Enzymatic properties of isozymes and variants of glucose dehydrogenase from Bacillus megaterium.
Three glucose dehydrogenases (GlcDH) from Bacillus megaterium, GlcDH-I, GlcDH-II and GlcDH-IWG3, were purified from Escherichia coli cells harboring one of the hybrid plasmids, pGDK1, pGDK2 and pGDA3, respectively, pGDK1 and pGDK2 contain two isozyme genes, gdhI and gdhII, respectively, from B. megaterium IAM 1030 and pGDA3 contains an isozyme gene from B. megaterium IWG3; GlcDH-IWG3 is a variant of GlcDH-I. GlcDH-I and GlcDH-II have similar pH/activity profiles and the profile for GlcDH-IWG3 is identical to that of GlcDH-I. The pH/stability profiles of these enzymes show that GlcDH-IWG3 is the most stable enzyme in the acidic region, while GlcDH-II is the most stable in the alkaline region, and GlcDH-I is the most unstable throughout the entire pH range examined. As for thermostability, GlcDH-II is the most resistant against heat inactivation at pH 6.5. The values of the first-order rate constant for heat inactivation at 50 degrees C are 0.27 min-1, 0.05 min-1 and 0.11 min-1 for GlcDH-I, GlcDH-II and GlcDH-IWG3, respectively. Kinetic studies show that these enzymes have similar kinetic constant values except that there are some differences in Kia for NAD(P) and Ka (the limiting Michaelis constant) for NAD; the values of the ratio of Kia for NAD and NADP are 11,340 and 8.7 for GlcDH-I, GlcDH-II and GlcDH-IWG3, respectively. GlcDH-I and GlcDH-IWG3 have very similar substrate specificities and GlcDH-II has a slightly higher specificity for D-glucose and 2-deoxy-D-glucose than the others. The results are discussed on the basis of the amino acid substitutions between the enzymes.
Mitamura
T
T
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
Urabe
I
I
Okada
H
H
D001410:Bacillus megaterium / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000032:analysis / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005948:Glucose Dehydrogenases / Q000032:analysis / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity
D007527:Isoenzymes; D002237:Carbohydrate Dehydrogenases; D005948:Glucose Dehydrogenases