title: Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum.
abstract: 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. beta-Mercaptoethanol and sodium diethyldithiocarbamate notably 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 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.
authors.affiliation: Department of Chemistry, College of Natural Sciences, Chung-Ang University, 221, Huksuk-Dong, Dongjak-Ku, Seoul 156-756, Korea.
authors.forename: K H
authors.initials: KH
authors.lastname: Kong
authors.forename: M P
authors.initials: MP
authors.lastname: Hong
authors.forename: S S
authors.initials: SS
authors.lastname: Choi
authors.forename: Y T
authors.initials: YT
authors.lastname: Kim
authors.forename: S H
authors.initials: SH
authors.lastname: Cho
mesh_terms: D000595:Amino Acid Sequence; D001709:Biotechnology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007526:Isoelectric Point; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D008623:Mercaptoethanol; D014442:Monophenol Monooxygenase