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

An over-expression and heat-resistant technology, applied in the fields of molecular biology, genetic engineering and enzymology, to achieve good decolorization effect, reduce dosage and improve the effect of color deepening

Inactive Publication Date: 2013-01-09
SHINE E BIOTECH CO LTD
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Laccase is a biocatalyst with wide application and high demand. In order to solve the key problems such as low yield and poor stability in the use of laccase, we have developed a coding The heat-resistant laccase gene was designed and modified to greatly increase the expression level of the gene in Escherichia coli to meet the needs of industrial production

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[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 laccase gene

[0047] (1) Use the data analysis software DNA2.0-Gene Designer to analyze the natural gene of laccase lcs The codons in -0 were anal...

Embodiment 2

[0049] (3) Take pHsh- lcs -0 is the template, and the above primers are used for PCR amplification. The PCR conditions were set as follows: 95°C, 5 min; pause timing, add Pyrobest polymerase, add 40 mL 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 electroporation E. coli After DH10B, apply to LB plates containing 0.1 mg / ml ampicillin and culture at 30°C. Pick a single colony from the LB plate, inoculate them into 4 ml LB liquid medium containing 0.1 mg / ml ampicillin, culture overnight at 30°C, and extract the plasmid according to standard methods.

[0052] (6) The plasmid was sent to Huada...

Embodiment ３

[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 ).

[0057] (2)-(6) are basically the same as Example 2, except that the target base segment for mutagenesis and the sequence of the reverse PCR primer: 5'-ATATACCCAT GCAGGGTCCG TCTTTCCCGG AACCGAAAGT TGTTCG-3' (SEQ ID NO: 12 ), 5'-CGAACAACTT TCGGTTCCGG GAAAGACGGA CCCTGCATGGG TATAT-3' (SEQ ID NO: 13). In order to achieve the cumulative effect of mutagenesis, this round of mutagenesis can be used as a template for the previous round of mutants.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap