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Expression of an Endo-β-1,4-glucanase Gene from Orpinomyces PC-2 in Pichia pastoris
Department of Chemical Engineering and Bioengineering, Zhejiang University, Hangzhou 310027, China
* Author to whom correspondence should be addressed.
Received: 11 April 2011; in revised form: 3 May 2011 / Accepted: 23 May 2011 / Published: 24 May 2011
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: endo-β-1,4-glucanase; Pichia pastoris; heterologous expression; neutral cellulase; induction medium
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Jin, X.; Meng, N.; Xia, L.-M. Expression of an Endo-β-1,4-glucanase Gene from Orpinomyces PC-2 in Pichia pastoris. Int. J. Mol. Sci. 2011, 12, 3366-3380.
Jin X, Meng N, Xia L-M. Expression of an Endo-β-1,4-glucanase Gene from Orpinomyces PC-2 in Pichia pastoris. International Journal of Molecular Sciences. 2011; 12(5):3366-3380.
Jin, Xin; Meng, Nan; Xia, Li-ming. 2011. "Expression of an Endo-β-1,4-glucanase Gene from Orpinomyces PC-2 in Pichia pastoris." Int. J. Mol. Sci. 12, no. 5: 3366-3380.