Further improvement of the thermal stability of a partially stabilized Bacillus subtilis 3-isopropylmalate dehydrogenase variant by random and site-directed mutagenesis

A thermostabilized mutant of Bacillus subtilis 3-isopropylmalate dehydrogen ase (IPMDH) obtained in a previous study contained a set of triple amino ac id substitutions. To further improve the stability of the mutant, we used a random mutagenesis technique and identified two additional thermostabilizi ng substitutions, Thr22--> Lys and Met256-->Val, that separately endowed th e protein with further stability. We introduced the two mutations into a si ngle enzyme molecule, thus constructing a mutant with overall quintuple mut ations. Other studies have suggested that an improved hydrophobic subunit i nteraction and a rigid type II beta-turn play important roles in enhancing the protein stability. Based on those observations, we successively introdu ced amino acid substitutions into the mutant with the quintuple mutations b y site-directed mutagenesis: Glu253 at the subunit interface was replaced b y Leu to increase the hydrophobic interaction between the subunits; Glu112, Ser113 and Ser115 that were involved in the formation of the turn were rep laced by Pro, Gly and Glu, respectively, to make the turn more rigid. The t hermal stability of the mutants was determined based on remaining activity after heat treatment and first-order rate constant of thermal unfolding, wh ich showed gradual increases in thermal stability as more mutations were in cluded.