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

Document Type

Article

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.