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Chitinase Chi6115 as well as coding gene and application thereof

A technology of chi6115, chitinase, applied in the field of bioengineering

Pending Publication Date: 2022-06-24
THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the chitinases found so far can only hydrolyze chitin pretreated with acid, while there are few reports of hydrolyzing enzymes that can directly hydrolyze unpretreated chitin

Method used

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  • Chitinase Chi6115 as well as coding gene and application thereof
  • Chitinase Chi6115 as well as coding gene and application thereof
  • Chitinase Chi6115 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of chitinase Chi6115

[0029] Proceed as follows:

[0030] Experimental Materials

[0031] Escherichia coli E.coil TOP10 and E.coil BL21 strains (purchased from ThermoFisher), expression vector pET-28a(+) (purchased from Novagen); restriction endonucleases BamHI and XhoI (purchased from Quanjinjin) ; T4 ligase (purchased from Takara); LB medium (1% peptone per liter, 0.5% yeast extract, 1% NaCl); binding buffer (phosphate buffer: 20 mM phosphate, pH 7.4 ); washing buffer (500 mM NaCl, 10-50 mM imidazole, 20 mM phosphate, pH 7.4); elution buffer (500 mM NaCl, 500 mM imidazole, 20 mM phosphate, pH 7.4); 3,5-dinitro Salicylic acid (DNS) (purchased from Shanghai Sangong); Kanamycin (purchased from Shanghai Sangong); Ni SepharoseTM6Fast Flow kit (purchased from Taijing Company); 30kDa ultrafiltration tube (purchased from Taijing Company) ; Chitin monosaccharide and chitobiose standard (purchased from Miragen); chitotriose, chitotetraose, chitopentaos...

Embodiment 2

[0042] Enzymatic properties of chitinase Chi6115

[0043] (1) Determination method of chitinase activity

[0044] Preparation of colloidal chitin: Slowly add 5 g of chitin powder to 50 mL of pre-cooled concentrated hydrochloric acid, stir evenly, and let stand at 4°C for 24 hours. Then the mixture was slowly added to 500 mL of pre-cooled deionized water, stirred evenly, and centrifuged at 4° C. and 5000 g for 10 min to collect the chitin precipitate. Repeatedly resuspend the precipitate with deionized water and centrifuge for collection, remove excess hydrochloric acid on the colloidal chitin until the pH value is about 6.5, and store at 4°C for future use.

[0045] The reducing sugar content was determined by DNS method. Mix 25 μL of enzyme solution and 100 μL of 1% colloidal chitin substrate, and react at 55° C. for 2 h. After the reaction was completed, 200 μL of DNS reagent was added, boiled for 5 min, immediately placed in ice water to cool, and the supernatant was tak...

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PUM

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Abstract

The invention discloses chitinase Chi6115 as well as a coding gene and application of the chitinase Chi6115. The enzyme sequence is shown in SEQ ID NO: 2. On the basis of metagenome sequencing data of a deep sea hydrothermal sulfide sample, a chitinase chi6115 gene sequence is obtained through an artificial synthesis method, enzyme cutting sites BamHI and XhoI are added, the gene is cloned into a pET-28a (+) expression vector, induced expression is carried out in escherichia coli E.coli BL21, and the recombinant chitinase chi6115 protein with high purity is obtained through purification. The optimum temperature of the enzyme is 55 DEG C, and the optimum pH value is 6.0; the enzyme activity is relatively high at the temperature of 45-60 DEG C and the pH value of 5.0-6.5, and the enzyme activity is kept above 85%; the enzyme not only can hydrolyze colloidal chitin, but also can hydrolyze an insoluble chitin substrate, and has important application value in the aspects of chitin resource development and utilization and chitosan oligosaccharide preparation.

Description

technical field [0001] The invention relates to a chitinase Chi6115 derived from deep-sea hydrothermal sulfide, its encoding gene and application, and belongs to the technical field of bioengineering. Background technique [0002] Chitin is the most abundant and renewable natural organic polymer in the marine environment, and its reserves ranks second in nature, second only to cellulose. It is widely found in crustacean shells, insect exoskeletons, The cell walls of fungi and algae, the global annual output of chitin produced by organisms can reach 10-100 billion tons. Chitin is a polymer composed of N-acetyl-D-glucosamine molecules covalently linked by β-(1-4) glycosidic bonds, which can be hydrolyzed by microbial chitinase β-(1-4) The glycosidic bond is decomposed, and finally oligosaccharides, chitobiose or N-acetylglucosamine are generated. [0003] Chitin has biological properties such as antibacterial, antiviral, promoting wound healing, lowering cholesterol, hemosta...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/70C12N9/42C12N1/21C12P19/26C12P19/14C12R1/19
CPCC12N9/2442C12N15/70C12P19/26C12P19/14C12Y302/01014Y02A50/30
Inventor 周梅先邵宗泽卢传琦王万鹏付晓腾
Owner THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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