Butanol-Ethanol Dehydrogenase and Butanol-Ethanol-Isopropanol Dehydrogenase: Different Alcohol Dehydrogenases in Two Strains of Clostridium beijerinckii (Clostridium butylicum)

Abstract

Alcohol-producing strains of Clostridium beijerinckii (Clostridium butylicum) produce, besides acetone, either n-butanol and ethanol or n-butanol, ethanol, and isopropanol as their characteristic products. Alcohol dehydrogenase has been isolated from a strain (NRRL B593) of C. beijerinckii producing isopropanol and from a strain (NRRL B592) not producing isopropanol. Butanol-ethanol dehydrogenase activities were present in both strains, but isopropanol dehydrogenase activity was present only in the isopropanol-producing strain. The butanol-ethanol dehydrogenase of strain NRRL B592 had M(r) 66,000 and a K(m) of 6 muM for butyraldehyde. In contrast, the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593 had a M(r) 100,000 and K(m)s of 9.5 and 1.0 mM for butyraldehyde and acetone, respectively. In a purification by four different types of separatory methods (DEAE-cellulose, hydroxyapatite, Sephacryl S-300, and Matrex Gel Red A), butanol-ethanol-isopropanol dehydrogenase activities of strain NRRL B593 were purified up to 200-fold (10 to 30% yield), and these activities were not separated. Gel electrophoresis followed by activity stain also revealed distinct mobilities for the butanol-ethanol dehydrogenase of strain NRRL B592 and the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593. In cell extracts from both strains, a higher alcohol dehydrogenase activity was measured with NADP(H) than with NAD(H). The 150- to 200-fold-purified alcohol dehydrogenase from strain NRRL B593 did not show any NAD(H)-linked activities. The K(m) for NADPH was 31 muM (with butyraldehyde as cosubstrate) and 18 muM (with acetone as cosubstrate) for the alcohol dehydrogenase of strain NRRL B593. This study showed that the alcohol dehydrogenases from two strains of C. beijerinckii differed significantly.