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Int J Mol Sci. 2011;12(5):3366-80. doi: 10.3390/ijms12053366. Epub 2011 May 24.

Expression of an endo-β-1,4-glucanase gene from orpinomyces PC-2 in Pichia pastoris.

Author information

  • 1Department of Chemical Engineering and Bioengineering, Zhejiang University, Hangzhou 310027, China; E-Mails: guyin018@gmail.com (X.J.); mengnan17@163.com (N.M.).

Abstract

The endo-β-1,4-glucanase gene celE from the anaerobic fungus Orpinomyces PC-2 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K, and integrated into the genome of a methylotrophic yeast P. pastoris GS115 by electroporation. The strain with highest endo-β-1,4-glucanase activity was selected and designed as P. pastoris egE, and cultivated in shaking flasks. The culture supernatant was assayed by SDS-polyacrylamide gel electrophoresis and showed a single band at about 52 kDa. Furthermore, the recombinant P. pastoris egE was proved to possess the ability to utilize sodium carboxymethyl cellulose as a carbon source. The recombinant endoglucanase produced by P. pastoris showed maximum activity at pH 6.0 and temperature 45 °C, indicating it was a mesophilic neutral endo-β-1,4-glucanase, suitable for denim biofinishing/washing. Further research was carried out in suitable fermentation medium in shaking flasks. The most favorable methanol addition concentration was discussed and given as 1.0%. After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 72.5 IU mL(-1). This is the first report on expression and characterization of endo-β-1,4-glucanase from Orpinomyces in P. pastoris. The endo-β-1,4-glucanase secreted by recombinant P. pastoris represents an attractive potential for both academic research and textile industry application.

KEYWORDS:

Pichia pastoris; endo-β-1,4-glucanase; heterologous expression; induction medium; neutral cellulase

PMID:
21686190
[PubMed - indexed for MEDLINE]
PMCID:
PMC3116196
Free PMC Article
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