Purification of the benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 of Pseudomonas putida MT53 and their preliminary comparison with benzyl alcohol dehydrogenase and benzaldehyde dehydrogenases I and II from Acinetobacter calcoaceticus

Abstract

Summary: A procedure was developed for purifying both the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 from a single batch of Pseudomonas putida MT53. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernate, followed by chromatography on DEAE-Sephacel, Matrex Gel Red A and Blue Sepharose CL-6B which separated the two enzymes, Phenyl Sepharose CL-4B and Matrex Gel Green A. The final preparations gave single bands on electrophoresis under denaturing and non-denaturing conditions. The subunit Mr values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are 43000 and 56300 respectively. Cross-linking studies with dimethylsuberimidate indicate that both enzymes are probably tetramers, although they run anomalously through gel-filtration columns. The benzyl alcohol dehydrogenase was fairly specific for NAD+ as cofactor but the benzaldehyde dehydrogenase had appreciable activity with NADP+ as well as with NAD+. The optimum pH values are 9.4 and 9.3 for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase respectively. Benzaldehyde dehydrogenase appears to require a monovalent cation for maximum activity. The apparent Km and maximum velocity values of the two plasmid-encoded dehydrogenases and of the chromosomally encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenases I and II from Acinetobacter calcoaceticus were determined for NAD+ and for the unsubstituted substrates and for the monomethyl ring-substituted analogues. The corresponding apparent specificity constants were then calculated. All three benzaldehyde dehydrogenases had very similar substrate profiles, as did the two benzyl alcohol dehydrogenases. The plasmid-encoded benzaldehyde dehydrogenase resembles benzaldehyde dehydrogenase I from A. calcoaceticus (which also requires monovalent cations for activity) in being much more heat-stable than benzaldehyde dehydrogenase II. Overall, the plasmid-encoded benzyl alcohol dehydrogenase from P. putida appears to be remarkably similar to the chromosomally encoded benzyl alcohol dehydrogenase from A. calcoaceticus, and the plasmid-encoded benzaldehyde dehydrogenase is very similar to the two chromosomally encoded benzaldehyde dehydrogenases, particularly benzaldehyde dehydrogenase I.