IMPROVING THE THERMOSTABILITY OF BACILLUS-STEAROTHERMOPHILUS NEUTRAL PROTEASE BY INTRODUCING PROLINE INTO THE ACTIVE-SITE HELIX

A proline residue was introduced into the N-terminus (Ile140 and Asp14 1), the middle (Leu147) and the C-terminus (Asp153) of the active site helix of Bacillus stearothermophilus neutral protease for comparing t he effects on the thermostability, Introduction of a proline residue i nto the N-terminus at sites 140 and 141 increased the half-survival te mperature (HST) by 7.5 and 2.8 degrees C, respectively, from 68.3 degr ees C of the wild-type enzyme, A proline residue at Leu147 decreased t he HST by 10.2 degrees C, while no change was observed by introducing a proline residue in the C-terminus, These results were coincidental w ith the CD data which indicated increases in T-m values of 4.4 and 2.3 degrees C for I140P and D141P, respectively. Susceptibility to a-chym otrypsin hydrolysis markedly decreased in mutants I140P and D141P, whi le increasing in L147P. Molecular modeling suggested that glycine resi dues on the N-terminus side of proline residues in I140P and D141P rel axed the possible strain caused by proline introduction, The thermosta bility can, therefore, be explained based on changes in the molecular rigidity.