Cloning and Expression of cel6B Cellobiohydrolase gene in Pichia pastoris

The Cel6B enzyme from Thermobifidia fusca, a CBHII, belongs to the family of cellulase B, which is highly resistant to heat. Due to the low level of production of this enzyme in this host, it cannot be used at industrial scale. For expression of cellobihydrolase in yeast, cel6B gene in pSZ143 plasmid was amplified by PCR and then was cloned in to pPICZαA vector. Recombinant construct contains cel6B gene transformed to E. coli Jm109 and recombinant were bacteria selected on LB medium with 0.5µg/ml Belleomycin. Recombinant plasmid was linearized and then transformed in Gs115 and KM71H yeast strains by electroporation method. Finally, the best concentration of methanol and the best day of expression of enzyme were determined by DNS method. The results in yeast indicated, the most CBH activity in GS115 and KM71H strains and on PC substrate in 50°C for 16h was 1.98 and 0.475 U (mmol/min)/ml, respectively. Only sample from high yield colony of GS115 strain for CBH expression was confirmed in SDS-PAGE and then the best methanol concentration and day for enzyme expression with same strain was obtained in 4th days after induction with three percent methanol. In this study, we did not optimize the codons of cel6B gene for expression in P. pastoris. Thus, its decreased expression level and cellobiohydrolase activity may be due to codon usage in Pichia.