Chitin deacetylase mutant with improved catalytic activity and preparation method thereof

A technology for improving catalytic activity and deacetylase, which is applied in the field of bioengineering to achieve the effects of strong penetrating power, favorable application and improved catalytic activity

Inactive Publication Date: 2019-05-21
JIANGSU AOXIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research on chitin deacetylase in my country mainly focuses on strain selection, enzyme purification, expression, enzymatic properties, and improvement of fermentation process. There are few reports on chitin deacetylase with high catalytic efficiency.

Method used

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  • Chitin deacetylase mutant with improved catalytic activity and preparation method thereof
  • Chitin deacetylase mutant with improved catalytic activity and preparation method thereof
  • Chitin deacetylase mutant with improved catalytic activity and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The construction of embodiment 1 mutant expression plasmid and the acquisition of recombinant Escherichia coli

[0028]Primers were designed according to the wild-type enzyme gene, and the restriction site was introduced. The primers used were as follows: P1: 5'-CGGAATTCATGAACCGTTACCCGCGTGAC-3' (introduced restriction site EcoR I); P2: 5'-CCCATTGTTAAGCCAGAGCTTCCAGACGC-3' (introduced Noc I restriction site).

[0029] Use the synthesized DNA sequence as a template for PCR, and the PCR reaction system is: ddH 2 O (17.5μL), buffer (2.5μL), Mg 2+ (2.5 μL), dNTP (0.5 μL), P1 (0.5 μL), P2 (0.5 μL), template (1 μL), Taq enzyme (0.2 μL). PCR program: pre-denaturation at 94°C for 5 minutes, followed by 35 cycles at 94°C for 30s, 54°C for 40s, and 72°C for 70s; extension at 72°C for 10 minutes. The amplified products were detected by 1.0% agarose gel electrophoresis and then sequenced.

[0030] The above PCR amplified product was subjected to 1.0% agarose gel electrophoresis, ...

Embodiment 2

[0048] Transformation of embodiment 2 mutant expression vector

[0049] The medium composition used: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH7.0.

[0050] The recombinant bacterium E.coli BL21(DE3)(pEtac-His6-cad) containing the gene encoding the mutant chitin deacetylase was activated and cultured and then inoculated into the fermentation medium containing 100 μg mE-1 ampicillin, and filled with liquid The amount is 30mL / 250mL, cultured at 37°C, 200r min-1; the bacteria grow to OD600=0.6, add final concentration of 0.5mM IPTG, 30°C, induce fermentation for 24h. 180r / min induced expression for 5h. Cells were collected by refrigerated centrifugation at 10,000 r / min at 4°C for 5 min.

[0051] Resuspend the bacteria in distilled water, crush the bacteria with an ultrasonic cell disruptor (200W, super 3s, stop 7s, ultrasonic 5min in total), centrifuge at 10000r / min at 4°C for 20min, and collect the supernatant as a crude enzyme solution.

[0052] According to Ni-NTA P...

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Abstract

The invention discloses a chitin deacetylase mutant with the improved catalytic activity and a preparation method thereof, and belongs to the technical field of bioengineering. Marine bacterium Roseivivax atlanticus chitin deacetylase as shown in SEQ ID NO.1 is mutated as below, 116 asparagine Q is mutated to arginine R, 179 asparagine D is mutated to glycine G, 194 histidine H is mutated to tyrosine Y, 254 valine V is mutated to leucine I, and 282 histidine H is mutated to aspartic acid. Compared with the specific activity before mutation, the specific activity of a mutant enzyme is 2.63 times that of a wild type enzyme. Accordingly, the chitin deacetylase catalytic efficiency is higher, and the mutant is more suitable for industrial production requirements.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a chitin deacetylase mutant with improved catalytic activity, a gene containing the mutant, a cell and a preparation method thereof. Background technique [0002] Chitin, also known as chitin, chitin, etc., is a linear polymer compound formed by N-acetyl-D glucosamine (GlcNAc) monomers linked by β-1,4 glycosidic bonds. Cellulose is the second largest natural organic compound in the world. Chitin is insoluble in water and common organic solvents, making it difficult to apply. Chitosan, formed after deacetylation of chitin, has good biocompatibility and biodegradability, and is widely used in food, medicine, light industry, printing and dyeing, environmental protection and agriculture. [0003] The treatment methods of deacetylation of chitin into chitosan are divided into chemical hot alkali method and biological enzyme method. The chemical method not only ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12N15/70C12N1/21
Inventor 詹小远房耀维王松叶詹金明
Owner JIANGSU AOXIN BIOTECHNOLOGY CO LTD
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