苯甲醇脱氢酶.pdfVIP

Journal of General Microbwlogy (1990), 136, 637-643. Printed in Great Britain 637 Purificationofthe benzyl alcohol dehydrogenase andbenzaldehyde dehydrogenase encodedby the TOLplasmid pWW53 ofPseudumonas putida MT53andtheir preliminary comparisonwith benzyl alcohol dehydrogenase and benzaldehyde dehydrogenasesI andI1from Acinetobacter calcoaceticus RONALDM. CHALMERS,ALANJ. Scornand CHARLESA. FEWSON* Department of Biochemistry, Universityof Glasgow,GlasgowG128QQ, UK (ReceivedI I September 1989 ;accepted 30 November 1989) A procedure was developed for purifying both the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase encoded by the TOLplasmid pWW53 from a single batch of PseudomonasputidtrMT53. The procedure involved disruptionof the bacteria in the French pressure cell and preparation of a high-speed supernate, followed by chromatographyon DEAE-Sephacel, Matrex Gel Red A and Blue SepharoseCL-6Bwhich separated the two enzymes, Phenyl SepharoseCL-4Band Matrex Gel Green A. The finalpreparations gave single bands on electrophoresis under denaturing and nondenaturing conditions. The subunit M values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are 43000and 56300respectively. Cross-linking studies with dimethylsuberimidateindicate that both enzymesare probably tetramers, although they runanomalously through gel-filtration columns. The benzyl alcohol dehydrogenase was fairly specific for NAD+ as cofactor but the benzaldehydedehydrogenasehad appreciableactivitywith NADP+aswell aswithNAD+.TheoptimumpH values are 9.4 and 9-3for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase respectively. Benzaldehyde dehydrogenaseappears to require a monovalent cation for maximum activity. The apparent Kmand maximum velocity values of the two plasmid-encoded d