Introduction of a stabilizing 10 residue β-hairpin in Bacillus subtilis neutral protease

Abstract

A 10 residue β-hairpin, which is characteristic of thermostable Bacillus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully active after introduction of the loop. However, the mutant protein exhibited autocatalytic nicking and a 0.4°C decrease in thermostability. Two additional point mutations designed to improve the interactions between the enzyme surface and the introduced β-hairpin resulted in reduced nicking and increased thermostability. After the introduction of both additional mutations in the loopcontaining mutant, nicking was largely prevented and an increase in thermostability of 1.1°C was achieved.