Abstract
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.
Highlights
Lignocellulosic biomass is an important source of the renewable energy for production of bioethanol
We report the cloning and expression of cbhII gene in pichia expression system
Using P. funiculosum NCL1 genomic DNA as template, PCR was performed with degenerate primers Cbh6F & Cbh6R and to amplify cbhII cellobiohydrolase gene
Summary
Lignocellulosic biomass is an important source of the renewable energy for production of bioethanol. The most abundant enzymes are two cellobiohydrolase, Cel7A and Cel6A, called CBHI and CBHII, respectively These are the most efficient enzymes on highly crystalline cellulose [4]. Genes coding cellobiohydrolase GH6 have been cloned and characterized from a variety of fungal sources, including Trichoderma reesei [7], Chaetomium thermophilum [8], Irpex lacteus MC-2 [9], Chaetomium thermophilum [10]. This is the first report on the expression of cbhII from Penicillium funiculosum in pichia pastoris. We report the cloning and expression of cbhII gene in pichia expression system
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