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Chitin binding domain and application thereof

A technology of chitin binding domain and identity, applied in the field of enzyme engineering, can solve the problems of increasing the investment of relevant reagents and equipment, increasing the cost of using immobilized enzymes, lack of specificity, etc., and achieving strong chitin specific binding ability. , Simplified immobilization process, easy separation effect

Active Publication Date: 2021-02-12
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problem of the above two methods in the process of enzyme immobilization is that there is often a lack of specificity between the enzyme and the solid material, and pretreatment such as extraction and purification of the enzyme is required.
These pretreatment processes will not only cause the loss of enzymes, but also increase the investment in related reagents and equipment, and increase the cost of immobilized enzymes.

Method used

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  • Chitin binding domain and application thereof
  • Chitin binding domain and application thereof
  • Chitin binding domain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of a general expression vector with a chitin-binding domain

[0052] A flexible linker peptide was selected to link with the chitin-binding domain to construct a universal expression vector. Wherein the amino acid sequence of the chitin-binding domain is shown in SEQ ID No.1, and the nucleotide sequence is shown in SEQ ID No.2; the amino acid sequence of the flexible linker peptide is shown in SEQ ID No.3, and the nucleotide sequence As shown in SEQ ID No.4. The gene connecting the peptide is located at the 5' end of the chitin binding domain gene and inserted into the pET22b vector to obtain the general expression vector pLChBD. The plasmid map of the general expression vector pLChBD is as follows: figure 1 shown. Plasmid pLChBD was maintained in E. coli DH5α.

Embodiment 2

[0053] Example 2: Construction of recombinant β-glucosidase with chitin binding domain

[0054] Using the genome of the acidophilic heat-resistant bacterium Acidothermus cellulolyticus 11B (ATCC 43068) as a template, it was designed according to the gene sequence of its β-glucosidase (the nucleotide sequence is SEQ ID No.5, and the amino acid sequence is SEQ ID No.6). For primers, select NdeI and XhoI restriction sites, and design primers P1 and P2 as follows:

[0055] P1: 5'-CGACTTCATATGATGACACAAATCGAAGAGCG-3'

[0056] P2: 5'-ACCGCTCGAGTCAGGGCGCCGCGATCGTGTTC-3'

[0057] The PCR amplified fragments with restriction enzymes NdeI and XhoI were also added to the general expression vector pLChBD with restriction enzymes NdeI and XhoI, and double enzyme digestion was carried out at 37°C for 3 hours. The product of the above double digestion was purified with a DNA purification kit. After the two purified products were mixed, T4 DNA ligase from Quanshijin Company was added, and l...

Embodiment 3

[0058] Example 3: Preparation of recombinant β-glucosidase with chitin binding domain

[0059] The recombinant plasmid pLChBD-AcBg prepared in Example 2 was transformed into Escherichia coli C43 (DE3), coated with LB plates containing ampicillin, cultured overnight at 37°C, and colonies in good condition were picked, and positive transformants were verified by PCR. And cultivated in liquid LB medium containing ampicillin, and selected recombinant Escherichia coli strains that meet the requirements. Take the above-mentioned preserved bacteria and inoculate them into 5 mL of liquid LB medium containing ampicillin for overnight culture at 37°C, transfer 100 mL of new LB liquid medium with 1% (v / v) inoculum amount, cultivate to OD600=1.0, add IPTG To a final concentration of 1mM / L, induce culture at 25°C for 24h. Centrifuge at 8000rpm / min for 10min to collect induced expression cells, resuspend the cells in 10mL of 20mM sodium phosphate buffer solution containing 40mM / L NaCl at p...

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Abstract

The invention discloses a chitin binding domain, which is constructed on a universal expression vector and is used for fusion expression of a target protein and synchronous completion of purificationand immobilization of the target protein. According to the invention, efficient immobilization of beta-glucosidase is realized by utilizing the specific affinity performance of chitin and fusion protein, and ginsenoside Rd is prepared by taking ginsenoside Rb1 as a substrate and utilizing the immobilized enzyme in a high-efficiency manner. The immobilization method provided by the invention has the advantages of low cost, high enzyme immobilization efficiency, small enzyme activity loss, strong specificity and the like, and fundamentally solves the problems of unrecoverability, poor stability,low repetition rate of the traditional whole-cell immobilization method and the like of the free enzyme.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to an enzyme immobilization dominated by a chitin binding domain and its application. Background technique [0002] β-glucosidase (β-D-glucoside glucohydrolase, EC3.2.1.21) has the function of specifically hydrolyzing oligosaccharides and aglycone analogs containing glycosidic bonds. In the energy industry, β-glucosidase can effectively hydrolyze oligosaccharides and cellobiose, thereby eliminating the inhibition of cellulase products in the process of cellulose hydrolysis, and can be used in conjunction with other cellulase to effectively hydrolyze cellulose ; In terms of food processing, β-glucosidase can process some substances in some green plants into spices, which can be used in fruit wine, fruit juice, and tea juice to enhance the flavor, enhance its taste, and improve the quality of the product; as a feed additive, β-glucosidase can hydrolyze the glycosidic bon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N11/10
CPCC12N9/2402C12Y302/01021C12N11/10
Inventor 张作明王丽梅杨玉环李玉卫
Owner JILIN UNIV
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