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 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 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 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 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 (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 appears to be remarkably similar to the chromosomally encoded benzyl alcohol dehydrogenase from , and the plasmid-encoded benzaldehyde dehydrogenase is very similar to the two chromosomally encoded benzaldehyde dehydrogenases, particularly benzaldehyde dehydrogenase I.


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