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A kind of chitosanase csnt and its application

A technology of chitosanase and chitosan, applied in the field of chitosanase CsnT and its application, can solve the problems of poor stability, high price, limiting the application prospect of chitosanase, etc.

Active Publication Date: 2021-08-20
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 csnt and its application
  • A kind of chitosanase csnt and its application
  • A kind of chitosanase csnt and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Sequence Analysis of Chitosanase CsnT

[0025] The enzyme-producing gene csnT of the chitosanase CsnT in the present invention is an artificially synthesized sequence. In the previous research, we found that the marine bacterium Renibacterium sp. T109 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. The chitosanase CsnT of the present invention contains 891 base sequences and encodes 297 amino acid sequences. Using the Conserved Domain (CDD) analysis in NCBI, it was found that the sequence contained a conserved region of the 46th family of polysaccharide hydrolase (GH-46). Multiple sequence comparison Basic Local Alignment Search To...

Embodiment 2

[0034] The preparation and purification method of embodiment 2 chitosanase CsnT

[0035] Cultivate the recombinant strain BL21(DE3) / pET22b-csnT in 100 mL of LB liquid medium (50 μg / mL ampicillin) in a shaker at 37°C at 180 rpm until 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 for chitosanase CsnT activity is: add 900 μL 0.3% chitosan substrate (20 mM acetic acid-sodium acetate, pH=6.0) to 100 μL enzyme solution, react at 50 °C for 10 min, add 750 μL DNS reagent was reacted in boiling water for 10 min for color development, and its absorbance was detected 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 796.3U / mL.

[0036] After the fermentation stopped, centrifuge at 12000 rpm for 10 min, discard the bacteria, and collect...

Embodiment 3

[0037] Embodiment 3 The influence of temperature on chitosanase CsnT

[0038] The chitosanase CsnT purified 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 (0-70°C) for 10 min, detect the effect of different reaction temperatures on enzyme activity, and calculate the relative enzyme activity of chitosanase CsnT at different temperatures with the highest enzyme activity as 100%. Such as figure 2 Shown, the optimal reaction temperature of chitosanase CsnT is 50℃.

[0039] The chitosanase CsnT purified in Example 2 was incubated at different temperatures (0-70°C) for 1 h, and after taking it out, its enzyme activity was detected at its optimum reaction temperature (50°C) to measure the activity before incubation. as 100%, such as image 3 The results showed that the temperature stability of chitosanase CsnT was good. After incubation at 40 °C...

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Abstract

The invention relates to a chitosanase CsnT and its application. The amino acid sequence of the endochitosanase CsnT is shown in SEQ ID NO.1. The invention provides a fermentation and purification method of chitosanase CsnT, the enzyme activity of the fermentation broth is as high as 796.3 U / mL, the purity of one-step purification is >95%, and the recovery rate is 88.9%. The chitosanase CsnT of the present invention is stable in nature, can maintain 52.4% of the enzyme activity when incubated at 50°C for 1 hour, and degrades the main products chitobiose and chitotriose, and has good industrial application potential.

Description

technical field [0001] The invention relates to a chitosanase CsnT and an application thereof, belonging to the field of biotechnology. Background technique [0002] Oligochitosan is known as the "sixth element of life", has a wide range of antibacterial, anti-inflammatory, anti-tumor effects, and is recognized as an effective health functional food. Oligochitosan is composed of N-acetyl-D-glucosamine (GLcNAc) and D-glucosamine (GLcN) linked by β-l,4-glucosidic bonds. It is very soluble in water and is used in medicine, health products and functional foods. And other fields have greater application value than macromolecular chitosan. [0003] The enzymatic degradation of macromolecular chitosan by chitosanase has the advantages of mild reaction conditions, single degradation product, easy control of the reaction process, and environmental friendliness, and has gradually become the mainstream method in the preparation of chitosan oligosaccharides. However, chitosanase sold ...

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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