ISOLATION AND CHARACTERIZATION OF TWO AROMATIC ALCOHOL DEHYDROGENASES FROM THE MICROORGANISM, PSEUDOMONAS ALCALIGENES

Abstract

Pseudomonas alcaligenes, wild-type strain N.C.I.B. 9867, synthesized two distinct NAD+-dependent alcohol dehydrogenases, designated as P-ADH I and P-ADH II, respectively, when grown on sodium lactate. P-ADH I was separated from P-ADH II by DEAE-Trisacryl M gel. P-ADH I and P-ADH II obtained from C.E.II were fractionated by ammonium sulphate (40-60 % ). P-ADH I was further purified by gel filtration (Sephacryl S-300), anion­ exchange chromatography (DEAE-Sephacel and DEAE­Trisacryl M), dye-ligand and affinity chromatography,whereas P-ADH II was partially purified using anion­ exchange chromatography (DEAE-Trisacryl M), gel filtration (Sephacryl S-300) and dye-ligand chromatography. Purification factors of P-ADH I and P-ADH II were 450 and 59, respectively. Both P-ADHs were stable for 7 days in 0.05 M KH2Po4 buffer, pH 7.0 containing 1 mM DTT and 2.5 mM MgC12. The assay condition for P-ADH I was 0.15 umole 4-hydroxybenzylalcohol, 1 umole NAD+, pH 9.6 at 30 °c for 1 min while that of P-ADH II was 2 umole benzyl­alcohol, 0.25 umole NAD+, pH 10.0 at 30 °c for 1 min. NAD+ could not be replaced by NADP+ The Km value of P-ADH I for 4-hydroxybenzylalcohol was O.015 mH and that of P-ADH II for benzylalcohol was o. 19 mM, whereas the Vmax values were 1293 U/mg and 45.7 U/ mg protein, respectively. The mechanism of the forward reaction catalyzed by P-ADH I and P-ADH II was sequential. P-ADH I catalyzed the reversed reaction by 12 % of that of the forward reaction.

For both P-ADHs ferrozine was a very potent inhibitor, whereas p-chloromercuribenzoate was a fairly potent inhibitor. From gel filtration and Ferguson plot the Mr of P-ADH I and P-ADH II were estimated to be 150,000, whereas the subunit Mr were 38,000 and 37,000,respectively, thus P-ADH I and P-ADH II were tetrameric. ADHs consisting of four identical subunits. The pI of pure P-ADH I was 4.9. P-ADH I and P-ADH II have fairly similar amino acid contents with the exception of isoleucine content. From N-terminal sequence analysis P-ADH I and P-ADH II were quite different, especially the N-terminal residues, but alignment of both sequences respect to each other showed some homology between them. The gene encoded for P-ADH II has been cloned in E. coli (DHI). From restriction analysis 1.2 kb DNA fragment was obtained. SOS-PAGE of the cell-free extract from clones gave rise to a 37,000 protein band,confirming the presence of Padh II gene in the clones.