Bacterial laccase mutant protein, recombinant expression plasmid, transformed engineered strain and fermentation preparation method thereof

A technology of mutants and engineering bacteria, which is applied in the field of enzyme genetic engineering technology and fermentation engineering, can solve the problems that hinder the industrial application process of bacterial laccase, and achieve the effect of facilitating large-scale industrial production and improving stability

Active Publication Date: 2014-10-08
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the properties of existing bacterial laccases cannot meet the series of requirements of modern industrial biotechnology for bacterial laccases, such as having temperature stability and chloride ion tolerance, which hinders the industrial application of bacterial laccases

Method used

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  • Bacterial laccase mutant protein, recombinant expression plasmid, transformed engineered strain and fermentation preparation method thereof
  • Bacterial laccase mutant protein, recombinant expression plasmid, transformed engineered strain and fermentation preparation method thereof
  • Bacterial laccase mutant protein, recombinant expression plasmid, transformed engineered strain and fermentation preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, utilize PCR method to carry out the construction of deletion mutant DNA coding sequence

[0026] A total of 10 amino acids (323GAMSRRMMQG332) upstream of the protein sequence including the glycine at position 332 of the break site were selected for site-directed deletion. Primers were designed as follows:

[0027]

[0028]

[0029]Using the expression vector pET-22b(+)-lac15 containing the coding sequence of the wild-type bacterial laccase Lac15 gene as a template, the first round of PCR was carried out with lac15_F and the deletion primer D(323-332)_R, and at the same time, lac15_R and the mutant primer D were used (323-332)_F performed the second round of PCR. The first-round and second-round PCR products were recovered as templates for the third round of PCR, and lac15_F and lac15_R were used for the third round of PCR to obtain the full-length sequence with deletion mutations. The obtained deletion mutant gene sequence was connected to pET22b...

Embodiment 2

[0031] Example 2. Induced expression and purification of mutant proteins

[0032] The recombinant engineered bacteria Escherichia coli BL21(DE3) / pET-22b(+)-lac15(Del) and the wild-type gene recombinant engineered bacteria Escherichia coli BL21(DE3) / pET-22b(+)-lac15 confirmed by sequencing were inoculated into the In 5ml of liquid LB medium, the inoculum size was 1%, and cultured with shaking at 220rpm at 37°C for 8-12h. Insert the overnight cultured recombinant engineered bacterium into 400ml liquid LB medium according to the inoculum size of 1%, 37 ℃, 200rpm shaking culture, when the fermentation broth OD 600 When it reaches 0.6-1.0, add IPTG to a final concentration of 0.2mM, and at the same time lower the temperature to 28°C to induce expression for 12-14h.

[0033] Collect the cells by centrifugation at 4°C at 8000g, add 0.1 times the volume of the bacterial solution in Binding buffer, break the cells by ultrasonication for 40min in a 350W ice bath, and collect the supe...

Embodiment 3

[0035] Embodiment 3, wild-type bacterial laccase and its mutant Lac15 (Del) stability experiment

[0036] The purified wild-type bacterial laccase and its mutant proteins were stored at 28°C with a buffer of 50mM Na 2 HPO 4 -KH 2 PO 4 (pH 7.5). Regularly detect the remaining enzyme activity of wild-type bacterial laccase and its mutant protein, and at the same time, carry out SDS-PAGE detection on the samples.

[0037] Such as figure 1 As shown, the SDS-PAGE electrophoresis pattern on the 4th day, 7th day, 15th day and 22th day showed that the wild-type protein was degraded by about 80% on the 4th day, and the mutant protein was degraded by about 50% on the 14th day. The half-life of the body protein at 28°C is 3-4 times that of the wild type.

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Abstract

The invention discloses a bacterial laccase mutant protein, which is characterized in that the mutant protein amino acid sequence is obtained by deletion mutation of the 323rd glycine residue to the 332rd glycine residue in the bacterial laccase amino acid sequence shown as SEQ ID No.1. Through a genetic engineering reconstruction method, a stability improved bacterial laccase protein coding gene, its expression plasmid and engineered bacteria can be obtained, and after large-scale fermentation and induced expression of the engineered bacteria, the stability improved bacterial laccase protein can be obtained. According to the invention, the marine uncultured microorganism source bacterial laccase Lac15 is taken as the foundation, and by means of genetic engineering reconstruction, mutant gene can be obtained. At the same time, a recombinant escherichia coli is employed to conduct high-density culture for high-efficiency expression of the bacterial laccase mutant protein. According to the invention, the stability and yield of the bacterial laccase are greatly improved.

Description

technical field [0001] The invention relates to a bacterial laccase mutant protein with improved stability constructed by a directional transformation method and a high-density culture production method in recombinant Escherichia coli, belonging to the fields of enzyme genetic engineering technology and fermentation engineering technology. Background technique [0002] Laccase (Laccase, EC 1.10.3.2) is a copper-containing polyphenol oxidase with a wide range of substrates, which can catalyze the oxidation of various phenolic and non-phenolic compounds. It plays an important role in the removal of toxicity of similar substances (such as phenoxy herbicides, etc.) and the synthesis of new compounds. Laccases are widely found in fungi and bacteria. Fungal laccases can be active under acidic or neutral to slightly acidic conditions, and some fungal laccases have been used in industrial production. Compared with fungal laccases, bacterial laccases can exert catalytic activity un...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0061C12Y110/03002
Inventor 方泽民肖亚中常飞周鹏张学成房伟袁璟
Owner ANHUI UNIVERSITY
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