Chitosanase OUC-CsnCA and application of chitosanase OUC-CsnCA

A technology of chitosanase and chitosan, applied in the field of functional enzymes, can solve the problems of low expression level, low activity, difficult industrial production, etc., and achieve the effect of high enzyme activity and enzyme production level

Active Publication Date: 2020-08-07
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing chitosanases generally have problems such as low activity, low expression level, and high cost of use, and are difficult to be used in industrialized production. For example, the expression level of recombinant chitosanase in the Chinese invention patent with publication number CN101397552A is 0.5 mg / ml, the specific enzyme activity can only reach 700U / mg, and the protein content of the crude enzyme liquid produced by the chitosan enzyme shake flask fermentation in the Chinese invention patent application whose publication number is CN 107586768A can only reach 0.77mg / ml

Method used

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Embodiment 1

[0025] Embodiment 1: the cloning of chitosanase gene

[0026] The inventors of the present invention analyzed some genome sequences through the method of genome database mining, and selected a batch of gene sequences predicted as chitosanase or putative chitosanase in some bioinformatics. Described excavation method is specifically: take the amino acid sequence of the chitosanase Csn-PD from Paenibacillus dendritiformis as template probe, carry out BLAST search in NCBI database, screen out and chitosanase Csn-PD homology in 30%-80% chitosanase or putative chitosanase sequences.

[0027] According to the above method, a candidate gene from Chromobacterium sp.ATCC 53434 was screened from the database, and according to the sequence information of the bacterium submitted in the Genbank database, the target gene sequence encoding chitosanase was used as the template sequence, and codon optimization was performed. Afterwards, total gene synthesis is performed.

[0028] The upstrea...

Embodiment 2

[0033] Embodiment 2: Contain the expression vector construction of chitosanase gene

[0034] The recovered target gene and plasmid pET-28a(+) were subjected to a seamless ligation reaction.

[0035] After the connection is completed, heat-induced transformation is used to transform the connected system into DH5α competent cells, and positive transformants are screened using LB plates containing kanamycin resistance, and the clones are verified by PCR using T7 universal primers , Agarose gel electrophoresis showed that there was a band near the position of 1000bp, and then the bacterial liquid was sequenced and compared, and the result was 100% consistent, indicating that the recombinant was successfully constructed.

Embodiment 3

[0036] Embodiment 3: expression and purification of recombinant chitosanase

[0037] The constructed recombinant plasmid was transferred into the BL21 expression strain for expression, and the LB plate containing kanamycin resistance was used for screening. After the positive transformant was activated, it was added to the ZYP-5052 medium at an inoculum size of 1%. 20°C, 220rpm shaking culture for 48h, induced expression of chitosanase, chitosanase named OUC-CsnCA. Centrifuge at 8000 rpm and 4°C for 20 min to collect the bacteria, add 10 mL of 50 mM Tris-HCl buffer solution with pH 8.0 to the bacteria pellet, and ultrasonically break for 30 min. Centrifuge at 8000rpm and 4°C for 20min, take the supernatant as the crude enzyme solution, and measure the enzyme activity of the supernatant.

[0038] Purify the recombinant expressed chitosanase with a nickel column, use 10mM imidazole solution (500mM NaCl, 50mM Tris-HCl) to equilibrate the column, after loading the sample, use 20mM,...

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Abstract

The invention discloses chitosanase OUC-CsnCA. The amino acid sequence of the chitosanase OUC-CsnCA is as shown in SEQ ID NO. 1. The invention also discloses a gene for encoding the chitosanase OUC-CsnCA, and the nucleotide sequence of the gene is shown as SEQ ID NO.2. The invention also discloses a preparation method of the chitosanase OUC-CsnCA. The invention also discloses application of the chitosanase OUC-CsnCA in degradation of chitosan / preparation of chitosan oligosaccharide, and further relates to application of the chitosanase OUC-CsnCA in preparation of an enzyme preparation for degrading chitosan. The chitosanase OUC-CsnCA disclosed by the invention is used for degrading chitosan to generate chitosan oligosaccharide, the optimum pH value of the chitosanase OUC-CsnCA is 8.0, theoptimum reaction temperature of the chitosanase OUC-CsnCA is 55 DEG C, and the specific enzyme activity of the chitosanase OUC-CsnCA is 1786.227 U / mg; in addition the enzyme activity and the enzyme production level are high, wherein the protein concentration after purification is 1.284 mg / ml, and compared with reported chitosanase, the chitosanase has certain advantages. The chitosanase OUC-CsnCAdisclosed by the invention has important industrial application value and economic value.

Description

technical field [0001] The invention relates to a high-expression chitosanase OUC-CsnCA and its application in degrading chitosan and preparing chitooligosaccharides, belonging to the technical field of functional enzymes. Background technique [0002] Chitosanase (EC 3.2.1.132), as a specific hydrolase, is the key enzyme for hydrolyzing chitosan to generate oligosaccharide products, which can specifically act on glycosidic bonds and break them, thus becoming shells with lower molecular weight. oligosaccharides. Chitosan oligosaccharides have strong activities in improving immunity, inhibiting tumor cell growth, lowering cholesterol and anti-oxidation, etc., and have unique and important applications in the fields of functional food, medicine, cosmetics and crop biological agents. [0003] At present, chemical methods, physical methods and enzymatic methods are mainly used to prepare chitosan oligosaccharides. Chemical method is the earliest and most commonly used method i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N1/21C12P19/26C12P19/14C12R1/19
CPCC12N9/2402C12P19/26C12P19/14C12Y302/01132
Inventor 毛相朝孙建安苏海鹏
Owner OCEAN UNIV OF CHINA
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