Characterization of tyrosinase from the cap flesh of portabella mushrooms.
Tyrosinase, purified from the cap flesh tissue of portabella mushrooms, was characterized with regard to its physical and biochemical properties. A native molecular size of 41 kDa for the enzyme was obtained by size exclusion chromatography, whereas SDS-PAGE indicated that the enzyme contained a single subunit with a size of approximately 48 kDa under reduced and nonreduced conditions. The purified enzyme showed a single immunological cross-reacting protein after Western blotting when probed with antibodies against Agaricus bisporus tyrosinase. Isoelectric focusing demonstrated that the enzyme preparation, apparently homogeneous by electrophoresis, still contained three isoforms of pI 5.1, 5.2, and 5.3. The purified enzyme was able to oxidize a variety of mono-, di-, and triphenolic compounds. An apparent K(m) of 5 mM was obtained using catechol as the substrate, and an apparent K(m) of 9 mM was found using L-Dopa as a substrate. Ascorbic acid, kojic acid, tropolone, mercaptobenzothiazole, and salicylhydroxamic acid inhibited the enzyme severely at 100 microM.
Zhang
X
X
Department of Chemistry, Indiana State University, Terre Haute, Indiana 47809, USA.
van Leeuwen
J
J
Wichers
H J
HJ
Flurkey
W H
WH
D000364:Agaricus / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D014442:Monophenol Monooxygenase