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Method for realizing over expression of thermostable laccase gene through location transformation

A technology of overexpression and heat resistance, applied in the fields of genetic engineering, enzymology, and molecular biology, can solve the problems of low yield and poor stability, and achieve the effect of increasing yield

Inactive Publication Date: 2011-05-18
SHINE E BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Laccase is a biocatalyst with a wide range of uses and a high demand. In order to solve the key problems of low yield and poor stability in the use of laccase, we have carried out multiple designs and modifications on a gene encoding an extremely heat-resistant laccase. Improve the expression level of this gene in Escherichia coli to meet the needs of industrial production

Method used

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  • Method for realizing over expression of thermostable laccase gene through location transformation
  • Method for realizing over expression of thermostable laccase gene through location transformation
  • Method for realizing over expression of thermostable laccase gene through location transformation

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Embodiment 1

[0041] (2) Using the Thermus thermophilus genomic DNA as a template, PCR amplification of the laccase gene was carried out with the above primers.

[0042] (3) with Pst I and Sph I process the PCR product and compare it with the Pst I and Sph I double-digested heat shock expression vector pHsh ligation.

[0043] (4) Imported by electroporation E. coli After DH10B, apply to LB plates containing 0.1 mg / ml ampicillin and culture at 30°C.

[0044] (5) Pick a single colony from the LB plate, inoculate them into 4 ml LB liquid medium containing 0.1 mg / ml ampicillin, culture at 30°C overnight, and extract the plasmid according to the standard method.

[0045] (6) The plasmid was sent to Huada Gene Technology Co., Ltd. for sequence determination. The plasmid with the same DNA sequence as TTC1370 was named pHsh- lcs -0, and used as starting material and experimental control for subsequent studies.

[0046] Example 2: Site-directed mutagenesis of the extremely thermostable ...

Embodiment 2

[0048] (2) Design and synthesize a pair of reverse long primers to replace the rare and non-dominant codons in the 1st to 27th codons. The sequence of the primers is: 5'-ACGAACAACT TTCGGTTCCG GAAAGCTCGG ACCCTGCATGG GTATATCTCC TTCTTG-3' (SEQ ID NO: 8), 5'-AGCCAGGGTG GTCTGCTGAG CCTGAAACTG AGCGCAACCC CGACCCCGCT TGCCCTGG-3' (SEQ ID NO: 9).

[0049] (3) Take pHsh- lcs -0 is the template, and the above primers are used for PCR amplification. The PCR conditions were set as: 95°C, 5 min; pause the timer, add Pyrobest polymerase, add 40 mL of paraffin oil for sealing; 35 cycles (94°C, 30 s; 60°C, 60 s; 72°C, 4 min); 72 ℃, 10 min; stop the reaction, keep warm at 4℃.

[0050] (4) Separate the PCR products and templates by agarose gel electrophoresis, and recover the DNA fragments in the PCR amplified bands by tapping the gel. After the PCR product was treated with T4 DNA kinase to phosphorylate it, T4 DNA ligase was added to self-circularize it.

[0051] (5) Imported by electroporat...

Embodiment ３

[0055] Example 4: Next round of site-directed mutagenesis of the extremely thermostable laccase gene

[0056] (1) In the experiment, the gene sequencing signal was often interrupted in a GC base-rich region (GC island) of the laccase gene, and the DNA online analysis software mfold (http: / / frontend.bioinfo.rpi.edu / applications / mfold) analysis found that GC islands lead to the formation of complex secondary structures in DNA, and the mutant designed in the present invention can reduce this structure ( figure 2 ).

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Abstract

The invention discloses a method for realizing over expression of the thermostable laccase gene through location transformation. Thermostable laccase produced from thermus thermophilus bacteria has great application potential in the industrial processes of pulp bleaching, textile decoloring, biomass hydrolysate detoxifying and the like, but the thermostable laccase gene has low expression level and can not become an enzyme source. In the invention, a carrier in a pHsh series is firstly applied to carrying out cloning, in-situ mutagenesis and induced expression on the laccase gene. By carryingout case analysis and multiple explorative site-directed mutagenesis on a special sequence of the thermostable laccase gene, the invention has the effects of reducing rare codons in the gene, lowering the GC base content and weakening formation of a nucleic acid secondary structure and the like, thereby enhancing the expression level of the gene in escherichia coli by 317 times compared with a natural gene.

Description

technical field [0001] The method for over-expressing the gene of extremely heat-resistant laccase through positioning transformation involves the fields of molecular biology, genetic engineering, enzymology and the like. Specifically, it relates to a method for carrying out positioning transformation of a base sequence of a gene encoding extremely heat-resistant laccase so as to realize high-efficiency expression of the laccase gene. technical background [0002] Laccase (Laccase, EC1.10.3.2) is a polyphenol oxidase, which belongs to the blue multi-copper oxidase family. The catalytic center of the enzyme protein generally contains 4 copper ions, so it is also called multi-copper oxidase . Laccase has a unique substrate catalytic mechanism. During the catalytic reaction, laccase can capture electrons from the reaction substrate and oxidize the substrate into active free radicals. At the same time, the captured electrons are transferred to the surrounding by copper ions O ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N1/21C12R1/19
Inventor 张永昌郑志强邵蔚蓝裴建军
Owner SHINE E BIOTECH CO LTD
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