Recombinant beta-glucosidase and expression and purification method and immobilization application thereof

A technology for expressing and purifying glucosidase, applied in chemical instruments and methods, biochemical equipment and methods, glycosylase, etc., can solve the problems of complicated separation and purification process, high application cost, and expensive production cost, and achieve purification Low cost, excellent effect

Active Publication Date: 2018-11-06
JIANGSU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the production and application of enzymes, there are mainly the following problems: first, the separation and purification process of enzymes is complicated, resulting in high production costs and large loss of enzyme activity, so it is necessary to develop a simple and efficient method for separation and purification of enzymes; second, The application cost of enzyme is high. In order to improve the efficiency of enzyme reaction process and reduce production cost, the development of enzyme immobilization technology is also a hot spot in the field of enzyme engineering research.
At present, there are few reports on GB polypeptides, mainly focusing on using it to modify the ends of some polypeptides so that they can be combined with hydrophobic materials

Method used

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  • Recombinant beta-glucosidase and expression and purification method and immobilization application thereof
  • Recombinant beta-glucosidase and expression and purification method and immobilization application thereof
  • Recombinant beta-glucosidase and expression and purification method and immobilization application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1: Prokaryotic expression of recombinant enzyme Glu-linker-ELP-GB

[0049] The gene encoding β-glucosidase is derived from Taiwan termites (NCBI database accession number GQ911585, as shown in SEQ.ID.NO.1), ELP is 50 pentapeptide repeating sequence units Val-Pro-Gly-Xaa-Gly Composed in series. Glu and ELP are connected with a connecting peptide linker, and the sequence of the GB polypeptide (the amino acid sequence is His-Asn-Trp-Tyr-His-Trp-Trp-Pro-His) is connected to the 3' end of the ELP gene to obtain a recombinant β- Glucosidase Glu-linker-ELP-GB (GLEGB), using T7 expression system, using the whole gene synthesis technology to synthesize DNA of recombinant enzyme (including recombinant β-glucosidase Glu-linker-ELP-GB (GLEGB) and control Glu-linker-ELP (GLE)), and then connect it with the empty vector pET28a to obtain the recombinant plasmid pET-Glu-linker-ELP-GB; as a control, the plasmids pET-Glu and pET-Glu-linker-ELP were constructed at the same tim...

Embodiment 2

[0059] Example 2: Prokaryotic expression of recombinant enzyme Glu-linker-ELP-GB

[0060] The gene encoding β-glucosidase is derived from the domestic termites of Taiwan, and ELP is composed of 50 pentapeptide repeating sequence units Val-Pro-Gly-Xaa-Gly in series. Glu and ELP were connected with a connecting peptide linker, and the GB polypeptide sequence was connected to the 3' end of the ELP gene to obtain a recombinant β-glucosidase Glu-linker-ELP-GB (GLEGB).

[0061] Using the T7 expression system, using pET28a as the carrier, using the whole gene synthesis technology to synthesize the DNA of the recombinase, and connecting it with the empty vector to obtain the recombinant plasmid pET-Glu-linker-ELP-GB; as a control, the plasmid pET-Glu was constructed at the same time and pET-Glu-linker-ELP (pET-GLE).

[0062] The successfully verified recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent. After the selected monoclonal BL21 strains were activa...

Embodiment 3

[0065] Embodiment 3: the prokaryotic expression of recombinant enzyme Glu-linker-ELP-GB

[0066] The gene encoding β-glucosidase is derived from the domestic termites of Taiwan, and ELP is composed of 80 pentapeptide repeating sequence units Val-Pro-Gly-Xaa-Gly in series. Glu and ELP were connected with a connecting peptide linker, and the sequence of the GB polypeptide was connected to the 3' end of the ELP gene.

[0067] Using the T7 expression system, using pET28a as the carrier, using the whole gene synthesis technology to synthesize the DNA of the recombinase, and connecting it with the empty vector to obtain the recombinant plasmid pET-Glu-linker-ELP-GB; as a control, the plasmid pET-Glu was constructed at the same time and pET-Glu-linker-ELP (pET-GLE).

[0068] The successfully verified recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent. After the selected monoclonal BL21 strains were activated in LB medium at 37 °C and 190 rpm for 12 h, th...

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Abstract

The invention relates to a recombinant beta-glucosidase and an expression and purification method and the immobilization application thereof, and belongs to the technical field of biological enzyme engineering. The recombinant beta-glucosidase is recombinant beta-glucosidase Glu-linker-ELP-GB (GLEGB) obtained by linking binary labels ELP and GB with beta-glucosidase (Glu) through a universal linking peptide linker, wherein the sequence of GB polypeptide is linked to the 3' end of an ELP gene. By virtue of a reversible phase change property of an ELP label on recombinant enzyme, separation andpurification of the enzyme can be achieved; the recombinant enzyme containing the ELP label can be precipitated only by adding a certain amount of (HN4)2SO4 for incubation for a period of time; by virtue of a temperature response behavior of the ELP label, one-step rapid separation and purification of a target enzyme molecule is achieved; the enzyme purifying effect of the expression and purification method is superior to that of an ammonium sulfate precipitation method, and the purification cost of the expression and purification method is much lower than that of a chromatography method.

Description

technical field [0001] The invention relates to a recombinant β-glucosidase, its expression and purification method and its immobilization application, and belongs to the technical field of biological enzyme engineering. Background technique [0002] Enzymes are a class of proteins that can catalyze chemical reactions under mild conditions, and have the characteristics of high substrate specificity, high product specificity and high catalytic efficiency. In the production and application process of enzymes, there are mainly the following problems: first, the separation and purification process of enzymes is complicated, resulting in high production costs of enzymes and large loss of enzyme activity, so it is necessary to develop simple and efficient methods for separation and purification of enzymes; second, The application cost of enzyme is high. In order to improve the efficiency of enzyme reaction process and reduce production cost, the development of enzyme immobilizatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/42C12N15/70
CPCC07K7/06C07K14/00C07K2319/00C12N9/2445C12N15/70C12Y302/01021
Inventor 戎军辉王赟韩娟李春梅王蕾倪良张文莉
Owner JIANGSU UNIV
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