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Pichia pastoris system coexpressed with aspergillus usamii (Aus) Cell2A and aspergillus oryzae (Aor Xyn11A)

A Pichia pastoris, co-expression technology, applied in the field of bioengineering, can solve problems such as unreported, and achieve the effect of reducing the preparation cost

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

AI Technical Summary

Problems solved by technology

The two enzyme genes from different sources have been cloned and expressed separately in Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and other expression systems, but co-expression in Pichia pastoris has not been reported

Method used

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  • Pichia pastoris system coexpressed with aspergillus usamii (Aus) Cell2A and aspergillus oryzae (Aor Xyn11A)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Construction, expression and recombinase activity determination of engineering bacteria GS115 / Aus cel12A

[0018] The recombinant plasmid pPIC9K-Aus cel12A was linearized with SalI, electrotransformed according to the Pichia expression manual, and high-copy Pichia recombinant GS115 / Aus cel12A was obtained by screening with different concentrations of G418. The engineering bacterium was induced at 2.0% methanol addition, 30°C, 220rpm for 96h, the fermentation supernatant was centrifuged at 8000rpm for 5min, and the centrifugation supernatant was recombinant endoglucanase; it was detected as a single band by SDS-PAGE, and It shows that the molecular weight of the recombinant endoglucanase is 27kDa. The optimal pH of the recombinant endoglucanase is 5.0.

Embodiment 2

[0019] Example 2 Construction and expression of engineering bacteria GS115 / Aus cel12A-Aor xyn11A

[0020] Use Sac I to linearize the recombinant expression plasmid pPICZαA-Aor xyn11A, refer to the Pichia pastoris expression manual, electrotransform Pichia pastoris recombinant GS115 / Aus cel12A, and obtain high-copy Pichia pastoris recombinant GS115 by screening with different concentrations of Zeocin / Aus cel12A-Aorxyn11A. This engineered bacterium is added amount of 1.0% methanol, 30 ℃, 220rpm induction 96h, SDS-PAGE shows endoglucanase and β-1, the molecular weight of 4-xylanase is respectively 27kDa and 24kDa, as figure 1 shown. The optimum action pH of the co-expressed recombinant endoglucanase was 5.0, and the optimum action temperature of the recombinant β-1,4-xylanase was 55°C.

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Abstract

The invention provides a method for constructing pichia pastoris engineering bacteria GS115 / Aus cell2A-Aor xyn11A coexpressed with endoglucanase cells and xylanase cells. According to the method, the mixed expression of endoglucanase and xylanase in the same engineering bacteria is realized by a double-plasmid expression system, so that a complex enzyme is prepared successfully, and the production cost of the enzyme is reduced. The two expressed enzymes have good zymologic properties and bright industrial application potentials.

Description

technical field [0001] The present invention relates to a co-expression endo-β-1,4-glucanase gene (Aus cel12A) derived from Aspergillus usamii (Aspergillus usamii) E001 and endo-beta-1,4-glucanase gene (Aus cel12A) derived from Aspergillus oryzae (Aspergillus oryzae) CICC 40186 The invention relates to the construction and expression of Pichia pastoris engineering bacteria of β-1,4-xylanase gene (Aor xyn11A), belonging to the technical field of bioengineering. Background technique [0002] Cellulose is the most abundant renewable resource on earth. It is the main component of plant cell walls, accounting for about 35%-50% of plant dry weight. It is a linear polymer formed by β-1,4-glucosidic bonds linking glucose. In water, it can be hydrolyzed into glucose by cellulase. Cellulose cannot be directly digested by humans and animals, and must be degraded into glucose before it can be used. At present, the degradation of cellulose in industry is mainly hydrolysis with concentr...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12R1/84C12R1/66C12R1/69
Inventor 邬敏辰刘高磊高树娟史红玲龚燕燕
Owner JIANGNAN UNIV