Expression and extensive characterization of a β-glycosidase from the extreme thermoacidophilic archaeon Sulfolobus solfataricus in Escherichia coli: Authenticity of the recombinant enzyme

• Published in: Enzyme and microbial technology 1995-11, Vol.17 (11), p.992-997
• Main Authors: Moracci, Marco, Nucci, Roberto, Febbraio, Ferdinando, Vaccaro, Carlo, Vespa, Nunzia, La Cara, Franco, Rossi, Mosè
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids - analysis; archaea; Bacteriology; Base Sequence; Biological and medical sciences; Biotechnology; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzyme engineering; Enzyme Stability; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial - genetics; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - metabolism; Hydrolysis; Kinetics; Lysine - analogs & derivatives; Lysine - analysis; lysine methylation; Metabolism. Enzymes; Methods. Procedures. Technologies; Microbiology; Miscellaneous; Molecular Sequence Data; Protein Processing, Post-Translational; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Analysis; Substrate Specificity; Sulfolobus - enzymology; Sulfolobus solfataricus; Temperature; Thermodynamics; β-glucosidase; β-Glycosidase
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/0141-0229(95)00012-7

The gene coding for the β-glycosidase from the archaeon Sulfolobus solfataricus has been overexpressed in Escherichia coli. The enzyme was purified to homogeneity with a rapid purification procedure employing a thermal precipitation as a crucial step. The final yield was 64% and the purification from the thermal precipitation was 5.4-fold. The expressed enzyme shows the same molecular mass, thermophilicity, thermal stability, and broad substrate specificity, with noticeable exocellobiase (glucan 1,4-β- d-glucosidase) activity, of the enzyme purified from S. Solfataricus. We provide evidence that the β-glycosidase can assume its functional state in E. coli without the contribution of N-ϵ-methylated lysine residues.