A p-nitrophenyl-alpha-D-maltoside-hydrolyzing alpha-glucosidase was purified and characterized from a Bacillus subtilis high-temperature growth transformant (H-17), previously generated by transformation of Bacillus subtilis 25S with Bacillus caldolyticus C2 DNA. The enzyme showed endo-oligo-1,4-glucosidase activity owing to its hydrolysis of linear malto-olgosaccharides to maltose and glucose, and pullulan hydrolase activity owing to its hydrolysis of pullulan to glucose, maltose, and (iso)panose. The enzyme was inactive against p-nitrophenyl-alpha-D-glucopyranoside, maltose, isomaltose, isomaltotriose, and panose, but slightly hydrolyzed starch. The native structure of the enzyme is a dimer composed of two identical subunits of M(r) 55,000. The enzyme had a pI of 4.8, pH optimum of 7.5, was 80% inhibited by 5 mM Tris-HCl, and had a K(m) value of 1.46 mM for the chromogenic substrate p-nitrophenyl-alpha-D-maltoside. The enzyme showed optimal activity between 65-degrees and 68-degrees-C, and retained 100% of initial activity after incubation at 65-degrees-C for 1 h. A minimum concentration of 0.02% 2-mercaptoethanol or 0.005 mM EDTA was required for thermostability. These physiochemical characteristics are similar to those for the previously described corresponding enzyme from B. subtilis 25S, except that the same enzyme from the transformed strain was thermolabile. Amino acid analysis showed higher levels of alanine, glycine, and proline residues in the H-17 enzyme, compared with 25S. This may account for the enhanced thermostability, owing to increased internal hydrophobicity.