Purification and characterization of two cold-adapted extracellular tannin acyl hydrolases from an Antarctic strain Verticillium sp. P9

Abstract

Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26% carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH of 5.5 and 25 and 20 degrees C, respectively. Both the enzymes were activated by Mg(2+)and Br(-) ions and 0.5-2.0 M urea and inhibited by other metal ions (Zn(2+), Cu(2+), K(+), Cd(2+), Ag(+), Fe(3+), Mn(2+), Co(2+), Hg(2+), Pb(2+) and Sn(2+)),[Formula: see text] anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, beta-mercaptoethanol, alpha-glutathione and 4-chloromercuribenzoate. Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E (a) of these reactions and temperature dependence (at 0-30 degrees C) of k (cat), k (cat)/K (m), DeltaG*, DeltaH* and DeltaS* for both the enzymes and substrates were determined. The k (cat) and k (cat)/K (m) values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II.