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Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum
Author(s) -
Kong KwangHoon,
Hong MinPyo,
Choi SangSook,
Kim YongTae,
Cho SungHye
Publication year - 2000
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba19990096
Subject(s) - tyrosinase , chemistry , catechol oxidase , enzyme , isoelectric point , molecular mass , biochemistry , amino acid , chromatography , pyrogallol , catechol , isoelectric focusing , guanidine , polyphenol oxidase , peroxidase
Tyrosinase, with an isoelectric point at pH 4.9, was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum . Gel filtration, N‐terminal amino acid sequencing and SDS/PAGE analysis indicate that T. roseum tyrosinase is composed of two identical subunits, each with a molecular mass of 43000 Da. The enzyme exhibited high substrate specificity towards catechol, chlorogenic acid, L‐3‐(3,4‐dihydroxyphenyl)‐L‐alanine (L‐DOPA) and pyrogallol. The K m value of the enzyme for L‐DOPA was 0.18 mM. β‐Mercaptoethanol and sodium diethyldithiocarbamate notably inhibited the enzymic activity. The activity of the enzyme was optimal at pH 9.5 and 70 °C, and was increased by addition of 1 mM Mg 2+ , K + or Cu 2+ . The enzyme was highly stable against high temperature and guanidine hydrochloride. The N‐terminal amino acid sequence of the enzyme was determined to be Asp‐Ile‐Asn‐Gly‐Gly‐Gly‐Ala‐Thr‐Leu‐Pro‐Gln‐Lys‐Leu‐Tyr. These facts indicate that T. roseum tyrosinase appears to be distinct from the tyrosinases so far purified from other sources.