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A kind of chitosanase csnq and its application

A technology of chitosanase and chitosan, applied in chitosanase CsnQ and its application field, can solve the problems of low activity, high price, poor stability and the like

Active Publication Date: 2020-10-27
枣庄全鼎生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, chitosanases currently on the market are expensive, have low activity, and poor stability, which severely limits the application prospects of chitosanases.

Method used

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  • A kind of chitosanase csnq and its application
  • A kind of chitosanase csnq and its application
  • A kind of chitosanase csnq and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The sequence analysis of embodiment 1 chitosanase CsnQ

[0026] The enzyme-producing gene csnQ of the chitosanase CsnQ of the present invention is an artificially synthesized sequence. In the previous study, we found that the marine bacterium Vibrio sp.Q108 has high chitosanase activity, and when it was sequenced, it was found that it contained a predicted chitosanase sequence. Under the condition that the amino acid sequence remains unchanged, we optimized the base sequence of the gene sequence according to the codon preference of the host (Escherichia coli), so as to facilitate its high-efficiency expression in Escherichia coli. Sequence synthesis was carried out at Huada Gene Company. The chitosanase CsnQ of the present invention contains 861 base sequences and encodes 287 amino acid sequences. Using the conserved domain analysis of the National Center for Biotechnology Information (NCBI) to analyze the Conserved domain (CDD) and the multiple sequence alignment Bas...

Embodiment 2

[0036] The preparation and purification method of embodiment 2 chitosanase CsnQ

[0037] Culture the recombinant strain BL21(DE3) / pET22b-csnQ in 100 mL of LB liquid medium (50 μg / mL ampicillin) in a shaker at 37°C at 180 rpm to OD 600 =0.6, add the inducer isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM, and induce at 20°C for 20 h. The standard assay method of chitosanase CsnQ activity is: add 900 μL 0.3% chitosan substrate (20mM acetic acid-sodium acetate, pH=5.9) to 100 μL enzyme solution, react at 60° C. for 10 min, add 750 μL DNS reagent, boil water React for 10 min to develop color, and detect its absorbance at OD520. Enzyme activity is defined as 1 U is the amount of enzyme required to produce 1 μM reducing sugar per min. After testing, the chitosanase activity in the fermentation broth can reach 1058.2U / mL.

[0038] After the fermentation stops, centrifuge at 12000rpm for 10min, discard the bacteria, and collect the supernatant; the fermentat...

Embodiment 3

[0040] Embodiment 3 temperature and the impact of pH on chitosanase CsnQ

[0041] The purified chitosanase CsnQ obtained in Example 2 was tested for enzyme activity under different conditions to detect the influence of different temperatures and pH on the enzyme activity. React at different temperatures (0-80° C.) for 10 minutes, detect the effect of different reaction temperatures on enzyme activity, and calculate the relative enzyme activity of chitosanase CsnQ at different temperatures with the highest enzyme activity as 100%. Such as image 3 As shown in A, the optimum reaction temperature of chitosanase CsnQ is 60℃.

[0042] The purified chitosanase CsnQ obtained in Example 2 was incubated at different temperatures (0-80° C.) for 1 h. After taking it out, its enzyme activity was detected at its optimum reaction temperature (60° C.), and the activity before incubation was used as 100%, as in image 3 As shown in B, chitosanase CsnQ has better temperature stability, and ...

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Abstract

The present invention relates to a novel chitosanase CsnQ and an application thereof. An amino acid sequence of the chitosanase CsnQ is shown in SEQ ID NO.1. A provided preparation method of the chitosanase CsnQ has a yield up to 1,058.2 U / mL. The chitosanase CsnQ has stable properties and an optimum reaction temperature of 60 DEG C, is stable between pH 4.0-6.0, still maintains 61.2% of activitywhen incubated at 40 DEG C for 1 h, and has a degradation mode of endo-cutting and main degradation products of chitobiose and chitotriose. The chitosanase CsnQ has high yield, good stability and goodindustrial application potential.

Description

technical field [0001] The invention relates to a chitosanase CsnQ and an application thereof, belonging to the field of biotechnology. Background technique [0002] Oligochitosan has important biological activities such as antibacterial, antitumor, and anti-inflammatory, and is known as the "sixth element of life". Oligochitosan is composed of N-acetyl-D-glucosamine (GLcNAc) and D-glucosamine (GLcN) connected by β-1,4-glucosidic bonds. Its molecular weight is less than 3900, and its degree of polymerization is less than 20. Water has greater application value than macromolecular chitosan in the fields of medicine, health care products, food, and breeding. [0003] Oligochitosan is produced by the degradation of macromolecular chitosan. Generally, degradation methods are divided into three types: physical method, chemical method and biological enzymatic method. Among them, biological enzymatic degradation has the advantages of mild reaction conditions, single degradation ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12P19/26
CPCC12N9/2442C12P19/26C12Y302/01014
Inventor 杨文钰
Owner 枣庄全鼎生物科技股份有限公司
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