Method for high-level expression of bacterial laccase in Escherichia coli

A high-efficiency expression and Escherichia coli technology, applied in the field of bioengineering, can solve the problems affecting the development and application of bacterial laccase, and achieve the effect of soluble expression and short fermentation time

Inactive Publication Date: 2012-03-28
HUBEI UNIV
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AI Technical Summary

Problems solved by technology

The low proportion of soluble enzyme protein seriously affects the development and application of bacterial laccase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: the expression of soil Klebsiella 601 bacterial laccase MCO in Escherichia coli

[0021] Firstly, restriction endonucleases BamHI and SalI were used to digest DNA encoding laccase gene of Klebsiella soil 601 bacteria and pMAL-c2x plasmid DNA respectively, and the enzyme digestion buffer was 10×T reaction buffer. Digest at 37°C for 4 hours. A 1.6 kb DNA fragment encoding the bacterial laccase gene of Klebsiella soil 601 and a 6.67 kb linear pMAL-c2x plasmid DNA were recovered after the digestion mixture was subjected to 1% agarose gel electrophoresis. Then the two recovered DNA fragments were mixed according to the ratio of 5 times laccase gene DNA fragment: 1 times linear pMAL-c2x plasmid DNA, and mixed with T 4 DNA ligase was ligated overnight at 16°C. Then the ligation reaction mixture was directly transformed into E.coli DH10B, and positive transformants were screened with LB plates containing 100 μg / ml ampicillin. The size of the recombinant plasmid...

Embodiment 2

[0022] Embodiment 2: the expression of soil Klebsiella 601 bacterium laccase MCO mutant in Escherichia coli

[0023]Using the same method in Escherichia coli DH10B to express the laccase MCO mutant α351-378 / β351-378 of Klebsiella soil 601 bacteria, the soluble fusion protein was 60% in the supernatant and 40% in the precipitate. However, the DNA encoding the laccase gene of Klebsiella soil 601 was connected to the ptac85 expression vector using the restriction endonuclease BamHI and SalI restriction sites and expressed in E.coli Top10 bacteria, and the soluble fusion in the supernatant 0 for protein, 100% for pellet.

Embodiment 3

[0024] Embodiment 3: the expression of soil Herbella 531 bacterial laccase MCO in Escherichia coli

[0025] Restriction endonucleases EcoRI and SalI were used to digest the gene DNA and pMAL-c2x plasmid DNA encoding the Herbella 531 bacterial laccase MCO respectively, and the digestion buffer was 10×T reaction buffer. Digest at 37°C for 4 hours. A 1.6kb DNA fragment encoding the laccase gene of Herstia soil 531 bacteria and a 6.67kb linear pMAL-c2x plasmid DNA were recovered after the digestion mixture was subjected to 1% agarose gel electrophoresis. Then the two recovered DNA fragments were mixed according to the ratio of 5 laccase gene DNA fragments: 1 linear pMAL-c2x plasmid DNA, and ligated overnight at 16°C with T4 DNA ligase. Then the ligation reaction mixture was directly transformed into E.coliDH10B, and positive transformants were screened with LB plates containing 100 μg / ml ampicillin. Express the target protein according to the above steps C-E, the soluble fusion ...

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Abstract

The invention provides a technical method for forming maltose binding protein-bacterial laccase fusion protein by fusing maltose binding protein and bacterial laccase and expressing the maltose binding protein-bacterial laccase fusion protein at low temperature. The method comprises the following steps: A, constructing a maltose binding protein-bacterial laccase gene; B, screening a positive converter; C, expressing fusion protein; D, identifying soluble fusion protein; E, purifying the fusion protein; and F, measuring parameters of enzyme kinetics of the bacterial laccase. The method realizes the high-level expression of the protein-bacterial laccase fusion protein in Escherichia coli cells. The soluble bacterial laccase can be expressed in high level and also maintains the same enzyme activity as unfused bacterial laccase. The problem that a large number of insoluble inclusion bodies are formed when the bacterial laccase is expressed in high level in the Escherichia coli cells is solved. The method has the advantages of simple operation, short fermentation time and low cost. The bacterial laccase also can be directly immobilized on maltose affinity chromatograph resin for various enzymatic reactions.

Description

technical field [0001] The invention relates to bioengineering technology, especially a method for highly expressing soluble bacterial laccase in Escherichia coli. Background technique [0002] Because laccase can oxidize aromatic compounds and some other non-aromatic organic substances, it has a wide range of substrate specificity, so it is used in pulp bleaching, textile dye decolorization, removal of toxic waste, bioremediation, medical diagnosis or preparation of pseudo-anticancer drugs Catalysts and biosensors have great application potential. However, the lack of abundant and cheap enzyme sources hinders the commercial application of laccases. A major approach to solve this problem is to obtain large quantities of laccases through heterologous expression of laccases. Because glycosylation modification is required to be active, fungal laccase is not suitable for expression in prokaryotic expression systems. Currently, it is mainly expressed in yeast and filamentous fu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12R1/19C12N15/70C12R1/22C12R1/01
Inventor 王行国李洋龚子君左文峰
Owner HUBEI UNIV
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