Effects of proline substitution of the active site helix on dipeptide synthesis mediated by Bacillus stearothermophilus neutral protease

Effects of structural stabilization on dipeptide synthesis mediated by Bacillus stearothermophilus neutral protease was investigated. A proline residue was introduced into the N-terminus (I140P and D141P), the middle (L147P) and C-terminus (D153P) of the active site helix (G138-Y154) as reported by Nakamura et al. (1997). Mutants I140P and D141P were found to be 60 and 39-fold more stable than the wild-type enzyme in anhydrous dimethylformamide (DMF), respectively. The addition of DMF to the reaction medium markedly improved the efficiency of forming a sweet aspartyl dipeptide analog of aspartame, Z-L-Asp-Met-OMe, except L147P. Mutants I140P and D141P greatly promoted the synthesis in 60% DMF, in which the dipeptide yields were 3- and 2-times that of the wild type, respectively.