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Method for artificially constructing chitin corpuscle multienzyme complex scoford-chiC-chiA-sg and application

A technology of scaford-chic-chia-sg and scaford-chbd is applied in the field of artificial construction of chitinosome multi-enzyme complexes, which can solve the problems of unfixed distance and intermediate product capture, so as to reduce production costs and achieve efficient degradation. , the effect of high monosaccharide yield

Pending Publication Date: 2021-03-19
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Based on comprehensive literature and patent reports, it can be concluded that in the traditional free multi-enzyme catalytic system, the distance between enzymes is not fixed, and the intermediate product cannot be captured by the next enzyme in time, thereby inhibiting other enzymes. is a common problem in multi-enzyme catalytic systems
In the chitinase system, there is also the phenomenon that disaccharide accumulation negative feedback inhibits the overall enzyme activity

Method used

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  • Method for artificially constructing chitin corpuscle multienzyme complex scoford-chiC-chiA-sg and application
  • Method for artificially constructing chitin corpuscle multienzyme complex scoford-chiC-chiA-sg and application
  • Method for artificially constructing chitin corpuscle multienzyme complex scoford-chiC-chiA-sg and application

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Embodiment 1

[0034] The following examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

[0035] The construction of embodiment 1 protein scaffold scaford-ChBD

[0036] CohesinI-1, cohesinII, and cohesinI-3 were derived from the strain Clostridium cellulovorans (ATCC 35296 T ), Clostridium cellulolyticum, (ATCC 35319 T ) and Clostridium thermocellum (ATCC 27405 T ) genome. The chitin-binding domain gene ChBD comes from the strain Chitinolyticbacter meiyuanensis SYBC-H1 (ATCC BAA-2140 T ) genome. The genes cohesinI-1, cohesinII, and cohesinI-3 were obtained by total gene synthesis from General Biosystems (Anhui) Co., Ltd.

[0037] The plasmid construction of the protein scaffold scaford-ChBD adopts a one-step cloning method, designing homology arms in the upper and lower primers of the genes cohesinI-1, cohesinII, cohesinI-3 and ChBD, and using the linearized vector pETDuet in homologous recombina...

Embodiment 2

[0038] The construction of embodiment 2 chitinase-dockerins

[0039] The anchoring proteins dockerin-I-1, dockerin-II, and dockerin-I-3 were derived from the strain Clostridium cellulovorans (ATCC 35296 T ), Clostridium cellulolyticum, (ATCC35319 T ) and Clostridiumthermocellum (ATCC 27405 T ). The genes dockerin-I-1, dockerin-II, and dockerin-I-3 were obtained by total gene synthesis from General Biosystems (Anhui) Co., Ltd. Chitinase chiC and chitinase chiA were derived from the strain Serratia marcescens (ATCC 13880 T ), N-acetylglucosaminidase sg from laboratory preservation strain Chitinolyticbacter meiyuanensis SYBC-H1 (ATCC BAA-2140 T ) genome.

[0040] The plasmid construction of chiC-doc1 adopts a one-step cloning method, and a homology arm is designed and added to the upper and lower primers of the gene chiC and dockerin-I-1, and the plasmid is constructed with the linearized vector pET22b under the action of homologous recombination enzyme (Vazyme CE113) pET22...

Embodiment 3

[0044] SDS-PAGE analysis of each enzyme element in the chitin body of embodiment 3

[0045] Preparation of the constructed protein scaffold scaford-ChBD, exonuclease chiC-doc1, endonuclease chiA-doc2, N-acetylglucosaminidase sg-doc3 and chitinase (chiC, chiA, sg) not connected to dockerins The seed liquid is LB medium, cultivated at 37°C and 200rpm for 6-7h, inoculated the seed liquid with a volume fraction of 2% in the fermentation medium containing 100ml LB, and cultivated to OD at 37°C and 200rpm was 0.6, and the inducer IPTG was added in an amount of 1‰, induced at 18°C ​​and 200rpm for 20h, and then the fermentation was terminated to collect the fermentation broth. The fermentation broth was centrifuged at 8000g to collect the bacteria, washed twice with distilled water, resuspended in 25mL TBS (50mM pH7.0), ultrasonically broken and centrifuged at 8000g, and the supernatant collected was the crude enzyme solution of each enzyme component , stored at 4°C for later use. ...

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Abstract

The invention provides a method for artificially constructing a chitin corpuscle multienzyme complex scoford-chiC-chiA-sg and an application. According to the multienzyme complex, interacting proteinpairs existing in cellosomes from the nature are used for artificially fusing to express cohesins in the cellosomes to construct a protein scaffold, and anchoring protein is fused and expressed on corresponding chitin incision enzymes, chitin excision enzymes and N-acetylglucosaminidase, and then extracellular self-assembly is carried out on the protein scaffold and the chitinase to obtain a chitinase multienzyme complex; and then the multienzyme complex is applied to degradation of chitin to prepare N-acetylglucosamine. The multienzyme complex not only realizes extracellular self-assembly offree chitinase, but also fixes the distance between enzymes, improves the synergistic effect of enzyme elements in a system and generates a substrate channel, so that the degradation efficiency of thechitin is improved, a single monosaccharide product is obtained, and the cost of production and subsequent separation is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an enzyme preparation, a method for artificially constructing a chitin body multi-enzyme complex scaford-chiC-chiA-sg and its application. Background technique [0002] According to statistics from the China Fisheries Association, in 2018, the total output of crayfish farming in the country was 1.415 million tons, and that of hairy crabs was 800,000 tons, and they are still showing a rapid growth trend. As one of the birthplaces of crayfish and hairy crab consumption in Jiangsu Province, Nanjing alone consumes at least 45 tons of crayfish every day. The leftovers such as heads and shells produced in the processing of shrimp and crab account for about 30% to 40% of the body weight. At present, the price of dried shrimp and crab shell powder is only 500-1000 yuan per ton, which is comparable to the price of agricultural lignocellulosic biomass waste. Valuation of shrimp and crab shells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/96C12N11/18C12N11/02C12N9/42C12N9/24C12P19/14C12P19/26
CPCC12N9/96C12N11/18C12N11/02C12N9/2442C12N9/2434C12N9/2402C12P19/14C12P19/26C12Y302/01014C12Y302/0105
Inventor 陈可泉周宁张阿磊陈雪曼陈燕王子豪刘鑫欧阳平凯
Owner NANJING UNIV OF TECH
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