Sarcosine oxidase from Arthrobacter ureafaciens: Purification and some properties.

Abstract

Sarcosine oxidase (EC 1.5.3.1) of Arthrobacter ureafaciens was purified to homogeneity by means of sequential chromatography on DEAE-Toyopearl 650M, Toyopearl HW-55, and hydroxyapatite. The purified enzyme was most active at pH 8.3 and was stable at pH 6.5-9.5 for 20h at 20°C. The molecular weight of the enzyme was estimated to be 185000 by gel filtration. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated that the enzyme was composed of four dissimilar subunits with molecular weights of 96000, 45000, 23000, and 14000 daltons. The isoelectric point was 4.5 as checked by isoelectrofocusing. The Km and kcat values for sarcosine were 6.4mM and 5.8s-1. The enzyme was strongly inactivated by heavy metal ions such as Ag+, Zn2+, Cu2+, Hg2+, Co2+, and Cd2+, and an SH-blocking regent, p-chloromercuribenzoate. Spectrophotometric and fluorogenic analyses suggested that a flavin cofactor associated with the 45000-dalton subunit participates in the enzyme reaction.