Single amino acid mutation alters thermostability of the alkaline protease from Bacillus pumilus： thermodynamics and temperature dependence

Dehairing alkaline protease （DHAP） from Bacillus pumilus BA06 has been demonstrated to have high catalytic efficiency and good thermostability, with potential application in leather processing. In order to get insights into its catalytic mechanism, two mutants with single amino acid substitution according to the homology modeling and multiple sequence alignment were characterized in ther- modynamics of thermal denaturation and temperature dependence of substrate hydrolysis. The re- sults showed that both mutants of V1491 and R249E have a systematic increase in catalytic efficiency （kcat/Km） in a wide range of temperatures, mainly due to an increase of kl （substrate diffusion） and k2 （acylation） for V1491 and of k2 and k3 （deacylation） for R249E. In comparison with the wild-type DHAP, the thermostability is increased for V1491 and decreased for R249E. Thermodynamic analysis indi- cated that the free energy （△Ga°） of activation for thermal denaturation may govern the thermostabil- ity. The value of △Ga°is increased for V1491 and decreased for R249E. Based on these data and the structural modeling, it is suggested that substitution of Va1149 with lie may disturb the local flexibility in the substrate-binding pocket, leading to enhancement of binding affinity for the substrate. In con- trast, substitution of Arg249 with Glu leads to interruption of interaction with the C-terminal of en- zyme, thus resulting in less thermostability. This study indicates that amino acid residues in the active center or in the substrate-binding pocket may disturb the catalytic process and can be selected as the target for protein engineering in the bacterial alkaline proteases.