title: Expression and characterization of a recombinant endoglucanase from western corn rootworm, in Pichia pastoris.
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.
authors.affiliation: Facultad de Ciencias Agropecuarias, Departamento de Producción Agropecuaria, Universidad de Caldas, Calle 6526-10, Manizales, Colombia.
authors.forename: Arnubio
authors.initials: A
authors.lastname: Valencia Jiménez
authors.affiliation: Department of Entomology, University of Nebraska, Lincoln, NE 68583-0816, USA.
authors.forename: Haichuan
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Entomology, University of Nebraska, Lincoln, NE 68583-0816, USA bsiegfried1@unl.edu.
authors.forename: Blair D
authors.initials: BD
authors.lastname: Siegfried
mesh_terms: D000818:Animals; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D001517:Coleoptera / Q000235:genetics* / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D011994:Recombinant Proteins; C056374:endoglucanase 1; D002480:Cellulase
keywords: Pichia pastoris; cellulase; glycosyl hydrolase; recombinant expression