A kind of novel chitosanase csnf and its application

A technology of chitosanase and chitosan, applied in the biological field, can solve the problems of limited application prospect of chitosanase, poor stability, high price, etc., and achieves good industrial application potential, good stability and simple preparation method. Effect

Active Publication Date: 2021-02-05
济南阿波罗甲壳素肥业有限公司
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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 novel chitosanase csnf and its application
  • A kind of novel chitosanase csnf and its application
  • A kind of novel chitosanase csnf and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Sequence Analysis of Chitosanase CsnF

[0025] The enzyme-producing gene csnF of the chitosanase CsnF of the present invention is an artificially synthesized sequence. The chitosanase CsnF full-length of the present invention contains 437 amino acids, N terminal (Phe 1 -Gly 152 ) is the third family carbohydrate binding domain (CBM3), the C-terminus (Arg 162 -Ile 437 ) is a chitosanase conserved region of the fungal polysaccharide hydrolase (GH75) family, which contains a Linker structure (Thr 153 -Glu 161 ), providing steric hindrance for the two-segment domains. 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 (Pichia pastoris), which is conducive to its high-efficiency expression in Pichia pastoris.

[0026] With the base sequence of chitosanase CsnF as restriction endonuclease EcoR I and Not I as restriction endonuclease site, design r...

Embodiment 2

[0035] The preparation and purification method of embodiment 2 chitosanase CsnF

[0036] Select the recombinant transformants identified as positive by PCR and inoculate them into BMGY liquid medium, culture at 29-30°C, 220-250rpm, and culture until OD600=0.6-0.7 after 50-fold dilution. Centrifuge at 6000rpm for 10min, collect the bacteria, and discard BMGY liquid culture. Add BMMY liquid medium (induction medium), add methanol at a concentration of 1.0%, and start methanol induction culture at 29-30°C and 220-250 rpm. Methanol was added every 24 hours, so that the final concentration of methanol in the medium was always maintained at 1.0%, and samples were taken every 24 hours. After the fermentation, centrifuge at 12000 rpm for 10 minutes to separate the bacterial cells and the supernatant of the fermentation broth. The standard assay method for chitosanase CsnF activity is: add 900 μL 0.3% chitosan substrate (20 mM acetic acid-sodium acetate, pH=6.0) to 100 μL enzyme solu...

Embodiment 3

[0038] Embodiment 3 The influence of temperature on chitosanase CsnF

[0039] The purified chitosanase CsnF obtained in Example 2 was tested for enzyme activity under different conditions to detect the influence of different temperatures on the enzyme activity. React at different temperatures (20-90°C) for 10 min, detect the effect of different reaction temperatures on enzyme activity, take the highest enzyme activity as 100%, and calculate the relative enzyme activity of chitosanase CsnF at different temperatures. Such as figure 2 As shown, the optimal reaction temperature of chitosanase CsnF is 70℃.

[0040] The chitosanase CsnF purified in Example 2 was incubated at different temperatures (0-80°C) for 1 h, and after taking it out, its enzyme activity was detected at its optimum reaction temperature (70°C) to measure the activity before incubation. as 100%, such as image 3 The results showed that the temperature stability of chitosanase CsnF was good. When incubated at ...

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Abstract

The invention relates to a novel chitosanase CsnF and an application thereof. The amino acid sequence of chitosanase CsnF is shown as SEQ ID NO. 1. The invention provides a preparation method for recombinant expression of chitosanase CsnF in Pichia pastoris, and a fermentation process of the recombinant bacteria, wherein the enzyme activity of the fermentation liquid is as high as 376.6 U / mL, theinvention provides a method for purifying the chitosanase CsnF, the purity is greater than 95%, and the recovery is as high as 92.2%. The chitosanase CsnF of the invention has good stability, and themain products of degradation are chitobiose and chitotriose, and the chitosanase CsnF has good industrial application potential.

Description

technical field [0001] The invention relates to a novel chitosanase CsnF and its application, belonging to the field of biotechnology. Background technique [0002] Oligochitosan has a wide range of antibacterial, anti-inflammatory and anti-tumor effects, and is recognized as an effective health-care 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. It has greater application value than macromolecular chitosan in other fields, and is known as "the sixth element of life". [0003] At present, the preparation methods of chitosan oligosaccharides are mainly divided into three kinds: acid hydrolysis method, physical degradation method and biological enzyme method. Among them, the physical degradation method (microwave method, ultrasonic crushing method) can only be used as an auxiliary degradation meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12P19/26C12P19/14C12P19/12
CPCC12N9/2402C12P19/12C12P19/14C12P19/26
Inventor 杨文钰
Owner 济南阿波罗甲壳素肥业有限公司
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