Expression and Characterization of a Recombinant Endoglucanase From Western Corn Rootworm, in Pichia pastoris

Authors

Arnubio Valencia Jimenez, Universidad de Caldas
Haichuan Wang, University of Nebraska-LincolnFollow
Blair D. Siegfried, University of Nebraska-LincolnFollow

Date of this Version

2014

Citation

Journal of Insect Science, 2014

Comments

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License

Abstract

ABSTRACT. The endoglucanase cDNA, Dvv-ENGase I, from western corn rootworm, Diabrotica virgifera virgifera LeConte was expressed using the GS115 methylotrophic strain of Pichia pastoris. The Dvv-ENGase I gene was cloned into the integrative plasmid pPICZαA under the control of AOX1, which is a methanol-inducible promoter. Positive clones were selected for their ability to produce the recombinant endoglucanase upon continuous methanol induction. The secreted recombinant insect endoglucanase Dvv-ENGase I has an apparent molecular mass of 29 kDa. The recombinant endo-1,4 -β-glucanase (ENGase) was able to digest the substrates: hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC), and Whatman No. 1 filter paper. A higher accumulation of reducing sugar was evident when the P. pastoris expression medium contained HEC (1%) instead of CMC (1%). An enzymatic activity band was detected after performing electrophoretic separation under nondenaturing conditions. The biological activity of the recombinant Dvv-ENGase I was influenced by the presence of protease inhibitors in the culture medium.