Glucoside hydrolase CmChi3 and application thereof in degradation of hydrolyzed colloid chitin

A technology of glycoside hydrolase and hydrocolloid, which is applied in the field of glycoside hydrolase CmChi3 and its application in degrading hydrocolloid chitin, which can solve the problems of easy-to-contaminate bacteria, low cost conversion rate, and industrial production that has not yet been realized.

Active Publication Date: 2021-03-26
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires the synergy of multiple chitinases in the process of degrading chitin, the products are not easy to separate and the degradation process is easy to be contaminated with bacteria
[0008] Based on some literature and patent reports, the enzymatic preparation of GlcNAc requires a variety of chitinases such as endo-chitinase (EC 3.2.1.14), exo-chitinase (EC 3.2.1.20) and (EC 3.2 .1.201) and N-acetylglucosaminidase (NAGase, EC 3.2.1.52) and other enzymes. Due to the high cost of extraction and purification of various enzymes and the low conversion rate, this method has not yet been realized. Industrial production

Method used

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  • Glucoside hydrolase CmChi3 and application thereof in degradation of hydrolyzed colloid chitin
  • Glucoside hydrolase CmChi3 and application thereof in degradation of hydrolyzed colloid chitin
  • Glucoside hydrolase CmChi3 and application thereof in degradation of hydrolyzed colloid chitin

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Embodiment 1

[0091] Source of bacteria

[0092] 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

[0094] Chitinase cm The construction of the Chi3 recombinant strain, the specific steps are as follows:

[0095] (1) Obtain chitinase cm Chi3 gene, its nucleotide sequence is shown in SEN ID NO:1.

[0096] (2) Design PCR amplification primers:

[0097] cm Chi3-F: 5'-GGGAATTCCATATGACCGAGATCGCCCCGTAC-3',

[0098] cm Chi3-R: 5'-CCGGAATTCGCGGCCGTTGCCCAGCAC-3' amplifies the full length of the gene;

[0099] (3) PCR amplification program:

[0100] Preheat at 95°C for 5 min; denature at 94°C for 45 s, anneal at 55°C for 45 s, extend at 72°C for 1.5 min, 30 cycles; extend at 72°C for 10 min; store at 4°C;

[0101] (4) Target gene after PCR amplification cm Chi3 and plasmid Pcold I both use restriction enzymes Nde I and EcoRI, incubate at 37°C for 1 hour. After digestion, the target gene is purified using a common DNA purification kit. After purification, it is stored at -20°C for later use. The recovery kit is recovered, and stored at -20°C after recovery;

[0102] (5) The...

Embodiment 3

[0105] recombinant chitinase cm Expression and purification of Chi3

[0106] (1) After the preserved recombinant expression strain was revived on the plate, a single colony was picked and inoculated in 5 mL of LB liquid medium containing 0.05 g / LAmp, and cultured at 200 rpm for 8-10 h at 37°C. Transfer the activated bacterial solution to a 500 mL Erlenmeyer flask containing 100 mL LB medium (0.05g / L Amp) at an inoculum volume of 1% by volume, and cultivate to OD at 37°C and 200 rpm 600 When the temperature was 0.6-0.8, the inducer IPTG was added for induction, the induction temperature was 18°C, and the induction time was 16 h.

[0107] (2) After the cultivation, the cells were collected by centrifugation at 6000 rpm for 10 min at 4°C. After crushing using a sonicator, centrifuge at 8000 rpm for 20 min at 4°C to collect the supernatant. The supernatant is the crude enzyme solution containing recombinant chitinase, which is stored at 4°C for future use.

[0108] (3) The col...

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Abstract

The invention discloses a glucoside hydrolase CmChi3 and an application thereof in degradation of colloid chitin. The invention comprises the following steps of performing genome sequencing analysis on a bacterial strain Chitinolyticbacter meiyuanensis SYBC-H1 screened from soil, cloning CmChi3, constructing a recombinant bacterial strain, expressing CmChi3 protein, purifying HIS-TAG, identifyingthat the recombinant protein has two catalytic structural domains and two chitin binding domains, has the properties of endonuclease and glucoside hydrolase, and can efficiently hydrolyze chitin and can take GlcNAc as a final chitin product. The research is expected to provide theoretical basis and basis for high-efficiency production of GlcNAc by a single-enzyme method.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a glycoside hydrolase cm Chi3 and its application in degrading hydrocolloidal chitin. Background technique [0002] Chitin is a natural high-molecular polysaccharide polymerized by N-acetylglucosamine through β-1,4-glycosidic bonds. It is the second largest carbohydrate polymer on earth after cellulose. The molecular formula is (C 8 h 13 o 5 N)n, the relative molecular weight is above 1 million. Chitin is widely found in fungal cell walls, insect shells, and shrimp and crab shells in nature. Chitin is mainly derived from shrimp and crab shells. Chitin with higher purity can be obtained after deproteinization and decalcification with 10% sodium hydroxide and concentrated hydrochloric acid. With the development of the shrimp and crab farming industry, more and more scraps such as shrimp and crab shells are produced, and these scraps are mostly discarded as solid waste. Afte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/26C12R1/19
CPCC12N9/2442C12N15/70C12P19/26C12Y302/01014
Inventor 陈可泉王成勇张阿磊周宁王莹莹陈燕陈雪曼欧阳平凯
Owner NANJING UNIV OF TECH
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