title: Identification of thermostabilizing residues in a Bacillus alkaline cellulase by construction of chimeras from mesophilic and thermostable enzymes and site-directed mutagenesis.
abstract: An alkaliphilic Bacillus sp. strain, KSM-64, produces a mesophilic alkaline endo-1,4-beta-glucanase that is suitable for use in detergents. The deduced amino acid sequence of the enzyme showed very high homology to that of a thermostable alkaline enzyme from alkaliphilic Bacillus sp. strain KSM-S237. Analysis of chimeric enzymes produced from the genes encoding the mesophilic and thermostable enzymes suggested that the lysine residues at positions 137, 179, and 194 are responsible for their thermal stabilization. Replacing the corresponding Glu137, Asn179, and/or Asp194 with lysine by site-directed mutagenesis made the mesophilic enzyme more thermostable. Analyses of the hydrophilicity of deduced amino acid sequences and isoelectric focusing of the modified enzymes suggested that these three specific lysine residues and their replacements are all located on the surface of the enzyme molecule. This fact further suggested that specific ionic interaction is involved in the thermal stabilization of the enzyme.
authors.affiliation: Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, 321-3497, Tochigi, Japan. 300475@kastanet.kao.co.jp
authors.forename: Y
authors.initials: Y
authors.lastname: Hakamada
authors.forename: Y
authors.initials: Y
authors.lastname: Hatada
authors.forename: T
authors.initials: T
authors.lastname: Ozawa
authors.forename: K
authors.initials: K
authors.lastname: Ozaki
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: S
authors.initials: S
authors.lastname: Ito
mesh_terms: D000468:Alkalies; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020740:Chimerin Proteins / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017422:Sequence Analysis, DNA; D013499:Surface Properties
chemical_list: D000468:Alkalies; D020740:Chimerin Proteins; D002480:Cellulase