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Cryogenic chitosanase and encoding gene and application thereof

A chitosanase and chitosan technology, applied in application, glycosylase, genetic engineering and other directions, can solve the problems of low enzyme production activity of natural strains and unfavorable industrial application, and achieve low energy consumption and mild reaction conditions. , the effect of important economic value

Active Publication Date: 2017-06-09
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on low-temperature chitosanase, and some disclosed low-temperature chitosanase-producing natural strains have low enzyme activity, which is not conducive to industrial application (publication number: CN102653749A)

Method used

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  • Cryogenic chitosanase and encoding gene and application thereof
  • Cryogenic chitosanase and encoding gene and application thereof
  • Cryogenic chitosanase and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Obtaining of gene GsCho46A and protein GsCho46A

[0066] According to the Gynuella sunshinyii whole genome sequence information submitted in the Genbank database (published in the document "Int.J.Syst.Evol.Micr.2015,65,1038-1043"), the Glycoside Hydrolase(GH)46 family is unknown. The target gene sequence of hypothetical protein (Hypothetical Protein; Genbank ID: AJQ97965) is the template sequence, and the target gene is synthesized from the whole gene.

[0067] Design the upstream primer GsCho46A-up(5’-ATTCTA GCTAGC ATGAATCCGTTCTGGCATTTTG-3’, the underline shows the Nhe I restriction site) and the downstream primer GsCho46A-down(5’-ATTCCG CTCGAG TTAACGGATCGGCAGGATGAAAAC-3', underlined shows the XhoI restriction site), using the target gene synthesized by the whole gene as a template, PCR amplification obtains the target DNA fragment.

[0068] The PCR amplification conditions were: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30s, 54°C annealing for 3...

Embodiment 2

[0072] Example 2. Expression and purification of recombinant chitosanase (GsCho46A) and detection of its properties

[0073] 1. Expression and purification of recombinant chitosanase (GsCho46A)

[0074] Transform and express the recombinant plasmid in Example 1 into host Escherichia coli BL21(DE3) (Beijing Bomed Gene Technology Co., Ltd., product number: BC201-01) to obtain recombinant bacteria, and inoculate them into 1L LB liquid medium (Containing 50μgmL -1 Kanamycin), cultivated to OD at 37°C and 200rpm 600 Between 0.6-0.8, add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 1 mM, and induce overnight at 30°C. After collecting the cells by centrifugation, the cells were resuspended in buffer A (20mM Tris-Hcl, 0.5M NaCl, 20mM imidazole, pH 7.9) at a ratio of 1:10 (v / v), and then placed in an ice water bath Mid-ultrasonic break (200W, ultrasonic 3s, intermittent 4s, 120 times), and then centrifuge to collect the supernatant is the crude enzyme solution. The crud...

Embodiment 3

[0085] Example 3. Application of recombinant chitosanase (GsCho46A) in the preparation of chito-oligosaccharides by enzymatic method

[0086] The reaction conditions of the recombinant chitosanase (GsCho46A) for hydrolysis of chitosan refer to the optimal reaction conditions of the enzyme: pH5.5, 30°C, substrate concentration 1%, enzyme amount 0.5U / mL, hydrolysis time 2h. The volume of the hydrolysate is 5L, and the stirring speed is 80-120rpm / min. Samples were taken at 0, 5, 10, 15, 30, 60, 120 min, and the reaction was terminated by incubating at 50°C for 10 min to inactivate the enzyme.

[0087] (1) Thin layer chromatography (TLC) to monitor hydrolysate

[0088] A Kieselgel 60 silica gel plate (Merck) was used to analyze the hydrolysate, and the spreading liquid was n-butanol: methanol: ammonia: water (5:4:2:1, v / v / v). Spot 2μL of the sample on the spot on the silica gel plate, spread the silica gel plate with a spreading agent, blow dry and evenly wet the surface with the color...

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Abstract

The invention discloses rhizosphere bacterium gynuella sunshinyii-sourced chitosanase (GsCho46A) and an encoding gene and application thereof. The chitosanase is a) or b) or c): a) protein of which the amino acid sequence is encoded by 1st-266th amino acid residues in SEQ ID NO.2; b) fusion protein obtained by connecting labels and / or a label to the N end and / or the C end of the protein shown by the 1st-266th amino acid residues in the SEQ ID NO.2; c) protein obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown by the 1st-266th amino acid residues in the SEQ ID NO.2 and having the same function. The chitosanase disclosed by the invention has excellent enzymatic properties: the activity under a cryogenic condition is relatively high and a requirement on enzymatic hydrolysis equipment is relatively low; through temperature control, high-polymerization chitooligosaccharide with the degree of polymerization (DP) 2-7 can be efficiently prepared; the reaction condition is mild, the energy consumption is low, and no pollutant is produced; during enzymatic preparation of the chitooligosaccharide, the chitosanase has an important application value and an important economic value.

Description

Technical field [0001] The invention belongs to the field of food biotechnology, and particularly relates to a low-temperature chitosanase (GsCho46A) derived from the rhizosphere bacterium Gynuella sunshinyii and its coding gene and application. Background technique [0002] Chitooligosaccharides (COS) are small molecular oligosaccharides produced by the degradation of chitosan derived from marine shrimp and crab shells. The D-glucosamine (or a small amount of N-acetyl-D-glucosamine) in the chitosan oligosaccharide molecule is connected by β-1,4-glycosidic bonds. The degree of polymerization (DP) is usually between 2-10, and the molecular weight is generally not More than 3kDa. Chitooligosaccharides have good water solubility and rich physiological activities. Different DPs have different physiological functions. Among them, the chitooligosaccharides of DP6-8 can improve the phagocytic ability of macrophages, inhibit tumor cell growth, reduce cholesterol, It has strong activity...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N1/21C12P19/26C12P19/14C12R1/19
CPCC12N9/2402C12P19/14C12P19/26C12Y302/01132
Inventor 赵黎明秦臻陈启明邱勇隽
Owner EAST CHINA UNIV OF SCI & TECH
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