Expression and extensive characterization of a beta-glycosidase from the extreme thermoacidophilic archaeon Sulfolobus solfataricus in Escherichia coli: authenticity of the recombinant enzyme.
The gene coding for the beta-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-beta-D-glucosidase) activity, of the enzyme purified from S. Solfataricus. We provide evidence that the beta-glycosidase can assume its functional state in E. coli without the contribution of N-epsilon-methylated lysine residues.
Institute of Protein Biochemistry and Enzymology, Naples, Italy.
M
M
Moracci
R
R
Nucci
F
F
Febbraio
C
C
Vaccaro
N
N
Vespa
F
F
La Cara
M
M
Rossi
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives / Q000032:analysis; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*; D013696:Temperature; D013816:Thermodynamics
D000596:Amino Acids; D011994:Recombinant Proteins; C005205:epsilon-N-methyllysine; D006026:Glycoside Hydrolases; D008239:Lysine