CLONING AND EXPRESSION OF BETA-GLUCOSIDASE GENE FROM THE YEAST PICHIA-ETCHELLSII

A 4.8-kilobase pairs DNA fragment from thermophilic yeast Pichia etche llsii was cloned into the vector plasmid pUC19 to form plasmid pBG55 a nd the encoded beta-glucosidase expressed in Escherichia coli. The eff ect of different carbon sources on growth and enzyme synthesis was stu died in the pBG55 transformant and 0.2% (w/v) cellobiose found to be t he most suitable carbon source for enzyme biosynthesis. The level of i ntracellularly produced beta-glucosidase was slightly reduced on 0.2% (w/v) glucose and 0.2% (w/v) maltose. The partially purified enzyme fr om the beta-glu transformant was active against a wide range of aryl b eta-glucosides and beta-linked disaccharides and the preferred substra tes were p-nitrophenyl-beta-D-glucoside (pNPG), cellobiose, gentiobios e, sophorose and sucrose. While maximum enzyme activity of 62 U/l was against pNPG at 50 degrees C, the activities in the range of 120-170 U /l mere against various beta-linked disaccharides at 37 degrees C. The enzyme displayed glucose tolerance and a temperature optima profile s lightly different from that exhibited by the native yeast glycosylated enzyme. The beta-glucosidase in the crude extract of pBG55 transforma nt was identified as a stably produced protein of 200 kDa by PAGE-Zymo gram analysis.