Molecular properties and enhancement of thermostability by random mutagenesis of glutamate dehydrogenase from Bacillus subtilis

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2005-10, Vol.69 (10), p.1861-1870
• Main Authors: Khan, Md.I.H.(Shimane Univ., Matsue (Japan). Faculty of Life and Environmental Science), Ito, K, Kim, H, Ashida, H, Ishikawa, T, Shibata, H, Sawa, Y
• Format: Article
• Language: English
• Subjects: BACILLUS SUBTILIS; Bacillus subtilis - enzymology; Biological and medical sciences; catalytic activity; CLONACION; CLONAGE; CLONING; Cloning, Molecular; CLOSTRIDIUM THERMOCELLUM; COFACTEUR; COFACTORS; Dimerization; Enzyme Stability; Escherichia coli; Fundamental and applied biological sciences. Psychology; GENE; GENES; GLUTAMATE DEHYDROGENASE; Glutamate Dehydrogenase - genetics; Glutamate Dehydrogenase - isolation & purification; Glutamate Dehydrogenase - metabolism; GLUTAMATE DESHYDROGENASE; GLUTAMATO DESHIDROGENASA; Glutamic Acid - metabolism; HEAT TOLERANCE; Hot Temperature; INDUSTRIAL USES; Ketoglutaric Acids - metabolism; Kinetics; Molecular Weight; MUTACION; Mutagenesis; MUTATION; Mutation, Missense; NAD; PROTEINAS RECOMBINANTES; PROTEINE RECOMBINANTE; PURIFICACION; PURIFICATION; random mutagenesis; RECOMBINANT PROTEINS; Substrate Specificity - genetics; thermostability; TOLERANCE A LA CHALEUR; TOLERANCIA AL CALOR; USAGE INDUSTRIEL; UTILIZACION INDUSTRIAL
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.69.1861

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 (Msub(r) 270 kDa) with strict specificity for 2-0xoglutarate and L-glutamate, requiring NADH and NADsup(+) as cofactors respectively. The enzyme showed low thermostability with Tsub(m)=41 deg 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 Tsub(m) values were 61 deg C and 49 deg C respectively. Furthermore, Q144R had a remarkably high ksub( cat) value (435ssup(-1)) for amination reaction at 37 deg 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.