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A kind of chitosanase and its application in the preparation of chitosan oligosaccharide

A chitosanase, chitosan technology, applied in the application, glycosylase, enzyme and other directions, can solve the problems of low specificity of chitosan, high equipment requirements, pollution of the environment, etc., to achieve good temperature stability, Efficient degradation and high enzymatic activity

Active Publication Date: 2021-05-25
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art of the advantages of low specificity, high equipment requirements, environmental pollution, and poor product quality of the traditional chemical hydrolysis of chitosan, and to provide a chitosanase and its preparation in chitosan. Application in sugar preparation, using the chitosanase to degrade large molecular weight chitosan to prepare small molecular weight chitosan oligosaccharides

Method used

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  • A kind of chitosanase and its application in the preparation of chitosan oligosaccharide
  • A kind of chitosanase and its application in the preparation of chitosan oligosaccharide
  • A kind of chitosanase and its application in the preparation of chitosan oligosaccharide

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Embodiment 1

[0017] Embodiment 1 produces the cloning of chitosanase gene fragment

[0018] The applicant analyzed the Streptomyces albolongus genome by molecular biology software, and found that there was a novel chitosanase gene, and by cloning the gene, the present invention was facilitated.

[0019] Using the genome of Streptomyces albolongus as a template, the upstream primer Csn-F 5'-CGCGGATCCATGGGCGCCGGCCGGGCC-3' and the downstream primer Csn-R respectively containing BamHI and HindIII restriction sites were designed on the upstream and downstream of the chitosanase gene 5'-CCCAAGCTTGTCGTACGGGTAGTGACG-3', the Csn gene fragment was amplified by PCR.

[0020] The PCR reaction system is: 2×PCR Buffer 25 μl, dNTP 5 μl, each primer 1 μl, template (Streptomyces albicans whole genome DNA) 1 μl, KOD Fx DNA polymerase (TOYOBO, KFX-101) 1 μl, add sterile water to a final volume of 50 μl. The reaction conditions of PCR were: pre-denaturation at 94°C for 2 min, denaturation at 98°C for 10 s, ...

Embodiment 2

[0022] Embodiment 2 Contains the expression vector construction of chitosanase gene

[0023] Recover PCR target gene fragments. Add BamHI enzyme and HindIII enzyme to the target gene and plasmid pET-28a, respectively, for double enzyme digestion. The enzyme-cut plasmid was recovered by electrophoresis, and the target gene was purified with a kit (Omega). Ligate the purified target gene fragment with the recovered plasmid.

[0024] After the connection is completed, the heat shock method is used to transform, and the connected system is transferred into DH5α competent cells. Use an LB plate containing ampicillin resistance to screen positive transformants, and use T7 universal primers to perform PCR verification on the clones. The consistency of the results is 100%, which proves that the chitosanase gene sequence has been completely cloned into the expression vector, which is a positive clone, and the recombinant plasmid is named pETC.

Embodiment 3

[0025] Embodiment 3 contains the expression of the recombinant plasmid of chitosanase gene and the construction of engineering bacterium

[0026] Extract the recombinant plasmids from the positive clones with correct sequencing and transform them into the host E.coli BL21 competent cells (the transformation process saves the concentration operation of the bacterial solution), and the positive transformants with ampicillin resistance are the goals of successful cloning Engineering bacteria.

[0027] The chitosanase-producing engineered strain was inoculated into 5 mL of resistant LB medium (100 μg / mL ampicillin resistance), and cultured overnight at 37° C. at 180 rpm for 12 hours to activate the strain. After the bacterial solution was activated, it was added to ZYP-5052 medium (100 μg / mL ampicillin resistance) according to 1% inoculation amount, and cultured at 20° C. and 220 rpm for 48 hours at low temperature to induce the expression of chitosanase.

[0028] After expressin...

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Abstract

The invention provides a chitosanase, the amino acid sequence of which is SEQ ID NO:1. The enzyme has good thermal stability, the optimum reaction temperature and pH are 50°C and 8.0 respectively, and the specific enzyme activity reaches 330U / mg. The chitosanase belongs to endocutting and exocutting mixed type, and its minimum degradable substrate is chitobiose, and can hydrolyze chitosan into glucosamine and chitobiose. It is expected that the chitosanase of the invention can be used in the production of chitosan oligosaccharides, and has practical application value.

Description

technical field [0001] The invention belongs to the technical field of functional gene cloning and expression, and in particular relates to a chitosanase and its application in the preparation of chitosan oligosaccharides. Background technique [0002] Oligochitosan is an oligosaccharide with a degree of polymerization of less than 20 and a molecular weight of less than 3900 after hydrolysis of chitosan, which is produced by N-acetyl-D-glucosamine (GLcNAc) and D-glucosamine (GLcN) linked by glycosidic bonds. Due to lower molecular weight and better solubility, chitosan has a wider range of applications than chitosan. In addition, chitosan oligosaccharides also have unique pharmacological functional activities such as anti-tumor, lowering blood pressure, and enhancing immunity. Jonge et al. reported that chitosan oligosaccharides can inhibit the growth of plant pathogenic bacteria and induce the production of related antibodies in higher plants; Park et al. It is found that...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12P19/26C12P19/14C12P19/12
CPCC12N9/2402C12P19/12C12P19/14C12P19/26C12Y302/01132
Inventor 毛相朝郭娜孙建安薛长湖
Owner OCEAN UNIV OF CHINA
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