Identification of thermostabilizing residues in a Bacillus alkaline cellulase by construction of chimeras from mesophilic and thermostable enzymes and site-directed mutagenesis

An alkaliphilic Bacillus sp. strain. KSM-64. produces a mesophilic alkaline endo-1,4-beta -glucanase that is suitable for use in detergents. The deduc ed amino acid sequence of the enzyme showed very high homology to that of a thermostable alkaline enzyme from alkaliphilic Bacillus sp. strain KSM-S23 7. Analysis of chimeric enzymes produced from the genes encoding the mesoph ilic and thermostable enzymes suggested that the lysine residues at positio ns 137, 179, and 194 are responsible for their thermal stabilization. Repla cing the corresponding Glu137, Asn179, and/or Asp194 with lysine by site-di rected mutagenesis made the mesophilic enzyme more thermostable. Analyses o f the hydrophilicity of deduced amino acid sequences and isoelectric focusi ng of the modified enzymes suggested that these three specific lysine resid ues and their replacements are all located on the surface of the enzyme mol ecule. This fact further suggested that specific ionic interaction is invol ved in the thermal stabilization of the enzyme. (C) 2001 Federation of Euro pean Microbiological Societies. Published by Elsevier Science B.V. All righ ts reserved.