Cloning of endo-β-glucanase I gene and expression in Pichia pastoris

Abstract

Total RNA of Thermoascus aurantiacus was isolated from its mycelium and acted as template for RT-PCR. The full-length cDNA encoding an endo-β-glucanase I was cloned via RACE-PCR method and the cDNA contained an ORF of 1005 bp encoding 305 amino acids. A recombinant plasmid, pPIC9k-egI, was constructed by inserting the ORF sequence of endo-β-glucanase I gene (egI) into the yeast expression vector pPIC9k and transformed to Pichia pastoris GS115. The results showed that the recombinant endo-β-glucanase I was excreted into the fermentation medium. The highest activity of endo-β-glucanase I and the protein content were up to 45.42 U/mL and 788.26 μg/mL at incubation time of 144 h. The optimal temperature and pH for the recombinant endo-β-glucanase I were found to be 70ºC and 3.5, respectively.

Keywords

Thermoascus aurantiacus / endo-1 / 4-β-glucanase / RACE / Pichia pastoris / gene expression

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Acknowledgements

This work was supported by the Science and Technology Project of Hebei Province China (Nos. 07225553 and 06220106D).

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg