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 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.
Kasieczka-Burnecka
Monika
M
Institute of Technical Biochemistry, Technical University of Lodz (ITB TUL), Stefanowskiego 4/10, Lodz, Poland. mkasieczka@poczta.onet.pl
Kuc
Karina
K
Kalinowska
Halina
H
Knap
Monika
M
Turkiewicz
Marianna
M
D000222:Adaptation, Physiological; D000864:Antarctic Regions; D001203:Ascomycota / Q000201:enzymology* / Q000254:growth & development; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D003080:Cold Temperature; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011522:Protons; D012965:Sodium Chloride / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology