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Chitosanase Csn-PT and application thereof

A technology of chitosanase and chitosan, applied in the field of functional enzymes, can solve the problems of low enzyme production level of wild-type strains, difficult to meet industrial production and application, etc., and achieve good biocatalysis efficiency, high yield and high efficiency. Effect

Active Publication Date: 2020-07-31
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme production level of the wild-type strain is low, and it is difficult to meet the requirements of industrial production and application. Heterologous expression can be used to increase the enzyme production level to prepare enzyme preparations for industrial production.

Method used

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  • Chitosanase Csn-PT and application thereof
  • Chitosanase Csn-PT and application thereof
  • Chitosanase Csn-PT and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The cloning of embodiment 1 chitosanase gene Csn-PT

[0026] The enzyme-producing gene of the chitosanase Csn-PT of the present invention is an artificially synthesized sequence. The inventor excavated the chitosanase fragment of Paenibacillus tyrfis preserved in the Marine Culture Collection of China (MCCC) preservation number MCCC: 1K01247, and the inventor obtained the gene sequence according to the host large intestine Bacteria codon preference, codon optimization for high-efficiency expression in E. coli. The chitosanase Csn-PT gene of the present invention includes a sequence of 897 bases, as shown in SEQ ID NO.2, and encodes a sequence of 299 amino acids, as shown in SEQ ID NO.1. According to the comparison of phylogenetic tree, it was found that the chitosanase belongs to the 46th family of polysaccharide hydrolase (GH-46).

[0027] Using the synthesized fragment as a template, primers for seamless connection were designed on the upstream and downstream of the...

Embodiment 2

[0034] Embodiment 2 Containing the expression vector construction of chitosanase gene

[0035]The gene fragment and the pET-28a cloning vector were connected by seamless cloning technology, and the connection product was transferred into E.coli DH5α competent cells, and spread on the (LB) medium solid plate containing 50 μg / m L kanamycin . After culturing in a 37°C incubator for 12-16 hours, pick a single clone into LB liquid medium containing 50 μg / mL kanamycin, culture overnight on a shaker at 37°C with a rotation speed of 220 rpm, sequence after positive verification, and name it pET28a -Csn-PT.

Embodiment 3

[0036] Embodiment 3 Containing the recombinant plasmid of chitosanase gene and the construction of engineering bacteria

[0037] 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 Csn-PT. The amino acid sequence of the chitosanase Csn-PT is shown as SEQ ID NO. 1. The invention further discloses a gene for coding the chitosanase Csn-PT, andthe nucleotide sequence of the gene is shown as SEQ ID NO. 2. The invention also discloses application of the chitosanase Csn-PT in degrading chitosan and application of the chitosanase Csn-PT in preparing an enzyme preparation for degrading chitosan. The chitosanase Csn-PT provided by the invention is used for degrading chitosan, has the optimum pH of 8, the optimum reaction temperature of 50 DEGC and the specific enzyme activity is 10163.826 U / mg, the chitosanase Csn-PT has high catalytic activity, and has obvious advantages in the aspects of catalytic activity and efficiency compared withreported chitosanase.

Description

technical field [0001] The invention relates to chitosanase Csn-PT and its application in degrading chitosan, belonging to the technical field of functional enzymes. Background technique [0002] Oligochitosan is the degradation product of chitosan, and its degree of polymerization (DP) is generally between 2 and 10. Cholesterol, lowering blood pressure, preventing infection and controlling arthritis, etc., which makes chitosan have greater application value in medicine, health products, food, agriculture and other fields. [0003] At present, the preparation methods of chitosan oligosaccharide mainly include chemical method, physical method and biological enzymatic method. The preparation of chitosan oligosaccharides by chemical and physical methods has the disadvantages of difficult control of process conditions, large environmental pollution, and low yield of oligosaccharides. When non-specific enzymes such as lipase and protease are used to prepare chitosan oligosaccha...

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
CPCC12N9/2434C12N15/70C12P19/26C12P19/14C12Y302/01132
Inventor 毛相朝孙建安苏海鹏
Owner OCEAN UNIV OF CHINA
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