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 chromatographic step. After 5.8-fold purification, the cell extract gave a homogeneous enzyme at 166 U/mg. The recombinant enzyme was functionally and structurally similar to the wild-type enzyme. Kinetic experiments showed the same wide substrate specificity; in fact, the expressed enzyme hydrolyzed beta-D-gluco-, fuco-, and galactosides and a large number of glucoside dimers and oligomers, linked beta 1 -> 4. Moreover, the molecular mass of the enzyme was estimated to be 60 kDa by SDS-PAGE and 240 kDa by gel filtration, glycerol gradient, and ultracentrifugation analyses, indicating that the enzyme has a tetrameric structure. The N-terminal amino acid sequence, the temperature dependent activity, and content of secondary structure were similar to those of the wild-type enzyme. CD spectral and kinetic analyses showed that the only differences from the wild-type enzyme consist of the absence of lysine methylation, the presence of some glycosylated amino acid residues, and lower thermostability. Furthermore, calorimetric analyses on the expressed protein indicated values of delta dH = 5072 kJ/ mol and delta (d)C(p)= 100 kJ/mol, appreciably lower than those of the wild-type protein.
D'Auria
S
S
Istituto di Biochimica delle Proteine ed Enzimologia, C.N.R., Naples, Italy.
Morana
A
A
Febbraio
F
F
Vaccaro
C
C
De Rosa
M
M
Nucci
R
R
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004795:Enzyme Stability; D005960:Glucosides / Q000494:pharmacology; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
D005960:Glucosides; D011994:Recombinant Proteins; D001617:beta-Glucosidase