Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Recombinant plasmid efficiently expressing xylanase and construction method for recombination Pichia pastoris

A Pichia pastoris and xylanase technology, which is applied in the field of expressing high-efficiency xylanase recombinant plasmids and the construction of recombinant Pichia pastoris, can solve the problems of rapid development and achieve high activity effects

Inactive Publication Date: 2014-09-17
NORTHEAST AGRICULTURAL UNIVERSITY
View PDF2 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the research on xylanase molecular biology started late, it developed rapidly

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
  • Recombinant plasmid efficiently expressing xylanase and construction method for recombination Pichia pastoris
  • Recombinant plasmid efficiently expressing xylanase and construction method for recombination Pichia pastoris
  • Recombinant plasmid efficiently expressing xylanase and construction method for recombination Pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Optimization of codons in xylanase gene

[0030] According to the codon preference of Pichia pastoris, the codon optimization of xylanase gene was completed with DNA works software. The optimization result is as figure 1 shown.

Embodiment 2

[0032] Construction of single-copy expression vector pPICZαA-XynB-opt

[0033] The optimized xylanase gene sequence XynB-optimized (XynB-opt) was synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd. Restriction sites were designed at both ends of the gene, EcoR I and Xba I respectively, and the gene was connected to the pPICZαA vector. The successfully constructed recombinant expression vector pPICZαA-XynB-opt was transformed into Escherichia coli competent E.coil top10, and positive clones were screened on LB low-salt culture plates.

Embodiment 3

[0035] pPICZαA-(XynB-opt) 2 Construction of double-copy expression vector

[0036] Double copies of XynB-opt were constructed by in vitro tandem method. The pPICZαA-XynB-opt recombinant plasmid stored in our laboratory was single-digested with Bgl II, double-digested with BamH I and Bgl II, and kept in a water bath at 37°C overnight. After the xynB gene fragment was recovered by 0.8% agarose gel electrophoresis, the double-digested product was ligated with the purified single-digested product with T4 DNA ligase overnight at 16°C. Build process like figure 2 shown.

[0037] Transform the ligation product into E. coil Top10 competent cells, incubate at 37°C for 1 h, spread the bacterial solution on LB solid medium containing 25 μL / 100mL Zeocin, and incubate overnight at 37°C. After visible colonies grow, use Cracking to quickly identify recombinant plasmids, and initially screen out double-copy recombinant plasmids. Pick double-copy recombinant plasmid colonies, inoculate ...

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 provides a recombinant plasmid efficiently expressing xylanase and a construction method for recombination Pichia pastoris. Based on the preference of Pichia pastoris codon, aspergillus niger xylanase gene XynB is optimized, linked to Pichia pastoris expression vector pPICZ alpha A and expressed in Pichia pastoris GS115. The enzyme activity of recombinase GS115 / pPICZ alpha A-XynBopt is improved by 1.8 times. Double-coppy GS115 / pPICZ alpha A-(XynBopt)2 of xylanase XynBopt gene is constructed by employing an in-vitro tandem way. The activity of double-copy xylanase is improved by 1.9 times than the activity of single-copy xylanase. After ten days of induction, the enzyme activity of the double-copy xylanase is 15158.23 + / - 45.11 U / mL. The enzyme activity of purified xylanase is 6853.00 + / - 20.08 U / mg.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and the field of hemicellulose decomposition, and specifically relates to a construction method of a recombinant plasmid expressing high-efficiency xylanase and recombinant Pichia pastoris. Background technique [0002] Plants contain 20% to 30% hemicellulose, and xylan is the main component of hemicellulose, and its content in plant cell walls is second only to cellulose, accounting for about 35% of cell dry weight. Xylan is difficult to be degraded and utilized under natural conditions. Xylan can be converted into xylooligosaccharides and monosaccharides by acid hydrolysis or enzymatic hydrolysis. The acid hydrolysis speed is fast, but it is accompanied by the production of toxic compounds, which are harmful to the subsequent microbial fermentation The process is affected, and the condition of enzymatic hydrolysis is mild, and no toxic by-products are produced. Therefore, the development...

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
IPC IPC(8): C12N15/81C12N1/19C12R1/84
Inventor 单安山高赫
Owner NORTHEAST AGRICULTURAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com