Rational design for stabilization and optimum pH shift of serine protease AprN
1998
Masui, A. (Osaka Inst. of Technology (Japan)) | Fujiwara, N. | Yamamoto, K. | Takagi, M. | Imanaka, T.
Rational shifting of the optimum pH toward alkalinity and enhancement of the 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 replaces by 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 as Pro residue into the beta-turn structure stabilizes the enzyme
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