Purification and Characterization of a Tyrosinase from Streptomyces glaucescens

TLDR: The purification of a tyrosinase from cell-free extracts of Streptomyces glaucescens is described, and the enzyme obtained is homogeneous according to the criteria of ultracentrifuge analysis and polyacrylamide gel electrophoresis, which shows optimal activity at pH 6.8.

Abstract: 1 The purification of a tyrosinase from cell-free extracts of Streptomyces glaucescens is described. The enzyme obtained is homogeneous according to the criteria of ultracentrifuge analysis and polyacrylamide gel electrophoresis. 2 An average molecular weight of 29100 was determined by dodecylsulfate-gel electrophoresis and by sedimentation equilibrium analysis in the ultracentrifuge. The amino acid composition of the pure enzyme is also reported. 3 The enzyme contains one atom of copper in the cuprous form per molecule and is strongly inhibited by KCN and 4-nitro-catechol. 4 The enzyme shows optimal activity at pH 6.8; it has an isoelectric point at pH 6.95 and is most stable at pH 8.0. 5 Variations of Km values between pH 5.0 and 8.9 when l-tyrosine methylester and 3,4-di-hydroxy-l-phenylalanine were used as substrate was interpreted by assuming that different ionizable groups are involved in the oxidation of these two substrates. 6 The Michaelis-Menten constants and KCat are strongly dependent on the oxygen concentration for 3,4-dihydroxy-l-phenylalanine and almost independent for l-tyrosine methyl ester. Kinetic parameters obtained for a series of mono- and o-diphenol substrates are also reported.