Expression and characterization of recombinant beta-xylosidase from Trichoderma reesei ATCC 58350 in Pichia pastoris

Abstract

B-xylosidase is one of the essential enzymes for complete breakdown of xylan since it degrades short xylo-oligosaccharides and hydrolyzed xylobiose by endoxylanases to xylose. B-xylosidase {Bxl 1) gene of Trichoderma reesei ATCC 58350 containing an open reading frame of 2, 331 nucleotides were amplified by polymerase chain reaction (PCR) and cloned into Pichia pPLCZaA vector. The 5x/l-pPICZaA construct was linearized and integrated at 5’ alcohol oxidase 1 (A0X1) locus in Pichia pastoris (P. pastoris) X33 genome. Effect of cultural conditions on recombinant Bxll production was studied. Production of recombinant Bxll by P. pastoris X33 was best achieved in buffered complex methanol medium (BMMY) pH 6 and induction with 3% methanol for every 24 hours at 30°C. The theoretical molecular weight of the recombinant Bxll was 88.8 kDa, but exhibited a molecular weight of 107.5 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for recombinant Bxll were pH 4 and 50°C, respectively. The enzyme was stable at pH 4 to pH 9 and retained 80% of its activity after incubation at 50°C for 30 minutes. The activity of recombinant Bxll was enhanced by Na2+, K+, Fe2+, Ca2+, Mg2+, Zn2+, NH2+, ethylenediamine-tetraacetate (EDTA), C02+, Ni2+, dimethyl sulfoxide (DMSO), urea, Triton X and Tween 80 and inhibited by Cu2+ and Fe2+. The Michaelis-Menten constant, Km and maximum velocity, Vmax for recombinant Bxll activities towards p-nitrophenyl p-D-xyloside (pNPX) were 8.98 mM and 8.51 (imol/min/mL, respectively. Recombinant Bxll alone can degrade pretreated oil palm empty fruit bunch and react better when combined with recombinant xylanase.