Cloning and expression of β-glucosidase gene from the yeast Pichia etchellsii

A 4.8-kilobase pairs DNA fragment from thermophilic yeast Pichia etchellsii was cloned into the vector plasmid pUC19 to form plasmid pBG55 and the encoded β-glucosidase expressed in Escherichia coli . The effect of different carbon sources on growth and enzyme synthesis was studied in the pBG55 transformant and 0.2% (w/v) cellobiose found to be the most suitable carbon source for enzyme biosynthesis. The level of intracellularly produced β-glucosidase was slightly reduced on 0.2% (w/v) glucose and 0.2% (w/v) maltose. The partially purified enzyme from the β- glu transformant was active against a wide range of aryl β-glucosides and β-linked disaccharides and the preferred substrates were p -nitrophenyl-β- d -glucoside ( p NPG), cellobiose, gentiobiose, sophorose and sucrose. While maximum enzyme activity of 62 U/ l was against p NPG at 50°C, the activities in the range of 120–170 U/ l were against various β-linked disaccharides at 37°C. The enzyme displayed glucose tolerance and a temperature optima prolile slightly different from that exhibited by the native yeast glycosylated enzyme. The β-glucosidase in the crude extract of pBG55 transformant was identified as a stably produced protein of 200 kDa by PAGE-Zymogram analysis.