] The invention relates to a Paenibacillus barenguez β-N-acetylglucosaminidase and its coding gene and application. Background technique [0002] Chitin is a linear polysaccharide formed by β-N-acetylglucosamine connected by β-1,4-glycosidic bonds. It exists widely in nature, and its content is second only to cellulose. Chitin is widely found in the crustaceans of shrimp and crabs, the shells of insects, and the cell walls of plants and fungi (LvY.M., Laborda P., Huang K., et al. Highly efficient and selective biocatalytic production of glucosamine from chitin . Green Chemistry, 2017, 19:527-535). Chitin degrading enzymes can be divided into endochitinase (EC 3.2.1.14), exochitinase (EC 3.2.1.14) and β-N - Acetylglucosaminidase (β-N-acetylglucosaminidase, EC 3.2.1.52). Endochitinase can randomly hydrolyze β-1,4-glycosidic bonds in the main chain of chitin to produce low-molecular-weight chitooligosaccharides, while β-N-acetylglucosaminidase The non-r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N1/21C12P19/26C12P19/14C12P19/02C12R1/19
CPCC12N9/2402C12P19/02C12P19/14C12P19/26C12Y302/01052
Inventor 江正强刘翊昊闫巧娟马帅杨绍青
Owner CHINA AGRI UNIV
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