title: Molecular properties and enhancement of thermostability by random mutagenesis of glutamate dehydrogenase from Bacillus subtilis.
abstract: The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in Escherichia coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined to have a hexameric structure (M(r) 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate, requiring NADH and NAD+ as cofactors respectively. The enzyme showed low thermostability with T(m) = 41 degrees C due to dissociation of the hexamer. To improve the thermostability of this enzyme, we performed error-prone PCR, introducing random mutagenesis on cloned GluDH. Two single mutant enzymes, Q144R and E27F, were isolated from the final mutant library. Their T(m) values were 61 degrees C and 49 degrees C respectively. Furthermore, Q144R had a remarkably high k(cat) value (435 s(-1)) for amination reaction at 37 degrees C, 1.3 times higher than that of the wild-type. Thus, Q144R can be used as a template gene to modify the substrate specificity of Bs-GluDH for industrial use.
authors.affiliation: Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Nishikawatsu, Matsue, Japan.
authors.forename: Md Iqbal Hassan
authors.initials: MI
authors.lastname: Khan
authors.forename: Kousuke
authors.initials: K
authors.lastname: Ito
authors.forename: Hyeung
authors.initials: H
authors.lastname: Kim
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ashida
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shibata
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sawa
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D006358:Hot Temperature; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D016296:Mutagenesis; D020125:Mutation, Missense; D009243:NAD; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D007656:Ketoglutaric Acids; D009243:NAD; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase