Rational shifting of the optimum pH toward alkalinity and enhancement of thermostability were investigated using an alkaline protease (optimum pH, 10.5-11.5) from the alkaliphilic Bacillus sp. B21-2. The protease gene (aprN) was cloned and the sequence analysis revealed an open reading frame of 379 amino acids that was composed of a putative signal sequence (27 amino acids), a prosequence (84 amino acids)? and a mature enzyme of 268 amino acids (MW 26,700). When Lys-230, which was thought to be located on the surface of the molecule, was replaced bg Arg, the optimum pH range was extended further to the alkaline side (11.5-12.0). Also, when the beta-turn positions, Ala-38, Ala-165, and Ala-187, were replaced by Pro, the residual activities of the resultant mutant enzymes treated at 55 degrees C for 30 min were higher than that of the wild-type enzyme (60 % as against 30%). These findings suggest that increasing the content of Arg residues and reducing the number of Lys residues increases the alkalinity of serine protease, while the introduction of a Pro residue into the beta-turn structure stabilizes the enzyme.