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Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene

A technology of metagenomic library and chitosanase is applied in the field of chitosanase, Escherichia coli engineering bacteria and their acquisition, and achieves the effects of simple acquisition method, good thermal stability and high catalytic activity

Inactive Publication Date: 2013-10-23
YANGZHOU RIXING BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, non-culturable microorganisms in the soil account for more than 99% of the total number of microorganisms, and the traditional isolation and culture of a single colony can only recognize less than 1% of the microorganisms in the soil, which has become a limiting factor for the development and utilization of microbial resources.

Method used

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  • Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene
  • Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene
  • Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene

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Experimental program
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Embodiment 1

[0031] The acquisition of the DNA sequence of embodiment 1 chitosanase (Meta chiA) gene

[0032] The obtaining of the DNA sequence of chitosanase (Meta chiA) gene comprises the following steps:

[0033] Take 1g of collected soil samples and apply Epicentre's Meta-G-Nome TM DNA Isolation Kit, strictly follow the instructions to extract soil DNA.

[0034] Take an appropriate amount of DNA, according to Epicentre's CopyControl TM Instructions for using the HTP Fosmid Library Production Kit, the DNA fragment was filled in at the end, after low melting point agarose gel electrophoresis ( image 3 ), cutting and separating DNA bands containing 30-60kb bases, recovering the DNA by sol method, ligation, packaging, transfection into EPI300 host cells, coating on a plate containing 12.5mg / L chloramphenicol, and culturing at 37°C 24h; Wash the colony library on the plate.

[0035] Spread the bacterial solution in the colony library on the chitosan-containing basal medium according ...

Embodiment 2

[0040] Embodiment 2 contains DNA sequence and is the preparation of the Escherichia coli engineering bacterium of the chitosanase gene shown in SEQ ID NO:1

[0041] Both the pMD-19T simple vector-chiA and the pET-28a plasmid prepared in Example 1 were double-digested with BamHI and HindIII, and the digested products recovered from rubber tapping were ligated under the action of T4 DNA ligase to obtain the recombinant plasmid pET28a-chiA , and sequenced the recombinant plasmid.

[0042] According to the molecular cloning manual, the recombinant plasmid pET28a-chiA was transformed into E.coli Rosetta-gami, and induced with IPTG (final concentration: 0.5mM) for 6-10h to obtain E. coli recombinant E.coli Rosetta-gami / / chiA.

Embodiment 3

[0043] Embodiment 3 measures the chitosanase activity of the escherichia coli engineering bacterium of embodiment 2

[0044] After sonicating Escherichia coli engineering bacteria, the supernatant was treated with Ni 2+ Metal chelation chromatography was used for separation and purification, and after purification, it was detected as a single band by SDS-PAGE, and it showed that the molecular weight of recombinant Meta ChiA was 27kDa, see figure 2 .

[0045] Measure chitosanase activity with DNS method, concrete operation is:

[0046] In 2ml EP tubes, add 0.8ml of colloidal chitosan solution with a mass concentration of 1% prepared by pH 6.0 and acetic acid-sodium acetate buffer solution, preheat at 50°C for 10min, and add 0.1ml of appropriate The diluted enzyme solution was accurately reacted at 50°C for 10 minutes. Immediately add 0.1ml of 1mol / L NaOH, shake well and centrifuge at 10,000rpm for 1min, absorb 0.5ml of the supernatant and add it to a 10ml stoppered test tube...

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Abstract

The invention provides chitosanase gene derived from soil metagenome library, and the DNA sequence of the chitosanase gene is shown in SEQID NO:1. The invention also provides chitosanase obtained by encoding of the chitosanase gene and recombinant plasmid including the chitosanase gene, and further provides escherichia coli engineering bacteria containing the chitosanase gene and an obtaining method thereof. The obtaining method of the chitosanase gene is simple, and the escherichia coli engineering bacteria containing the chitosanase gene is provided. The chitosanase can be obtained through high-efficiency expressionof the escherichia coli engineering bacteria. The bioinformatics analysis shows that the obtained chitosanase gene belongs to the forty-sixth family of glycoside hydrolase. The chitosanase gene has high catalytic activity and good thermal stability, with optimal action temperature being 55 DEG C and pH being 6.0, and has great industrial value and economic value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and particularly relates to a chitosanase gene derived from the glycoside hydrolase 46 family of a soil metagenomic library and an acquisition method thereof, and also relates to the chitosanase encoded by the gene, and also relates to a chitosanase comprising The recombinant plasmid of the gene also relates to Escherichia coli engineering bacteria containing the gene and its obtaining method. Background technique [0002] Chitosan is the product of deacetylation of chitin, which has the functions of lowering cholesterol, lowering blood pressure, preventing and treating diabetes, strengthening liver function, and treating burns and scalds. Oligochitosan is a degradation product of chitosan. In addition to enhancing immune function, it also has antiseptic and antibacterial effects, so it is widely used in many fields such as medicine, food, cosmetics, materials, chemical industry, agricultu...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/10C12N9/42C12N15/70C12N1/21
Inventor 张超许正宏戚善龙史劲松费忠李恒丁振中
Owner YANGZHOU RIXING BIO TECH
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