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Chitosanase mutant G21K and application thereof

A chitosanase and chitosan technology, applied in the directions of glycosylase, enzyme, hydrolase, etc., can solve the problems of high price, cumbersome purification process, environmental pollution, etc., and achieve simple separation, improved product singleness, high yield effect

Active Publication Date: 2021-01-05
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fundamental reason is that the preparation of single-component chitosan oligosaccharides mainly adopts the method of strong acid hydrolysis at present, which has serious environmental pollution problems, and the obtained hydrolyzate has complex components, and the purification process is cumbersome. Low, expensive, 97% chromatographically pure chitobiose is sold at a price as high as 130 yuan / mg, which greatly limits the functional research and application of chitooligosaccharide single component

Method used

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  • Chitosanase mutant G21K and application thereof
  • Chitosanase mutant G21K and application thereof
  • Chitosanase mutant G21K and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment one: construct chitosanase mutant

[0025] 1. Construct a mutant plasmid using a method dependent on DpnI enzyme: use the plasmid pET28a / BsuCsnMY002 as a template, add forward and reverse primers to amplify, obtain a single-stranded plasmid containing the mutation site, and digest it with DpnI enzyme at 37°C for 4 hours. For the template plasmid, mix the mutant single-stranded plasmids, boil in a boiling water bath for 10 minutes, and cool to room temperature. The plasmid was transformed into the host strain E.coil DH5α, spread on the LB plate containing Kanna resistance, cultured overnight at 37°C, picked a single colony on the plate, and sequenced after pre-cultivation.

[0026] 2. Transform the plasmid with correct sequencing into the expression host strain E.coil BL21 for the expression of the mutant protein. Transfer the transformed host bacteria to the LB liquid medium containing Kanna resistance according to the inoculum amount of 1%, culture at 37°C ...

Embodiment 2

[0027] Example 2: Purification and Enzyme Activity Determination of CsnMY002 Wild Type and G21K

[0028] 1. Obtaining purified protein: collect the two bacterial liquids in Example 1 by centrifugation, and after ultrasonic crushing, obtain the supernatant by centrifugation. A nickel column was used for purification, the loading buffer was (50mM MES, 500mM NaCl, pH=6.0), and the elution buffer was (50mM MES, 500mM NaCl, 100mM imidazole, pH=6.0), eluted to G-250 detects until there is no blue color. The eluted protein samples were filtered with a 0.22 μm filter membrane, and the obtained filtrate was concentrated to 10 mg / ml by ultrafiltration, and stored at -80°C for future use. The chitosanase after nickel column purification is carried out SDS-PAGE electrophoresis detection, the result is as follows figure 1 As shown, the molecular weight of the wild type and the mutant is about 27KDa. figure 1 Among them, lane 1 is the protein molecular weight standard, lane 2 represents ...

Embodiment 3

[0034] Example 3: TLC and ELSD-UPLC analysis of CsnMY002 wild type and G21K hydrolyzate

[0035] 20mg of chitosan powder was dissolved in 1mL of 0.1M ammonium acetate buffer (pH=5.5), and the amount of chitosanase added was 5 μg / mg (the amount of enzyme required for each mg of chitosan), respectively, at 37°C. Reaction 1, 3, 5, 10, 30, 60, 90min, using the DNS method to measure the reducing sugar content, reducing sugar content per milligram of chitosan can produce reducing end calculation (μmoL / mg).

[0036] TLC analysis: add sodium hydroxide with a final concentration of 1M to the enzymatic hydrolysis product to stop the reaction, centrifuge at 12000rpm for 5min, draw 2μL of the supernatant and spot it on a TLC plate (10×5cm), pre-prepare the TLC chromatography plate in a chromatography tank Equilibrate for 30 minutes, and develop the layer for about 2 hours using the development system, which is: by volume, ethyl acetate: ethanol: water: 28% ammonia water = 5:5:4:0.5. Afte...

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Abstract

The invention belongs to the technical field of enzymes, and particularly relates to a chitosanase mutant and application thereof. The chitosanase mutant is characterized in that glycine at the 21 position of CsnMY002 wild type chitosanase is mutated into lysine. Compared with a wild strain, the chitosanase mutant has the advantages that chitosan oligosaccharide can be converted into chitobiose ina more targeted mode, separation is simpler, and the yield of a target product is higher.

Description

technical field [0001] The invention belongs to the technical field of enzymes, and in particular relates to a chitosanase mutant G21K and an application thereof. Background technique [0002] In nature, chitin is the second most abundant polysaccharide, its natural existence is second only to cellulose, and its sources are very extensive. Chitin is composed of N-acetylglucosamine (GlcNAc), which is a polymer linked by β-1,4 glycosidic bonds. Chitosan can be obtained by enzymatically or chemically N-deacetylating chitin (deacetylation degree>50%). Chitosan is degraded by chitosanase to obtain chitooligosaccharides (CHOs) with a degree of polymerization of 2-20. Oligochitosan is a class of biologically active small molecular substances, which have important applications in many fields such as the treatment and prevention of human diseases, the induction of plant disease resistance, animal husbandry, and the food industry. [0003] At present, in the research and applica...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12P19/26C12P19/14C12P19/12
CPCC12N9/2434C12P19/26C12P19/14C12P19/12C12Y302/01132
Inventor 王刚刚李玉斌
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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