Molecular Cloning and Characterization of an Enzyme Hydrolyzing p-Nitrophenyl α-D-Glucoside from Bacillus stearothermophilus SA0301

Abstract

Bacillus stearothermophilus SA0301 produces an extracellular oligo-1,6-glucosidase (bsO16G) that also hydrolyzes p-nitrophenyl α-D-glucoside (Tonozuka et al., J. Appl. Glycosci., 45, 397–400 (1998)). We cloned a gene for an enzyme hydrolyzing p-nitrophenyl α-D-glucoside, which was different from the one mentioned above, from B. stearothermophilus SA0301. The k₀⁄K_m values of bsO16G for isomaltotriose and isomaltose were 13.2 and 1.39 s⁻¹·mM⁻¹ respectively, while the newly cloned enzyme did not hydrolyze isomaltotriose, and the k₀⁄K_m value for isomaltose was 0.81 s⁻¹·mM⁻¹. The primary structure of the cloned enzyme more closely resembled those of trehalose-6-phosphate hydrolases than those of oligo-1,6-glucosidases, and the cloned enzyme hydrolyzed trehalose 6-phosphate. An open reading frame encoding a protein homologous to the trehalose-specific IIBC component of the phopshotransferase system was also found upstream of the gene for this enzyme.