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Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum

2000; Wiley; Volume: 31; Issue: 2 Linguagem: Inglês

10.1042/ba19990096

1470-8744

Kwang‐Hoon Kong, Min‐Pyo Hong, Sang‐Sook Choi, Yong‐Tae Kim, Sung‐Hye Cho,

RNA regulation and disease

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.