CLONING AND EXPRESSION OF CHAETOMIUM THERMOPHILUM XYLANASE 11-A GENE IN PICHIA PASTORIS

Abstract

The various thermophilic fungi like Chaetomium thermophile has potential to secrete xylanase and cellulase enzymes. Inthe present study eukaryotic expression system of Pichia pastoris (yeast) was used to express xylanase gene. Thexylanase (Xyn 11-A) gene was isolated from C. thermophile strain NIBGE-1. Primers were designed to amplify the gene,ligated into P. pastoris pPIC3.5K vector, the resultant recombinant clone pSSZ810 was transformed into the genome ofP. pastoris GS115 strain through electroporation. Transformants were selected on yeast peptone dextrose medium(YPD) plates containing antibiotic geneticin (100 mg/mL) upto final concentration of 0.75 mg/mL. The maximum activityof xylanase 2.04 U/mL after incubation of 2 hrs at 50ºC was observed in the presence of 100% methanol inducer uptofinal concentration of 30μL (0.5%) as compared to control. HPLC analysis represented high peak of xylose as comparedto control. SDS-PAGE indicated approx. 28 kDa protein of expressed xylanase gene.