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Chitinase gene chiC and encoded protein and application thereof

A chitinase gene and chitinase technology, applied in the field of genetic engineering, can solve problems such as environmental pollution and waste, and achieve the effects of simple preparation process, low production cost and high degradation activity

Active Publication Date: 2015-07-08
DALIAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the chitin resource is not handled properly, it will not only cause waste but also pollute the environment

Method used

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  • Chitinase gene chiC and encoded protein and application thereof
  • Chitinase gene chiC and encoded protein and application thereof
  • Chitinase gene chiC and encoded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Amplification of chiC gene

[0041] The present invention designs primers according to the gene sequence of Pseudoalteromonas chitinase reported on NCBI, and obtains pseudoalteromonas (Pseudoalteromonas sp.DL-6) several The conserved sequence of the chitinase chiC. The bacterial strain has been preserved in the General Microorganism Center of the China Committee for the Collection of Microbial Cultures. The preservation date is December 13, 2013, and its preservation number is CGMCC No.8580.

[0042] Utilize PCR technology to use the genomic DNA of pseudoalteromonas (Pseudoalteromonas sp.DL-6) strain as a template, and use Primer5.0 software to design primers, which are: upstream primer chiC-F (SEQ ID No.2): 5' -CCC GGATCC GGCGCCTTCTACACCAAGCATTAATTGG-3' is designed with a BamHI restriction site; downstream primer chiC-R (SEQ ID No.3): 5'-CCG CTCGAG AAGTGCTAACCATACATCGG-3' is designed with XhoI restriction site.

[0043] PCR reaction system:

[0044] ...

Embodiment 2

[0046] Example 2 Preparation of Escherichia coli Cloning Vector pMD19-T-chiC

[0047] 1. Connection between chiC gene and pMD19-T

[0048] The PCR product obtained in Example 1 was purified and recovered, and the recovered chiC PCR product was ligated with the pMD19-T simple cloning vector using a TaKaRa company kit (TaKaRa DNA Ligation Kit ).

[0049] The connection reaction system is:

[0050] PCR product of chiC

2μL

pMD19-TSimple (50ng / μL)

1μL

Ligation Mix (50ng / μL)

5μL

dH 2 o

2μL

[0051] The conditions of the ligation reaction were: overnight ligation at 16°C to construct the cloning vector pMD19-T-chiC.

[0052] 2. Transformation of cloning vector (pMD19-T-chiC)

[0053] (1) Take 100 μL of Escherichia coli competent cells E.coli DH5α frozen and thawed on ice, and mix them gently in the above 10 μL of the ligation reaction solution;

[0054] (2) After being placed in ice for 30 minutes, heat shock treatment at 42°...

Embodiment 3

[0060] Example 3 Preparation of Escherichia coli expression vector pET28a-chiC

[0061] 1. Preparation of chiC gene

[0062] The constructed recombinant plasmid pMD19-T-chiC was identified by digestion with two restriction endonucleases BamHI and XhoI, and the reaction system was as follows:

[0063] pMD19-T-chiC (30ng / μL)

10 μL

BamHI (5U / μL)

2μL

XhoI (5U / μL)

2μL

10×M buffer

10 μL

dH 2 O up to

100μL

[0064] The reaction system was hydrolyzed in a water bath at 37°C for 2-3 hours, and the product was detected by 1% agarose gel electrophoresis. The enzymatic hydrolysis product was recovered by using the TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 from TaKaRa Company to recover the gene chiC.

[0065] 2. Preparation of expression vector pET-28a

[0066] The expression vector pET-28a was identified by double digestion with the same restriction enzymes BamHI and XhoI, and the reaction system was as follows: ...

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Abstract

The invention relates to a chitinase gene chiC and encoded protein and application thereof, and belongs to the technical field of genetic engineering. According to the chitinase gene chiC and the encoded protein and the application thereof, pseudoalteromonas sp. DL-6 serves as a research object, the chitinase gene chiC with a nucleotide sequence showed in SEQ ID No 1 is cloned for the first time, and a high-activity chitinase is obtained. The preparation method of the high-activity chitinase is simple, the production cost is low, a product is easy to conserve, the appropriate hydrolysis temperature is 30 DEG C, The enzyme-decomposed PH is 9.0, high degradation activity of a colloidal chitin can reach to 1.569 + / - 0.017 U / ml, the chitinase chiC degrades an aipha chitin and a beta chitin to generate a chitobiose, and industrial production prospect is possessed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a chitinase gene chiC, in particular to a chitin derived from a new strain of Pseudoalteromonas sp.DL-6 (CGMCC No.8580) Mass enzyme gene chiC and its encoded protein and application. Background technique [0002] Chitin, commonly known as chitin, is a natural polysaccharide that can be extracted from waste such as shrimp and crab shells. Its structure is N-acetyl-D-glucosamine (50-100%) and a small amount of D-amino Glucose is connected by β-1,4-glucosidic bonds (0-50%). In nature, chitin is synthesized by organisms every year at 10-100 billion tons, which is the most abundant in the marine environment and second only to chitin in nature. The second largest biomass source of cellulose. Chitin degradation products are high value-added chitooligosaccharides, chitomonosaccharides and their derivatives, etc., which have biological activities such as enhancing immunity, ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/10C12P19/26
Inventor 王晓辉张庆芳迟乃玉
Owner DALIAN UNIVERSITY
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