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A kind of chitosanase csn4 and its coding gene and application

A chitosanase, catalyzing chitosan technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of limiting the exploration and utilization of microbial resources, breeding of rare chitosanase strains, etc. Prospects for industrial application, mild reaction conditions, high activity effect

Inactive Publication Date: 2018-07-20
一好(山东)海洋生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cultivable microorganisms in various environments are an important source of new enzymes and bioactive molecules, but in a microbially diverse earth, less than 1% of microorganisms can be obtained using cultivable technologies, which limits the exploration and utilization of microbial resources
The method of metagenomics has been successfully used in the development of various industrial enzymes, but there are few reports on the selection of chitosanase strains

Method used

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  • A kind of chitosanase csn4 and its coding gene and application
  • A kind of chitosanase csn4 and its coding gene and application
  • A kind of chitosanase csn4 and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Obtaining the gene csn4 derived from deep-sea silt chitosanase CSN4

[0023] The method for obtaining the gene csn4 of chitosanase CSN4 of the present invention includes the following steps:

[0024] (1) Construction of a deep-sea silt metagenomic library

[0025] Take 10g of deep-sea silt samples collected and apply Epicentre's Meta-G-Nome TM DNA IsolationKit, operate in strict accordance with the instructions to extract and purify sample DNA.

[0026] Take appropriate amount of purified DNA and use Epicentre's CopyControl TM Fosmid LibraryProduction Kit with pCC1FOS TM Vector vector is ligated, and the ligation solution is packaged and transferred into EPI300TM-T1R E.coli host bacteria. Then spread it on a plate containing 12.5 mg / L chloramphenicol and culture it overnight at 37°C; wash the colonies on the plate with sterile LB, add glycerol (20%, v / v) and store at -80°C.

[0027] (2) Screen positive clones of chitosanase

[0028] Spread the preserved bacterial...

Embodiment 2

[0040] Example 2. Expression and purification of recombinant chitosanase CSN4

[0041] The recombinant plasmid csn4 / pET-28a in Example 1 was transformed into E. coli BL21 (DE3) according to conventional methods to obtain a genetically engineered strain for producing chitosanase CSN4.

[0042] The above-mentioned genetically engineered strains were inserted into LB medium and cultured at 37°C to the logarithmic growth phase, IPTG was added to a final concentration of 0.1 mmol / L, and the culture was continued at 20°C for 20 hours. The cells were collected by centrifugation, washed with saline, and NPI-0 buffer (50mM Na 2 HPO 4 , 0.3M NaCl, pH 8.0) suspended. Then, ultrasonically break in an ice water bath (power 200-400W, work 3s, gap 5s, ultrasonic 20min), centrifuge to collect the supernatant, which is the crude enzyme solution of chitosanase CSN4. The crude enzyme solution contains recombinant protein, namely chitosanase CSN4.

[0043] The recombinant chitosanase CSN4 was purified...

Embodiment 3

[0044] Example 3. Enzymatic properties detection of recombinant chitosanase CSN4

[0045] The chitosanase CSN4 pure enzyme prepared in Example 2 was diluted with citrate buffer (100mM, pH3.5), and then reacted at 25-55°C. The reaction conditions were: take 10μL of pure enzyme and add it to In 990 μL of 100 mM, pH 3.5 citric acid buffer, after incubating at different temperatures for 10 minutes, 100 μL of 1% chitosan was added to react for 20 minutes, and then the reducing sugar content in the reaction solution was determined by DNS method. Taking the highest value of enzyme activity as 100%, the result is as figure 2 Shown. The results showed that the optimal temperature of recombinant chitosanase CSN4 was 35℃, and it had high activity at 25-40℃.

[0046] The pure chitosanase CSN4 prepared in Example 2 was diluted with 100 mM citrate buffer with a pH of 2.5-5.0, and then reacted at 30°C. The reaction conditions were: take 10 μL of pure chitosanase CSN4 After adding to 990 μL of ...

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Abstract

The invention provides a chitosanase CSN4, an encoding gene and an application thereof. The amino acid sequence of the chitosanase CSN4 is shown as SEQ ID NO:1; the nucleotide sequence of the gene csn4 of the chitosanase CSN4 is shown as SEQ ID NO:2; when the chitosanase CSN4 is used for degrading chitosan and generating chitosan oligosaccharide at low polymerization degree, the reaction conditionis mild, the energy consumption is less and no pollutant is generated; and the chitosanase CSN4 has an excellent industrial application prospect in the enzymatic preparation of chitosan oligosaccharide and has high economic value.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a chitosanase CSN4 and its coding gene and application. Background technique [0002] Chitosan (Chitosan) is obtained by deacetylation of chitin, which is widely present in nature. A large number of studies have shown that chitosan has broad application prospects in the fields of food, chemical industry, medicine and agriculture. However, due to the solubility problem of chitosan, the effective utilization of its resources is seriously hindered. Therefore, degrading chitosan into oligosaccharides and then using them is one of the current research hotspots in this field. Chitooligosaccharide is a kind of low polymerization degree (generally 2-10), water-soluble amino sugar compound, and it is also the only known basic oligosaccharide. Studies have shown that oligochitosan has unique physiological activity and functionality, and is easily absorbed by the human body. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/26C12R1/19
Inventor 孙慧慧刘淇曹荣赵玲
Owner 一好(山东)海洋生物科技有限公司
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