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

概要

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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 trasformant 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 ρ-nitrophenyl-β-D-glucoside (ρNPG), cellobiose, gentiobiose, sophorose and sucrose. While maximum enzyme activity of 62 U/l was against ρNPG at 50℃, the activities in the range of 120-170 U/l were against various β-linked disaccharides at 37℃. The enzyme displayed glucose tolerance and a temperature optima profile 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.
1995-11-25