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Chitin endonuclease CmChi4 as well as cloning expression and application thereof

A chitin and endonuclease technology, applied in the field of genetic engineering, can solve the problems of unsuitability for industrialization, high fermentation cost, long cycle and the like, and achieve the effects of low cost, simple preparation process and simple operation

Inactive Publication Date: 2021-06-11
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A highly active endochitinase was reported in the paper "The Properties and Applications of Glycoside Hydrolase 19 Family Chitinases from Streptomyces meliloti Sa Chi19B, under the action of a combination of N-acetylglucosaminidase, degrades chitin for 6 hours to obtain a higher yield of GlcNAc; patent CN111996205A discloses a preparation of chitin oligosaccharides by expressing endochitinase from Pichia pastoris Sugar method, this method induces 120h overexpression of chitinase by methanol, the fermentation cost is high and the cycle is long, and it is not suitable for industrialization

Method used

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  • Chitin endonuclease CmChi4 as well as cloning expression and application thereof
  • Chitin endonuclease CmChi4 as well as cloning expression and application thereof
  • Chitin endonuclease CmChi4 as well as cloning expression and application thereof

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Experimental program
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Embodiment 1

[0036] Embodiment 1 strain source

[0037] Bacterial strains used in the present invention Chitinolyticbacter meiyuanensis SYBC-H1 was screened by our laboratory and preserved in China General Microorganism Collection (CGMCC 3438) and American Type Microorganism Collection (ATCC BAA-2140).

Embodiment 2

[0038] Example 2 Construction of recombinant plasmid pCOLDI- CmChi4 ,Specific steps are as follows:

[0039] Step 1, design PCR amplification primers

[0040] CmChi4- F-5'-GAAGGTAGGCATATGATGTTTAGCAAGGCT-3';

[0041] CmChi4- R-5'-CAGGTCGACAAGCTTGCGTACGCCGGACAT-3',

[0042] Amplify the full length of the gene;

[0043] Step 2, the reaction conditions for the above PCR amplification are: pre-denaturation at 95°C for 5 min; 30 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 40 sec; and finally extension at 72°C for 10 min;

[0044] Step 3, performing PCR amplification using the primers in step 1, and recovering the obtained amplification product;

[0045] Step 4, use restriction enzymes NdeI and HindIII to double-digest the vector pCOLDI, and recover the vector backbone (4378bp);

[0046] Step 5, performing homologous recombination ligation of the amplified product obtained in step 3 and the vector backbone of step 4 to obtain the recombinant plasmid pCOLDI- C...

Embodiment 3

[0047] Example 3 Endochitinase cm The clone expression of Chi4 gene comprises the following steps:

[0048] In the first step, the recombinant plasmid pCOLDI- CmChi4 Introduce Escherichia coli BL21 (DE3) to obtain recombinant plasmid pCOLDI- Cmchi4 Escherichia coli BL21 (DE3), named recombinant bacteria;

[0049] In the second step, select recombinant bacteria (expressing pCOLDI- CmChi4 ) single clones were connected to 5 mL LB medium containing 100 μg / mL ampicillin and cultured overnight in a shaking shaker at 37 °C and 200 rpm;

[0050] The third step is to inoculate the above-mentioned bacterial solution into 100 mL LB liquid medium containing 100 μg / mL ampicillin at a volume ratio of 1:100, and culture it at 37 ° C and 200 rpm until OD 600 Reach 0.6-0.8;

[0051] In the fourth step, add IPTG (inducer) to a concentration of 1mmol / L, shake and culture at 20°C and 200rmp for 20h;

[0052] Step 5: Collect the above-mentioned bacterial solution into a centrifuge tube, ...

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Abstract

The invention discloses a chitin endonuclease CmChi4 as well as the cloning expression and the application of the chitin endonuclease CmChi4. A chitinase gene CmChi4 is constructed on a plasmid pCOLDI through a genetic engineering means, the plasmid pCOLDI is transferred into E.coli BL21 (DE3) for expression, and the molecular weight of the recombinase is 72.1 kDa. Researches on enzymatic properties of CmChi4 show that the activity is optimal when the temperature is 50 DEG C and the pH value is 6; fe <2+>, K<+>, Al<3+> and Zn <2+> have relatively strong activation on the enzyme CmChi4. In addition, the products of the chitin endonuclease CmChi4 for hydrolyzing the colloid chitin are (GlcNAc) 2, (GlcNAc) 3 and (GlcNAc) 4, and the chitin endonuclease CmChi4 is good in stability and suitable for industrialization.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a chitin endonuclease cm Chi4 and its clone expression and application. Background technique [0002] Chitin (Chitin) molecular formula is (C 8 h 13 o 5 N) n , is a polymer made of N-acetyl-D-glucosamine (GlcNAc) linked by β-1,4-glycosidic bonds. As an important biomass resource, chitin is second only to cellulose in the earth's content, with an annual natural synthesis of about 10 11 Ton. It widely exists in shells of marine organisms such as shrimp, crab and shellfish, insect exoskeletons and fungal cell walls. However, a huge amount of crustacean resources have not been well utilized so far, and are usually piled up or discarded as waste, causing environmental pollution and waste of space resources. In recent years, the efficient and green refining of crustacean resources has gradually been put on the agenda as a scientific research hotspot. [0003] Chitosan oligos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/26C12P19/14C12R1/19
CPCC12N9/2442C12N15/70C12P19/26C12P19/14
Inventor 陈可泉陈雪曼张阿磊陈燕周宁魏国光欧阳平凯
Owner NANJING UNIV OF TECH
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