The p-nitrophenyl-alpha-D-maltoside hydrolyzing alpha-glucosidase from the mesophile Bacillus subtilis 25S and the obligate thermophile Bacillus caldolyticus C2 was purified, characterized, and compared in order to determine the molecular mechanisms that may confer thermostability of starch-degrading enzymes. Both enzymes showed endo-oligo-1,4-glucosidase activity owing to their identical hydrolysis of linear malto-oligosaccharides to maltose and glucose as determined by thin-layer chromatography. Neither enzyme showed activity against p-nitrophenyl-alpha-D-glucopyranoside, maltose, isomaltose, isomaltotriose, or panose. The enzymes may tentatively be classified as a panose-producing pullulanase owing to their hydrolysis of pullulan. The 25S and C2 enzymes were composed of two identical subunit of M(r) 55,000 and 60,000 respectively. Both the 25S and C2 enzymes have a pI of 4.85, pH optimum of 7.5 and 7.0, and K(m) values for the chromogenic substrate p-nitrophenyl-alpha-D-maltoside of 2.96 mM and 1.31 mM respectively. The 25S enzyme exhibited optimal activity between 35 and 37-degrees-C, and complete inactivation after 10 min at 45-degrees-C, while the C2 enzyme showed optimal activity at 60-degrees-C and retained 100% of initial activity at 60-degrees-C for 2 h. The C2 enzyme required a minimum of 0.02% 2-mercaptoethanol or 0.01 mM EDTA for thermostability. A comparison of the amino acid compositions showed an increase in the number of proline, alanine, and leucine residues for the thermostable C2 enzyme. These alterations in hydrophobicity may influence enzyme thermostability; this may be a factor in the design of engineered proteins for industrial use.