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Chitin enzyme and application thereof

A technology of chitin enzyme and chitin, applied in the direction of application, enzyme, enzyme, etc., can solve the problems of low product yield, environmental burden, and large consumption of chemical reagents

Active Publication Date: 2018-07-31
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical method mainly uses chemical reagents such as hydrochloric acid and hydrogen peroxide to degrade chitin to obtain oligosaccharides with a molecular weight of <3000. However, due to the severe reaction conditions, the reaction process cannot be controlled, the purity of the reaction product is low, and a large amount of chemical reagents are consumed. Not only cause a lot of waste of resources, but also easily bring a burden to the environment
The physical method uses energy such as ultrasonic wave, microwave, γ-ray and light to degrade chitin, but the yield of the product prepared by the physical method is low and the distribution of the product is not uniform, so it has not been widely used in industrial production
The enzymatic preparation of chitosan oligosaccharides has mild reaction conditions, the reaction process is easy to control, and the product is relatively pure and easy to separate, which is of great significance for the large-scale production of chitosan oligosaccharides; Sulfase

Method used

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  • Chitin enzyme and application thereof
  • Chitin enzyme and application thereof
  • Chitin enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Cloning of embodiment 1 chitinase gene saChiA4

[0022] The applicant used Streptomyces albolongus (ATCC 27414) to ferment chitin, and found that the amount of chitobiose in its product was significantly higher than that of other strains. Therefore, the genome of the strain was analyzed, and the chitinase obtained in the present invention was found.

[0023] According to the obtained full-length sequence of the chitinase gene, the upstream and downstream primers for amplification were designed, respectively SaChiA4F 5'-CCGGAATTCATGGAACGCGTACTACCC-3' and SaChiA4R5'-CCCAAGCTTGCAGGCGCCGTTGTCCGC-3'. The genome was used as a template for PCR amplification.

[0024] The PCR reaction system is: 2×PCR Buffer 25 μl, dNTP 5 μl, upstream and downstream primers 1 μl, template 1 μl, KOD Fx DNA polymerase (TOYOBO, KFX-101) 1 μl, add sterile water to a final volume of 50 μl.

[0025] The PCR reaction conditions were: pre-denaturation at 94°C for 2 min, denaturation at 98°C for 10 s,...

Embodiment 2

[0028] Expression of embodiment 2 recombinant protein

[0029] The correct recombinant plasmid pET28a::saChiA4 was transformed into competent cells of Escherichia coli BL21(DE3), spread evenly on LB plates with kana resistance (50ug / ml), and cultured overnight at 37°C. A single colony was picked and inserted into a test tube containing kana resistance (50ug / ml), and cultured at 37°C and 220rpm for 12h. Inoculate 1% to 2% of the inoculum into ZYP-5052 self-induction medium and induce at 20°C for 48 hours. After induction, transfer the bacterial solution to a 50mL centrifuge tube and centrifuge at 4°C for 10min to collect the cells.

[0030] ZYP-5052 medium: 1.0% peptone, 0.5% yeast powder, 0.7% Na 2 HPO4, 0.7%KH 2 PO4, 0.3% (NH4) 2 SO4, 0.05% MgSO 4 , 0.5% glycerin, 0.05% glucose, 0.2% α-lactose.

[0031] Using the principle of affinity chromatography, a Ni column was used to purify the protein of the tag expression vector containing 2 histidines. The cell pellet was firs...

Embodiment 3

[0032] The mensuration of embodiment 3 enzyme activity

[0033] The chitinase activity was determined using the DNS method.

[0034] Reaction system: 195 μL colloidal chitin (0.1g / mL), 5 μL pure enzyme (5U / mL), incubate at 55°C for 30 minutes, after the reaction, add 300 μL DNS in a boiling water bath for 10 minutes, boil for 10 minutes, and develop color. Centrifuge at 10000rpm for 5min, take 200μL supernatant, and measure OD 540 . Protein concentration was measured according to the Bradford method using bovine serum albumin (BSA) as a standard. Definition of enzyme unit: An enzyme activity unit (U) refers to the amount of enzyme required to release 1 μmol of reducing sugar per minute under the optimal reaction conditions of the enzyme.

[0035] Determination of the optimal reaction conditions of chitinase.

[0036] Take an equal amount of enzyme solution (5 μL) to react at 55° C. under different pH conditions (pH 3.0-10.0), measure the enzymatic activity of chitinase, an...

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Abstract

The invention aims at providing a novel chitin enzyme with a high-efficiency degradation rate. The high-activity soluble chitin enzyme is obtained through cloning a gene, which is used for coding thechitin enzyme, in a genome of streptomyces albolongus ATCC (American Type Culture Collection) 27414, constructing a recombinant vector and transferring the recombinant vector into escherichia coli BL21 (DE3) to carry out inducible expression. Through Ni column affinity chromatography, the chitin pure enzyme is purified and obtained; the enzymatic property and hydrolysis mechanism of the chitin pure enzyme are researched; the optimum temperature and optimum pH (potential of Hydrogen) of the enzyme are 55 DEG C and 5.0 respectively; the specific enzyme activity is 66.2U / mg; a main product of degraded chitin is chitobiose, and the chitin enzyme is expected to be applied to the preparation of a chitooligosaccharide, and has potential application value.

Description

technical field [0001] The invention belongs to the technical field of functional gene screening, and in particular relates to a chitinase and its application. Background technique [0002] Chitin, also known as chitin, chitin, etc., is a linear high-molecular polysaccharide polymer connected by N-acetyl-D-glucosamine (GlcNAc) through β-1,4-glycosidic bonds. Chitin widely exists In crustacean shells, fungal cell walls, insects, algae cells, and cartilage or inner shells of molluscs, it is the second largest polysaccharide polymer in nature after cellulose. However, the insolubility of chitin is the biggest limiting factor for the application of chitin, and the degradation product of chitin, chitooligosaccharides (polymerization degree is 2 to 10), has a wider range of biological activities, such as; anti-oxidation, anti-tumor, improving immunity In addition, chitosan is also a growth-promoting factor for intestinal beneficial bacteria, and can be used as a supplement for fu...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12P19/26C12P19/14C12P19/12
CPCC12N9/2442C12P19/12C12P19/14C12P19/26C12Y302/01014
Inventor 毛相朝高丽孙建安薛长湖
Owner OCEAN UNIV OF CHINA
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