Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum

Tyrosinase, with an isoelectric point at pH 4.9, was purified to electropho retic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. Gel filtration, N-terminal amino acid sequencing and SDS/PAGE anal ysis indicate that T. roseum tyrosinase is composed of two identical subuni ts, each with a molecular mass of 43 000 Da, The enzyme exhibited high subs trate specificity towards catechol, chlorogenic acid, L-3-(3,4-dihydroxyphe nyl)-L-alanine (L-DOPA) and pyrogallol. The K-m value of the enzyme for L-D OPA was 0.18 mM. beta-Mercaptoethanol and sodium diethyldithiocarbamate not ably inhibited the enzymic activity. The activity of the enzyme was optimal at pH 9.5 and 70 degrees C, and was increased by addition of I mM Mg2+, K or Cu2+. The enzyme was highly stable against high temperature and guanidi ne hydrochloride. The N-terminal amino acid sequence of the enzyme was dete rmined to be Asp-lle-Asn-Cly-Gly-Gly-Ala-Thr-Leu-Pro-Gln-Lys-Leu-Tyr. These facts indicate that T. roseum tyrosinase appears to be distinct from the t yrosinases so far purified from other sources.