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Chitinase CmChi6 gene and cloning expression and application thereof

A technology of chitinase and colloidal chitin, which is applied in the field of genetic engineering, can solve the problems of high energy consumption in the process, poor biological activity of the product, and low yield of the product, and achieve good repeatability, good protein expression effect, and high yield. The effect of degradation activity

Active Publication Date: 2019-05-28
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this method consumes a lot of energy, the product yield is low, and the biological activity of the product is poor.

Method used

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  • Chitinase CmChi6 gene and cloning expression and application thereof
  • Chitinase CmChi6 gene and cloning expression and application thereof
  • Chitinase CmChi6 gene and cloning expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Chitinase cm Amplification of the Chi6 gene

[0046] The present invention designs primers based on the reported gene sequence of SYBC-H1 chitinase, and obtains SYBC-H1 chitinase by polymerase chain reaction (PCR) cm Conserved sequence of Chi6.

[0047] Using PCR technology, donated by Hao Zhikui of Jiangnan University Chitinolyticbacter meiyuanensis Genomic DNA of SYBC-H1 was used as a template, and primers were designed using Primer5.0 software, respectively: upstream primers Chi -F (SEQID No.2): 5'-CCCAAGCTTCTACTTTGCCGCCGGAAT-3' designed with NdeI restriction site; downstream primer Chi -R (SEQ ID No.3): 5'-CCCAAGCTTCTACTTTGCCGCCGGAAT-3' is designed with a HindIII restriction site.

[0048] PCR reaction system:

[0049]

[0050] PCR reaction conditions: 95°C pre-denaturation, 2min; 95°C denaturation for 20s, 52°C annealing, 20s, 72°C extension, 2min, a total of 30 cycles; 72°C extension, 5min, and finally 4°C incubation.

[0051] After the PCR r...

Embodiment 2

[0052] Example 2 Plasmid vector pColdI- Cmchi6 and the cloned strain pColdI- Cmchi6 - E. coli Preparation of Trans1T1

[0053] 1. Purify and recover the PCR product obtained in Example 1, using a TaKaRa company kit (TaKaRa DNALigation Kit);

[0054] 2、 Construction of plasmid vector pColdI- Cmchi6

[0055] The DNA sequence amplified by PCR and the pColdI vector were double-digested with the same restriction enzymes NdeI and HindIII, and the digested products were recovered and purified, and used T 4 DNA ligase was connected to obtain the plasmid vector pCooldI- Cmchi6 ;

[0056] 3. Construction of cloning strain pColdI- Cmchi6 - E. coli Trans1T1

[0057] The plasmid vector pColdI- Cmchi6 convert to E. coli Trans 1T1: (1) Take 20 μl of competent cells Trans 1T1 frozen and thawed on ice, and mix them gently in the above 10 μl connection reaction solution; (2) After standing on ice for 30 minutes, heat shock at 42°C for 45 seconds , and then quickly put it ...

Embodiment 3

[0062] Construction of recombinant expression strain pColdI- Cmchi6 - E. coli BL21(DE3)

[0063] 1. Strain cultivation: the constructed recombinant plasmid pColdI- Cmchi6 extracted from the cloning host and retransformed into E. coli. BL21(DE3) competent cells. The conversion operation was as the conversion step in the above-mentioned Example 2. Finally, 1-2 single colonies were picked and inoculated into 5ml LB medium containing ampicillin with a final concentration of 0.2%, and cultivated overnight at 37°C.

[0064] 2. Main culture: After 12 hours of culture, transfer to 100ml LB / Amp medium according to the inoculum size of 1%, and shake culture at 37°C until OD 600 =0.4-0.6.

[0065] 3. Induced expression: Add IPTG at a final concentration of 0.05mM to induce bacterial cells, and induce at 18°C ​​and 200rpm for 20h at low temperature.

[0066] 4. Collect whole cells: Centrifuge at 4°C, 6000rpm for 8-10min to collect the bacteria, mix with 10ml PBS, and then ultr...

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Abstract

The invention relates to chitinase CmChi6 gene and cloning expression and application thereof. In the invention, exochitinase CmChi6 gene with the nucleotide sequence shown in SEQ ID No.1 is firstly cloned, and exochitinase with high activity is obtained. The preparation method is simple, the production cost is low, the product is easy to preserve, the suitable enzymolysis temperature is 45 DEG C,the optimum pH for enzymolysis is 7.4, the thermal stability is good at 25-50 DEG C, and the enzyme activity is maintained at 60% or above for 33 h; the optimum pH is 7.4, and the enzyme activity ismaintained at 75% or above for 33 h at pH of 7.0-10.0; Ba<2+> has a slight activation effect on the activity of CmChi6; CmChi6 has high degradation activity on colloid chitin, and can be used to prepare chitobiose, with high conversion rate, low energy consumption, environmental friendliness, and important economic value in application of industrial degrading of chitin.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a chitinase cm Chi6 gene and its clone expression and application. Background technique [0002] Chitin: also known as chitin, chitin, shell protein, chitin, clear cutin, etc., is a nitrogen-containing polysaccharide, the second most abundant biopolymer in nature, and is widely distributed. It is an important component of the shells of many lower animals, especially arthropods such as shrimps, crabs, insects, etc. It is also a component of the cell membranes of lower plant fungi, and it is also contained in lichens, green algae, yeast, jellyfish and squid. It is estimated that the annual biosynthesis is more than 10 billion tons, which is a huge renewable resource. [0003] Chitin is a linear polymer connected by monomer N-acetylglucosamine (GlcNAc) through β-1,4-glycosidic bonds, and its molecular weight is mostly above 1 million. The degradation products of chiti...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/26C12P19/14C12R1/19
Inventor 陈可泉王莹莹张阿磊莫晓芳周宁杨赛魏衍鹏曹飞欧阳平凯
Owner NANJING UNIV OF TECH
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