title: Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation.
abstract: Random mutagenesis was used to enhance the thermal stability of Streptomyces cholesterol oxidase. Four thermostable mutants were isolated and the following amino acid substitutions were identified: Ser103 to Thr (mutant S103T), Val121 to Ala (mutant V121A), Arg135 to His (mutant R135H) and Val145 to Glu (mutant V145E). The wild-type and mutant enzymes were purified and characterized. The properties of mutants S103T, V121A and R135H were similar to those of the wild type but they showed improved thermal stability. When the V145E mutation was introduced, the thermal stability of the enzyme was markedly increased and the optimum pH was desirably changed to encompass a broad range from acid to alkali. Analysis of multiple mutants constructed by site-directed mutagenesis showed that all the mutations except that of R135H had an additive influence on the other mutations. These mutational effects are discussed in terms of a three-dimensional structural model of the enzyme constructed on the basis of homology modelling.
authors.affiliation: Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., Fukui, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Nishiya
authors.forename: N
authors.initials: N
authors.lastname: Harada
authors.forename: S I
authors.initials: SI
authors.lastname: Teshima
authors.forename: M
authors.initials: M
authors.lastname: Yamashita
authors.forename: I
authors.initials: I
authors.lastname: Fujii
authors.forename: N
authors.initials: N
authors.lastname: Hirayama
authors.forename: Y
authors.initials: Y
authors.lastname: Murooka
mesh_terms: D002784:Cholesterol / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D002784:Cholesterol; D002789:Cholesterol Oxidase