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Recombinant aspergillus flavus chitosanase and encoding gene, as well as preparation and application

A kind of Aspergillus flavus chitosanase and coding gene technology, applied in the fields of application, glycosylase, genetic engineering, etc., can solve the problems that the production and activity of chitosanase are difficult to meet the application, and the activity needs to be improved, so as to achieve the goal of fermentation enzyme Vitality improvement, high-efficiency secretion expression, and high-efficiency degradation effects

Active Publication Date: 2017-12-01
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the chitosanase gene of the Aspergillus flavus strain was recombinantly expressed in Pichia pastoris X-33, the endochitosanase activity reached 286.8±5.4U / mL (Zhang T et al, Process Biochemistry, 2009,44:1335- 1339), but the yield and activity of recombinant chitosanase are still difficult to meet its application in food and pharmaceutical industries
Chitosanase in Aspergillus flavus has been secreted and expressed in Pichia pastoris, but the expression level and activity are still far from industrial production (Xiaomei Chen et al, Biotechnol Lett (2012) 34:689–694), and the activity needs to be improved

Method used

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  • Recombinant aspergillus flavus chitosanase and encoding gene, as well as preparation and application
  • Recombinant aspergillus flavus chitosanase and encoding gene, as well as preparation and application
  • Recombinant aspergillus flavus chitosanase and encoding gene, as well as preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Aspergillus flavus endo-chitosanase 3D model construction

[0025] Using the homologous modeling server Swiss-Model (http: / / swissmodel.expasy.org), the chitosanase mutants are automatically modeled, and the 3D structure is as follows figure 1 . The N-terminal of the chitosanase mutant is composed of 3 sheets and 3 helical sheets, and the C-terminal is composed of 2 sheets and a linear structure, connected by a linker in the middle. from figure 1 It can be seen that the chitosanase mutant position 95D, away from the catalytic active center (D143 / E152), will not affect the catalytic activity of the enzyme

Embodiment 2

[0026] Embodiment 2 mature chitosanase mutant gene cloning

[0027] Taking the Aspergillus flavus chitosanase gene as a reference (the sequence was synthesized by Suzhou Jinweizhi Co., Ltd., GenBank number AY190324, the mature recombinant chitosanase gene was mutated at two sites A286G and C287T, and the corresponding amino acid residue 95D was mutated to 95G ), designed amplification primers, C-F: 5'-TCT CTCGAG TACAACCTACCCAAC-3' and C-R:5'-CTC GCGGCCGC TTAAGCCTTCAAACCAGCAAC-3', wherein the underline is respectively XhoI, NotI enzyme cutting site, and italics is the Kex2 signal cutting site, then carry out polymerase chain reaction (PCR) amplification to obtain recombinant chitosanase mutant gene (without signal Peptide sequence), where the underlines are the XhoI and NotI restriction sites, and the oblique line is the Kex2 signal cleavage site. PCR reaction was carried out with LATaq DNA polymerase. The reaction conditions were: pre-denaturation at 94°C for 2 min, then ...

Embodiment 3

[0051] Embodiment 3 Construction of recombinant plasmid pPICZαA-N-chitosanase mutant

[0052] The construction mode of the recombinant plasmid pPICZαA-N-chitosanase mutant is as follows figure 2 , the α-factor secretion signal sequence gene on the vector pPICZαA is directly connected to the N-terminal recombinant chitosanase mutant gene (without signal peptide sequence), and the Kex2 signal peptide cleavage site on the vector pPICZαA is used to cut off the secretory expression The chitosanase mutant signal peptide sequence was obtained to obtain a mature recombinant chitosanase mutant. The specific construction process of the pPICZαA-N-chitosanase mutant is as follows.

[0053]The purified PCR product was TA-ligated with the T vector pMD19-T, and the ligated product was transformed into E.coli Top10. Positive clones were obtained through blue-white screening and colony PCR identification. At the same time, the plasmid was extracted and sent to Beijing Liuhe Huada Gene Techno...

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Abstract

The invention provides preparation of a high-effective aspergillus flavus (Aspergillus sp.Y2K) chitosan endonuclease mutant. The mutation enzyme can be used for hydrolyzing chitosan to produce oligosaccharide or monosaccharide. The method mainly includes the following steps: 1) cloning a DNA sequence of a matured recombinant chitosanase gene, with mutation at two loci A286G and C287T, onto an expression vector pPICZ[alpha]A of pichia pastoris; and 2) integrating the recombinant plasmid to pichia pastoris host bacteria X-33 through an electric-conversion process to form the recombinant chitosanase mutant which can high-effectively secrete and express and high-effectively degrade chitosan and is high in purity. Compared with a non-mutation enzyme, the chitosan endonuclease mutant is increased in activity by three times. The chitosan endonuclease is widely applied to the fields of foods, medicines, bio-resources, etc.

Description

technical field [0001] The invention provides the preparation and application of a high-efficiency recombinant Aspergillus flavus chitosanase mutant. The recombinant chitosanase mutant in the supernatant prepared by this method reaches 1.02mg / ml, and the activity of the fermented enzyme is as high as 4400U / ml. The purity reaches 90%. The recombinant chitosanase mutant prepared by the invention has potential industrial application value and can be widely used in the fields of food, medicine, biomass transformation and the like. Background technique [0002] Chitosan (chitosan) was first discovered in the 19th century. It is obtained by deacetylation of chitin widely present in nature. This natural polymer has good safety, biocompatibility and microbial degradation. A large number of studies have shown that chitosan has broad application prospects in the fields of food, chemical industry, medicine and agriculture (Gao Wei, Ding Wenping. Preparation and application of low mol...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/81C12P19/26
CPCC12N9/2402C12N15/81C12N2800/102C12P19/26C12Y302/01132
Inventor 尹恒谭海东陈玮李悝悝曹海龙王文霞王江
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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