144509-68-2Relevant articles and documents
Molecular properties and enhancement of thermostability by random mutagenesis of glutamate dehydrogenase from Bacillus subtilis
Khan, Md. Iqbal Hassan,Ito, Kousuke,Kim, Hyeung,Ashida, Hiroyuki,Ishikawa, Takahiro,Shibata, Hitoshi,Sawa, Yoshihiro
, p. 1861 - 1870 (2005)
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 (Mr 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate, requiring NADH and NAD + as cofactors respectively. The enzyme showed low thermostability with Tm = 41°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 Tm values were 61°C and 49°C respectively. Furthermore, Q144R had a remarkably high kcat value (435 s-1) for animation reaction at 37°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.
