A kind of chitosanase and its application

A technology of chitosanase and chitosan, applied in the direction of glycosylase, enzyme, hydrolase, etc., to achieve good biocatalytic activity, easy control, and efficient degradation effect

Active Publication Date: 2022-04-12
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most of the degradation products of chitosanase are (GlcN) 2 -(GlcN) 4 and (GlcN) 2 -(GlcN) 6 , and some products are rare GlcN-(GlcN) 5 and GlcN-(GlcN) 3 , but has not been able to degrade to produce GlcN-(GlcN) 4 Chitosanase

Method used

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  • A kind of chitosanase and its application
  • A kind of chitosanase and its application
  • A kind of chitosanase and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Cloning of embodiment 1 chitosanase gene Csn-gly

[0024] In order to mine a GlcN-(GlcN) 4 Chitosanase, the inventors passed to be able to generate (GlcN) 2 -(GlcN) 4 The chitosanase derived from Streptomyces griseus HUT 6037 was used as a template, and a chitosanase with an amino acid sequence similarity of 88.28% to the template was discovered by means of molecular biology. The chitosanase was derived from Streptomyces bacillaris SDUM420012 (purchased from Weihai Marine Microorganism Resource Center of Shandong University), by cloning the gene expressing the enzyme, thereby contributing to the present invention. Its nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.1. Blast results showed that Csn-gly is a new member of glycoside hydrolase class 5 family.

[0025] Using the genome of Streptomyces bacillaris SDUM420012 as a template, primers for seamless connection were designed on the upstream and...

Embodiment 2

[0032] Embodiment 2 Contains the expression vector construction of chitosanase gene

[0033] The gene fragment was connected with the PET-28a cloning vector using seamless cloning technology, and the connection product was transferred into E.coli DH5α competent cells. The LB plates containing kanamycin sulfate resistance were screened for positive transformants, the clones were verified by colony PCR using T7 universal primers, and positive clones were picked for sequencing.

Embodiment 3

[0034] Embodiment 3 Contains the recombinant plasmid of chitosanase gene and the construction of engineering bacterium

[0035] The recombinant plasmids with correct sequencing were extracted and transformed into host E.coli BL21 competent cells, and the constructed engineering bacteria were grown on kanamycin sulfate-resistant plates.

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Abstract

The invention discloses a chitosanase, the amino acid sequence of which is shown in SEQ ID NO.1. The nucleotide sequence of the gene encoding the chitosanase is shown in SEQ ID NO.2. Chitosanase of the present invention, derived from the GH5 family of glycoside hydrolase, can degrade chitosan to produce GlcN-(GlcN) 4 , can be used to prepare new components of the enzymatic COS mixture. The chitosanase of the present invention has good biocatalytic activity, can hydrolyze chitosan to produce glucosamine, chitobiose, chitotriose and chitotetraose, and the product has high purity; at pH 10 and 50°C, its relative The enzyme activity is 898.6U / mg, which is higher than most other known chitosan enzymes. It can efficiently degrade chitosan. The reaction conditions are mild, easy to control, high efficiency, and environmentally friendly. It has the ability to produce a new type of enzymatic chitosan oligosaccharide mixture industrial application value.

Description

technical field [0001] The present invention relates to a chitosan enzyme and application thereof, in particular to a chitosan enzyme capable of degrading chitosan to generate GlcN-(GlcN) 4 The chitosanase and its application in the preparation of chitosan oligosaccharides belong to the technical field of functional gene discovery, cloning and characterization. Background technique [0002] Chitosan is derived from the complete or partial deacetylation of chitin, and is randomly composed of D-glucosamine (GlcN, D) and N-acetyl-D-glucosamine (GlcNAc, A) through β-1,4 glycosidic bonds. connected. Although chitosan has unique and excellent polymer structure diversity, its high molecular weight and low bioavailability greatly limit its application. However, the degradation product of chitosan, chitooligosaccharide (COS), is widely used in food industry, medicine, agriculture and cosmetic industry due to its superior solubility and biological function. [0003] Chitosanase (EC...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12P19/00C12P19/04C12P19/12C12P19/14C12P19/26
CPCC12N9/2402C12Y302/01132C12P19/12C12P19/00C12P19/04C12P19/26C12P19/14
Inventor 毛相朝孙建安苏海鹏薛长湖
Owner OCEAN UNIV OF CHINA
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