| Research Abstract |
Recently we have reported that the mutants, G82K and M101S of roc G glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) , were specific for oxaloacetate with Kcat values 3.45 and 5.68 s^<-1>, which were 265 and 473 folds higher than those for 2-oxoglutarate, respectively. Using recently accomplished NAB-dependent thermostable single mutant GluDH Q144R, as a template, a series of double mutant enzymes was constructed in the effort to change the substrate specificity from 2-oxoglutarate to oxaloacetate. In the reductive amination reaction, among the double mutant GluDHs Q144R/G82K and Q144R/M101S showed higher; preferences for oxaloacetate producing aspartate. The Kcat values of Q144R/G82K and Q144R/M101S were 199 and 2.14 s^<-1> respectively for oxaloacetate, and 5.73 and 2.69 s^<-1> respectively for pyruvate.
The malate dehydrogenases (MDH) have been cloned and characterized from Escherichia coli, Bacillus subtilis and Nostoc sp.PCC7120. The EcMDH was the only dehydrogenase which
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showed slightly L-aspartate deamination activity The comparison of biochemical parameters between EcMDH and archeal AspDH (AfAspDH) were analyzed. The Inc value of EcMDH for L-Asp oxidative deamination was higher than that of AfAspDH at 37-50℃, while Km value was also much higher than that of AfAspDH. The Km value of AfAspDH for oxaloacetate (OAA) changed depending on coexistent ammonium concentrations. lb improve AspDH activity of EcMDH, new mutants were tried to be designed by using MOE MD calculation. Various EcMDH loop mutants (N119S, N119A, A80P, P83V, G84V, D86G, R87G, S222G, V213F, V214F, E215D, E215R) were constructed to have a strong AspDH activity. Mutants E215D and E215R showed higher k, values for L-Asp deamination than wild type although Km value was also much high than wild type. However, these two mutants showed no reductive amination activity for OAA. It is necessary to apply the molecular evolutionary engineering techniques for improving the aspartate dehydrogenase activity of EcMDH. Less
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