Purification and Some Properties of Sarcosine Oxidase from Corynebacterium sp. U-96

During the course of studies on creatinine metabolism by bacteria, a newly isolated bacterial strain, Corynebacterium sp. U-96, showed the highest potency in the production of sarcosine oxidase. The enzyme was purified from the cell-free extract to a homogeneous state by successive procedures involving polyethyleneimine treatment and chromatographies on DEAE-cellulose, QAE-Sephadex A-50, DEAE-Sephadex A-50, Ultrogel AcA34, and creatine-AH-Sepharose 4B. The absorption spectrum of the enzyme had maxima at 276, 369, and 451 nm, being characteristic of a flavoprotein. This enzyme contains both 1 mol of non-covalently bound FAD and 1 mol of covalently bound FAD per mol of enzyme. Sedimentation experiments gave an $s20,W0$ value of 9.35 and the molecular weight was calculated to be 174,000 by the meniscus depletion method. The enzyme was shown to be composed of one each of four non-identical subunits; subunit A 110,000; B 44,000; C 21,000; D 10,000. One molecule of FAD is covalently bound to subunit B. The enzyme acted preferentially on sarcosine rather than on N -methyl-L-alanine or N -ethyl-glycine, but did not act at all on other N -methyl-L-amino acids tested. The K_m and k_cat values of the enzyme were estimated to be 3.4 mM and 5.8 s ⁻¹ for sarcosine at pH 8.3 and 37°C, 8.7 mM 2.1 s ⁻¹ for N -methyl-L-alanine at pH 8.3 and 37°C, and 11.4 mM and 2.2 s ⁻¹ for N -ethyl-glycine at pH 7.0 and 37°C, respectively. The K_m and V_max values for oxygen as a hydrogen acceptor were found to be 0.13 mM and 12.81 μmol/min/mg of enzyme. Oxygen can be replaced by 2,6-dichlorophenolindophenol, phenazine methosulfate, ferricyanide, but not by methylene blue or cytochrome c . The enzyme activity was strongly inhibited by Zn ²⁺ , Cu ²⁺ , Cd ²⁺ , Hg ²⁺ , Ag ⁺ , iodoacetamide, p -chloromercuribenzoate, or N -ethylmaleimide. Acetate, propionate, formaldehyde, and acetaldehyde also had inhibitory effects on the enzyme activity. The stoichiometry data showed that oxygen consumption was accompanied by the formation of equimolar amounts of formaldehyde, glycine, and hydrogen peroxide from sarcosine.