Purification and characterization of tyrosinase from gill tissue of Portabella mushrooms

A group of tyrosinase isoforms with isoelectric points between 4.9 and 5.2 was isolated from gill tissue of Portabella mushrooms. Use of protease inhibitors was not able to increase the amount of latent forms significantly in crude extracts or to preserve latent tyrosinase activity during purificati...

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Bibliographic Details
Published in:Phytochemistry (Oxford) 2004-03, Vol.65 (6), p.671-678
Main Authors: Fan, Yan, Flurkey, William H.
Format: Article
Language:English
Subjects:
Agaricus - enzymology
Agaricus bisporus
Biological and medical sciences
Chromatography, DEAE-Cellulose
Electrophoresis, Polyacrylamide Gel
Enzymes
Fundamental and applied biological sciences. Psychology
Gill tissue
Hydrogen-Ion Concentration
Isoelectric Focusing
Isoenzymes
Kinetics
Metabolism
Monophenol Monooxygenase - chemistry
Monophenol Monooxygenase - genetics
Monophenol Monooxygenase - isolation & purification
Monophenol Monooxygenase - metabolism
Phenols - chemistry
Phenols - metabolism
Plant physiology and development
Portabella
Portabella mushrooms
Protease Inhibitors - pharmacology
Purification
Sodium Dodecyl Sulfate - pharmacology
Substrate Specificity
Tyrosinase
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Summary:A group of tyrosinase isoforms with isoelectric points between 4.9 and 5.2 was isolated from gill tissue of Portabella mushrooms. Use of protease inhibitors was not able to increase the amount of latent forms significantly in crude extracts or to preserve latent tyrosinase activity during purification. The tyrosinase in gill tissue extracts showed latent activity above pH 5.5 and suppressed or displayed no latent activity below pH 5.5 when assayed in the presence of SDS. The purified isoforms showed monophenolase activity toward 4-hydroxyanisole but practically no activity toward tyrosine or tyramine. The purified isoforms showed greater activity toward catechol than either 4-methylcatechol, dopa, dopamine, chlorogenic acid, t-butylcatechol, or catechin. The Km for catechol was similar for the group of isolated isoforms (4.3 mM) compared to the isoforms in crude extracts (5.3 mM). Crude extracts showed several isoforms ranging from 50 to 230 kDa after partially denaturing SDS PAGE, while the purified isoforms showed molecular weights of 70 kDa. A group of tyrosinase isoforms, isoelectric points 4.9–5.2, was isolated from gill tissue and characterized with regard to isoform composition molecular weight, substrate specificity, latency, and kinetic constants.
ISSN:0031-9422
1873-3700
DOI:10.1016/j.phytochem.2004.01.008