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Yeast system for co-expressing faeA and xyn11A

An expression system and co-expression technology, applied in the field of Pichia pastoris new expression system, to achieve the effect of reducing the preparation cost

Inactive Publication Date: 2013-03-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Construction, expression and product activity determination of embodiment 1 engineering bacterium GS115 / faeA:

[0017] Linearize pPICZαA-faeA with Sac I, perform electrotransformation of GS115 competent cells according to the Pichia expression manual, and screen with Zeocin to obtain high-copy Pichia recombinant GS115 / man5A. The engineered bacteria were induced with 1.0% methanol for 72 hours, using methyl ferulate as a substrate, the amount of ferulic acid released was analyzed by HPLC, and the activity of recombinant ferulic acid esterase in the fermentation broth was determined to reach 7.04U / mL. SDS-PAGE electrophoresis showed that the molecular weight of the recombinant mannanase was 36.0kDa.

Embodiment 2

[0018] Example 2 Construction, expression and product activity determination of engineering bacteria GS115 / faeA-xyn11A:

[0019] pPIC9k-xyn11A was linearized with Sal I, and GS115 / faeA competent cells were electrotransformed according to the Pichia expression manual, and high-copy Pichia recombinant GS115 / man5A-xyn11A was obtained by screening with G418. The engineering bacterium was induced with 1.0% methanol for 72 hours, and the activity of recombinant xylanase in the fermentation broth was up to 600IU / mL as measured by DNS method, and the activity of recombinant ferulic acid esterase was still 7.04U / mL. SDS-PAGE electrophoresis showed that the molecular weights of recombinant xylanase and ferulic esterase were 20.7kDa and 36.0kDa, respectively.

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Abstract

The invention provides a building method of a novel pichia pastoris coexpression system for co-expressing engineering bacteria GS115 / faeA-xyn11A originated from aspergillus usamii E001 feruloyl esterase A gene and beta-1, 4-xylanase gene. The built pichia pastoris coexpression system can be used for the industrial production of the feruloyl esterase and the beta-1, 4-xylanase. The prepared two enzymes have the characteristics of being high in substrate affinity and high in catalytic efficiency, and are higher in industrial production potential and economic value.

Description

technical field [0001] The present invention relates to a new expression of Pichia pastoris that co-expresses ferulic acid esterase A mature peptide gene (faeA) and β-1,4-xylanase gene (xyn11A) derived from Aspergillus usamii E001 The system 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. It has been reported that ferulic acid esterase can cooperate with other hemicellulase (such as xylanase) to make microorganisms degrade hemicellulose in plant cell walls to the greatest extent. Ferulic acid esterase (E.C.3.1.1.73, ferulic acid esterase, FAE), also known as cinnamic esterase, is a subclass of carboxylic acid hydrolase, ...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66C12N9/42C12N9/18C12R1/84C12R1/66
Inventor 邬敏辰龚燕燕殷欣曾妍李剑芳
Owner JIANGNAN UNIV
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