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Pichia pastoris system capable of co-expressing An Man5A and XynII

A Pichia pastoris, expression system technology, applied in the field of bioengineering, to achieve the effect of reducing production costs

Inactive Publication Date: 2012-07-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, co-expression studies in the Pichia pastoris system have not been reported

Method used

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  • Pichia pastoris system capable of co-expressing An Man5A and XynII

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Construction, expression and product activity determination of engineering bacteria GS115 / man5A

[0016] Linearize pPICZαA-man5A with Sac I, perform electrotransformation of GS115 competent cells according to the Pichia expression manual, and use Zeocin to screen to obtain high-copy Pichia recombinant GS115 / man5A. The engineered bacterium was induced with 1.0% methanol for 96 hours, and the recombinant mannanase activity in the fermentation broth was measured by DNS method up to 29.05IU / mL. SDS-PAGE electrophoresis showed that the molecular weight of the recombinant mannanase was 52.0kDa.

Embodiment 2

[0017] Example 2 Construction, expression and product activity determination of engineering bacteria GS115 / man5A-xynII

[0018] Linearize pPIC9k-xynII with Sal I, perform electrotransformation of GS115 / man5A competent cells according to the Pichia expression manual, and use G418 to obtain high-copy Pichia recombinant GS115 / man5A-xynII. The engineering bacterium was induced with 1.0% methanol for 96 hours. The activity of recombinant xylanase in the fermentation broth was 112.10IU / mL and the activity of recombinant mannanase was still 29.05IU / mL as measured by DNS method. SDS-PAGE electrophoresis showed that the molecular weights of recombinant xylanase and mannanase were 24.1kDa and 52.0kDa, respectively, as figure 1 shown.

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Abstract

The invention provides a novel construction method of engineering bacteria GS115 / man5A-xyn II for co-expressing a beta-mannase gene derived from Aspergillus niger LW-1 and a beta-1,4-xylanase gene derived from Aspergillus usamii E001. A constructed Pichia pastoris co-expression system can be used for industrial production of beta-mannase and beta-1,4-xylanase. The prepared two enzymes have the characteristics of strong substrate appetency and high catalytic efficiency and have large industrial production potentials and economic values.

Description

technical field [0001] The present invention relates to a co-expression of β-mannanase gene (An man5A) derived from Aspergillus niger LW-1 and β-1,4-xylan derived from Aspergillus usamii E001 The invention relates to a new expression system of Pichia pastoris for an enzyme gene (xyn II), which belongs to the technical field of bioengineering. Background technique [0002] Hemicellulose is the second largest polysaccharide polymer in nature, and it is widely present in plant cell walls as a link between lignin and cellulose. The degradation products of hemicellulose have a wide range of applications, so how to make full use of the abundant hemicellulose resources has become a research hotspot. The complete degradation of hemicellulose requires the synergistic action of enzymes such as β-1,4-endoxylanase, β-mannanase, galactosidase and deacetylase. β-mannanase (β-1,4-D-mannan mannohydrolase, EC 3.2.1.78) is a kind of enzyme that can degrade β-1,4-mannoside from the interior ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N9/42C12R1/84
Inventor 李剑芳魏喜换赵顺阁邬敏辰殷欣
Owner JIANGNAN UNIV
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