EFFECTS OF PROLINE SUBSTITUTION OF THE ACTIVE-SITE HELIX ON DIPEPTIDESYNTHESIS MEDIATED BY BACILLUS-STEAROTHERMOPHILUS NEUTRAL PROTEASE

Effects of structural stabilization on dipeptide synthesis mediated by Bacillus stearothermophilus neutral protease was investigated. A prol ine residue was introduced into the N-terminus (I140P and D141P), the middle (L147P) and C-terminus (D153P) of the active site helix (G138-Y 154) as reported by Nakamura et al. (1997). Mutants I140P and D141P we re 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 s weet aspartyl dipeptide analog of aspartame, Z-L-Asp-Met-OMe, except L 147P. Mutants I140P and D141P greatly promoted the synthesis in 60% DM F, in which the dipeptide yields were 3- and 2-times that of the wild type, respectively.