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Chitin histone deacetylase mutant

A technology of deacetylase and chitin, applied in the field of bioengineering

Active Publication Date: 2016-08-24
JIANGSU AOXIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the expression level is high, the enzymatic properties such as the catalytic efficiency of the enzyme need to be further improved
The research on chitin deacetylase in my country mainly focuses on strain selection, enzyme purification, expression, enzymatic properties, and improvement of fermentation process. There is still a lack of chitin deacetylase with high catalytic efficiency.

Method used

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Examples

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Embodiment 1

[0028] Implementation example 1, construction of mutant expression plasmid and acquisition of recombinant Escherichia coli

[0029]Primers were designed according to the wild-type enzyme gene, and the restriction site was introduced. The primers used were as follows: P1: 5'-CGGAATTCATGGCGCGTGGTGTCCGCAT-3' (introduced into EcoR I restriction site); P2: 5'-CCAAGCTTCTATGCCGTGGCTTCACCGGA-3' (introduced into HindIII Restriction sites). PCR was performed using Salinispora arenicola genomic DNA as a template. The PCR reaction system was: ddH2O (17.5 μL), buffer (2.5 μL), Mg2+ (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 5 minutes. The amplified products were detected by 1.0% agarose gel electrophoresis and then sequenced. The above-mentioned PCR amplification products were subjected to 1.0% ag...

Embodiment 2

[0038] Implementation example 2, transformation of mutant expression vector

[0039] The medium composition used: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH7.0. 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 mL-1 ampicillin, and filled with 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. 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. According to Ni-NTA Purification System (...

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Abstract

The invention discloses a chitin histone deacetylase mutant, and belongs to the technical field of bioengineering. 52nd-bit valine V of chitin histone deacetylase of obligate Salinispora arenicola is mutated to obtain glycine G, and a 66th-bit proline point P is mutated to obtain serine S. The chitin histone deacetylase mutant has the advantages that the specific activity of enzymes of the chitin histone deacetylase mutant is 3.92 times the specific activity of wild type enzymes as compared with the enzymes of the chitin histone deacetylase mutant prior to mutation; the chitin histone deacetylase is applicable to industrial production requirements, and social production requirements can be met.

Description

technical field [0001] The invention discloses a mutant of chitin deacetylase, which belongs to the technical field of bioengineering. 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. A natural polymer next to cellulose. Chitin is almost insoluble in water and general organic solvents, which limits the application of chitin (. Chitin is deacetylated to form chitosan, the chemical name is polyglucosamine (1-4)-2-amino-B-D Glucose. Chitosan is soluble in acidic and neutral aqueous solutions, has good biocompatibility and biodegradability, and is widely used in medicine, food, chemical industry, cosmetics, water treatment, metal extraction and recovery, biochemical and biomedical engineering and many other fields (Pradip Kumar Dutta. Chitin and Chitosan for Regenerative Medicine [M]. Springer India, 2016). [0003] At present, the industr...

Claims

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Application Information

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IPC IPC(8): C12N9/44C12N15/56C12N15/70
CPCC12N9/2457C12N15/70C12N2800/101C12Y302/01041
Inventor 王松叶詹金明陈延静陈进利赵艳凯
Owner JIANGSU AOXIN BIOTECHNOLOGY CO LTD
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