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Purification, gene cloning, and enzymatic characteristic identification of chitosanase

A technology of chitosanase and chitosan, used in genetic engineering, plant genetic improvement, biochemical equipment and methods, etc.

Inactive Publication Date: 2014-06-25
SUZHOU ZHONGTONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chitosan currently used in production is basically the deacetylation product of chitin.

Method used

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  • Purification, gene cloning, and enzymatic characteristic identification of chitosanase
  • Purification, gene cloning, and enzymatic characteristic identification of chitosanase
  • Purification, gene cloning, and enzymatic characteristic identification of chitosanase

Examples

Experimental program
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Effect test

Embodiment 1

[0012] Cultivation of chitosanase-producing strains: We isolated a miscellaneous bacterium from the contaminated yeast culture solution, Gram staining and morphological observation showed that it was a Gram-positive bacillus ( figure 1 ), tentatively named Bacillussp.TK-02. The strain was first cultured overnight on LB solid medium (30°C), and then a single clone was picked and inoculated in LB liquid medium for shaking culture at 30°C for 24 hours. The bacterial solution was centrifuged (5000g, 10min), and the supernatant was collected.

Embodiment 2

[0014] Purification of chitosanase and determination of N-terminal amino acid sequence: the composition of the supernatant of the fermentation broth of this bacterium is relatively simple, and the secreted expression product is mainly the chitosanase ( figure 2 A). After the culture supernatant is concentrated, it is separated and purified. First, the chitosanase is separated from the large-volume fermentation supernatant by hydrophobic chromatography. The SDS-PAGE identification shows that the chitosanase obtained in this step is purified. It mainly contains a heteroprotein with a large molecular weight ( figure 2 B, lane 1). Accordingly, purifying by molecular sieves, high-purity chitosanase ( figure 2 B, lane2). The purity of the chitosanase was further identified by C3 reverse phase HPLC. Such as image 3 As shown, the enzyme has only one peak on HPLC, and its purity is greater than 95%. The molecular mass of the eluted chitosanase was determined by electrospray m...

Embodiment 3

[0017] Cloning of chitosanase gene: According to the DNA sequence of other chitosanases, take the conserved gene sequence before the signal peptide and the termination signal TAA, and design primers as follows: Primer 1:

[0018] 5'-tta-aaaggagctgacaacataat3'; Primer 2: 5'-ttaatatcgtatccttcataaattgca-3'. The bacterial genome DNA was used as a template, and the above primers were used for PCR amplification. A sequence of about 1.3 kb was amplified, and then cloned into a T-vector for DNA sequence determination. Through searching online (http: / / www.ncbi.nlm.nih.gov / BLAST), its sequence has high homology with other 11 kinds of chitosanases, reaching 96%-98%, and their numbers in GenBank are respectively: AAQ 75085, ZP00240544, AAK 07481, YP 084010, NP 832437, NP845032, BAB 19277, AAQ 75084, AAQ 19679, AAO49750, and AAQ 19678. They all belong to the glycoside hydrolase 8 family, and all are derived from the genus Bacillus. The gene encodes 453 amino acids, of which the N-termin...

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Abstract

The invention relates to purification, gene cloning, and enzymatic characteristic identification of chitosanase. A strain is separated from a contaminated yeast culture solution, and is identified to be a gram-positive bacillus; a main protein product secreted by the strain is purified, and subjected to N-end amino acid sequence determination, and the result shows that the protein product is quite similar to some known endo-chitosanases. Based on the homologous DNA sequence, a full-length gene is cloned by a PCR method. The strain grows quite rapidly, and the constitutive secretion product is mainly endo-chitosanase. The enzyme can be purified by two steps of hydrophobic chromatography and molecular sieve. The enzymatic characteristics of the enzyme and the conditions for catalyzing the degradation of chitosan into chitosan oligosaccharide by the enzyme are further studied. When the yield of the chitosanase is increased, the chitosanase is expected to be used for chitosan oligosaccharide production, and the chitosanase has actual application value.

Description

technical field [0001] The invention relates to a Gram-positive bacillus which secretes a chitosanase, in particular to the purification, gene cloning and identification of the chitosanase of the chitosanase. Background technique [0002] Chitin (chitin) is mainly a chain polymer formed by N-acetylglucosamine through β-1,4-glucosidic bonds, and is mainly found in the shells of aquatic crustaceans, molluscs and arthropods (such as shrimp, crab, etc.) etc.), with an annual output of more than 1 billion tons, second only to cellulose, and is a class of abundant natural resources. However, chitin is insoluble in common solvents, so it is very difficult to use directly. After the butin is treated with lye, the deacetylation product (the degree of deacetylation is 65% to 99%, and 70% to 80% is the most common) is obtained, that is, chitosan. Chitosan also occurs in nature, mainly in the cell walls of some fungi and some green algae. However, the chitosan currently used in produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/42C12N15/56C12Q1/34C12P19/14C12R1/07
Inventor 杨艳菊何胜祥
Owner SUZHOU ZHONGTONG BIOTECH
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