FUNCTIONAL AND STRUCTURAL-PROPERTIES OF THE HOMOGENEOUS BETA-GLYCOSIDASE FROM THE EXTREME THERMOACIDOPHILIC ARCHAEON SULFOLOBUS-SOLFATARICUS EXPRESSED IN SACCHAROMYCES-CEREVISIAE

A protein with beta-glycosidase activity from Sulfolobus solfataricus (S beta gly) was expressed in the yeast Saccharomyces cerevisiae. The purification procedure was made fast and easy by employing a single ch romatographic step. After 5.8-fold purification, the cell extract gave a homogeneous enzyme at 166 U/mg. The recombinant enzyme was function ally and structurally similar to the wild-type enzyme. Kinetic experim ents showed the same wide substrate specificity; in fact, the expresse d enzyme hydrolyzed beta-D-gluco-, fuco-, and galactosides and a large number of glucoside dimers and oligomers, linked beta 1 --> 4. Moreov er, the molecular mass of the enzyme was estimated to be 60 kDa by SDS -PAGE and 240 kDa by gel filtration, glycerol gradient, and ultracentr ifugation analyses, indicating that the enzyme has a tetrameric struct ure. The N-terminal amino acid sequence, the temperature-dependent act ivity, and content of secondary structure were similar to those of the wild-type enzyme. CD spectral and kinetic analyses showed that the on ly differences from the wild-type enzyme consist of the absence of lys ine methylation, the presence of some glycosylated amino acid residues , and lower thermostability. Furthermore, calorimetric analyses on the expressed protein indicated values of Delta(d)H = 5072 kJ/mol and Del ta(d)C(p) = 100 kJ/mol, appreciably lower than those of the wild-type protein. (C) 1996 Academic Press, Inc.