Achromobacter protease I (API), a lysine-specific serine protease, shows one order of magnitude higher activity than bovine trypsin, while its optimum pH is in the alkaline region at about pH 9. To improve the optimum pH range, mutant enzyme His210 replaced by Ser(H210S), Ala (H210A), and Lys (H210K) were constructed. The optimum pH of H210S shifted from about pH 9 (wild-type enzyme) to about pH 7, retaining its high activity. The putative electrostatic interaction between His 210 and catalytic Asp 113 was elucidated by the optimum-pH shift of H210K and H210A. These results indicate that this unconserved His 210 in API, which plays a key role in generating the useful peptidase, broadened the optimum-pH range without decreasing lysylendo-peptidase activity.