Characterization of a beta-glycosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius.
In cell free extracts of the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius ATCC27009, we have identified beta-gluco- and galactosidase activities showing a specific activity of 0.1 and 12 U/mg, respectively. The two enzymatic activities are associated with different polypeptides and we show here the functional cloning, the expression in Escherichia coli and the characterisation of the beta-glucosidase (Aabeta-gly). The enzyme, which is optimally active and stable at temperatures above 65 degrees C, belongs to glycoside hydrolase family 1 (GH1) and shows wide substrate specificity on different aryl-glycosides and cello-oligosaccharides with k (cat)/K (M) for 4-nitrophenyl-beta-D-glucoside and cellobiose of 2,976 and 185 s(-1)mM(-1), respectively. Interestingly, upstream to the beta-glycosidase gene, we identified a second ORF homologous to the ATPase subunit of the bacterial ABC transporters (abc1) that is co-transcribed with the beta-glycosidase gene glyB and that could be involved in the carbohydrate import. The activity of the enzyme on cello-oligosaccharides of up to five glucose units strongly indicates that the enzyme could be involved in vivo in the degradation of glucans together with endoglucanase enzymes previously described. This, together with the co-expression of the two genes, suggests a role for the glyB-abc1 cluster in A. acidocaldarius in the degradation of cellulose and hemicelluloses.
Di Lauro
Barbara
B
Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131, Naples, Italy.
Rossi
Mosè
M
Moracci
Marco
M
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005959:Glucosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013696:Temperature
D005959:Glucosidases