Cloning and Expression of P-Glucosidase Gene from the Yeast Pichia etchellsii

Abstract

umerical Modelling of Coastal Upwelling In The Bay of Bengal Pergamon,21,(5),667-670

A 4%kilobase pairs DNA fragment from thermophilic yeast Pichia etchellsii was cloned into the vector plasmid pUC19 to form plasmid pBG55 and the encoded ,Beta-glucosidase expressed in Escherichiu 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 Beta-glucosidase was slightly reduced on 0.2% (w/v) glucose and 0.2% (w/v) maltose. The partially purified enzyme from the Beta-glu transformant was active against a wide range of aryl Beta-glucosides and B-linked disaccharides and the preferred substrates were p-nitrophenyl-Beta-o-glucoside (PNPG), cellobiose, gentiobiose, sophorose and sucrose. While maximum enzyme activity of 62 U/1 was against pNPG at SO’C, the activities in the range of 120-170 U/1 were against various Beta-linked disaccharides at 37’C. The enzyme displayed glucose tolerance and a temperature optima profile slightly different from that exhibited by the native yeast glycosylated enzyme. The Beta-glucosidase in the crude extract of pBG55 transformant was identified as a stably produced protein of 200 kDa by PAGE-Zymogram analysis.