Three glucose dehydrogenases (GlcDH) from Bacillus megaterium, GlcDH-I, GlcDH-I1 and GlcDH-IWG3, were purified from Escherichia coli cells harboring one of the hybrid plasmids, pGDK1, pGDK2 and pGDA3, respectively, pGDKl and pGDK2 contain two isozyme genes, gdhl and gdhZI, respectively, from B. megaterium IAM 1030 and pGDA3 contains an isozyme gene from B. megaterium IWG3; GlcDH-IWG3 is a variant of GlcDH-I. ClcDH-I and GlcDH-I1 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-11 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-I1 is the most resistant against heat inactivation at pH 6.5. The values of the first-order rate constant for heat inactivation at 50°C are 0.27 min-', 0.05 min-' and 0.11 min-' for GlcDH-I, GlcDH-I1 and GlcDH-TWG3, respectively. Kinetic studies show that these enzymes have similar kinetic constant values except that there are some differences in Ki, for NAD(P) and K, (the limiting Michaelis constant) for NAD; the values of the ratio of K,, for NAD and NADP are 11 340 and 8.7 for GlcDH-I, GlcDH-I1 and GlcDH-IWG3, respectively. GlcDH-I and GlcDH-IWG3 have very similar substrate specificities and GlcDH-TI has a slightly higher specificity for u-glucose and 2-deoxy-u-glucose than the others. The results are discussed on the basis of the amino acid substitutions between the en7ymes.Glucose dehydrogenase catalyzes the oxidation of D-glucose to u-glucono-1,5-lactone using NAD or NADP as a coenzyme. This enzyme has been purified from sporulating cell extracts ofBacilluscereu.s [l, 21, Bacillusmegaterium M1286 [3] and Bucillu~~ suhtilis [4], and their enzymatic properties have been investigated [l -51. The amino acid sequences have also been reported for the enzymes from B. megaterium M1286 [6 -81 and B. subtilis [9]. However, the effects of amino acid substitutions on the enzymatic properties of glucose dehydrogenase have not been studied.Recently, we have cloned one glucose dehydrogenase gene from B. megaterium IWG3 [lo] and we have also cloned two isozyme genes from B. megaterium IAM1030 (Mitamura, T., Ebora, R. V., Nakai, T., Makino, Y., Negoro, S., Urabe, I. and Okada, H., unpublished results). These enzymes are naturally prepared mutant enzymes and their comparison should provide valuable information on the structure/function relationship of glucose dehydrogenase. In this work, wc have purified from Escherichia coli cells the two isozymes of B. megaterium 1AM1030, as well as the enzyme of B. megaterium IWG3, and investigated their enzymatic properties.