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

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 Mg2+ and Br- ions and 0.5-2.0 M urea and inhibited by other metal ions (Zn2+, Cu2+, K+, Cd2+, Ag+, Fe3+, Mn2+, Co2+, Hg2+, Pb2+ and Sn2+ ), CO32- 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, Delta G*, Delta H* and Delta S* 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.