摘要
Aim: Lignocelluloytic enzymes are the largest class of hydrolase enzyme which utilizes the plant biomass to produce renewable sources. Hence practices for larger production of these enzymes at lower cost received much attention for industrial use. Hence this paper deals with expression and purification of cellobiohydrolase gene from Penicillium funiculosum NCL1. Methods & Results: A cellobiohydrolase gene, cbhII of Penicillium funiculosum NCL1 was cloned and expressed in Pichia pastoris X33. Two exons of the cbhII gene were amplified separately and fused by overlap extension PCR. The fused product was cloned in yeast expression vector pPICZαA and expressed in P. pastoris under the control of the AOX1 promoter. P. pastoris transformants expressing recombinant cellobiohydrolase were selected on CMC agar plate and their ability to produce the cellobiohydrolase was evaluated in flask cultures. P. pastoris X33 (pPICbh6) efficiently secreted the recombinant cellobiohydrolase into the medium and produced the cellobiohydrolase activity (5 U/ml) after 96 h of growth. The recombinant cellobiohydrolase produced by P. pastoris (pPICBH6) showed maximum activity at pH 4.0 and temperature 50°C and higher specificity in hydrolysis of filter-paper.
Aim: Lignocelluloytic enzymes are the largest class of hydrolase enzyme which utilizes the plant biomass to produce renewable sources. Hence practices for larger production of these enzymes at lower cost received much attention for industrial use. Hence this paper deals with expression and purification of cellobiohydrolase gene from Penicillium funiculosum NCL1. Methods & Results: A cellobiohydrolase gene, cbhII of Penicillium funiculosum NCL1 was cloned and expressed in Pichia pastoris X33. Two exons of the cbhII gene were amplified separately and fused by overlap extension PCR. The fused product was cloned in yeast expression vector pPICZαA and expressed in P. pastoris under the control of the AOX1 promoter. P. pastoris transformants expressing recombinant cellobiohydrolase were selected on CMC agar plate and their ability to produce the cellobiohydrolase was evaluated in flask cultures. P. pastoris X33 (pPICbh6) efficiently secreted the recombinant cellobiohydrolase into the medium and produced the cellobiohydrolase activity (5 U/ml) after 96 h of growth. The recombinant cellobiohydrolase produced by P. pastoris (pPICBH6) showed maximum activity at pH 4.0 and temperature 50°C and higher specificity in hydrolysis of filter-paper.
