INTRODUCTION OF A STABILIZING 10-RESIDUE BETA-HAIRPIN IN BACILLUS-SUBTILIS NEUTRAL PROTEASE

A 10 residue beta-hairpin, which is characteristic of thermostable Bac illus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully act ive after introduction of the loop. However, the mutant protein exhibi ted autocatalytic nicking and a 0.4-degrees-C decrease in thermostabil ity. Two additional point mutations designed to improve the interactio ns between the enzyme surface and the introduced beta-hairpin resulted in reduced nicking and increased thermostability. After the introduct ion of both additional mutations in the loop-containing mutant, nickin g was largely prevented and an increase in thermostability of 1.1-degr ees-C was achieved.