Purification and Characterization of Sarcosine Oxidase of Bacillus Origin

Abstract

Sarcosine oxidase (EC 1.5.3.1) produced by Bacillus sp. B-0618 was purified by ion exchange chromatography on diethyl aminoethyl-cellulose and gel filtration on Sephadex G-100 and G-150. The molecular weight of the enzyme was estimated to be 42000 by gel filtration on Sephadex G-150 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited an absorption spectrum characteristic of flavoprotein. The enzyme showed the maximum activity at pH 8.5—9 and was stable at pH 7—10. The pl value was 4.7 as determined by isoelectric focusing. Although sarcosine is the preferred substrate, the enzyme also oxidized N-methyl-DL-alanine N-methyl-L- and N-methyl-DL-valine to lesser extents. The apparent Kmvalues of sarcosine, N-methyl-DL-alanine N-methyl-L-leucine and N-methyl-DL-valine were 12.2, 6.8, 106 and 173 mM, respec-tively. The enzyme was inactivated by N-bromosuccinimide Zn2+, Fe3+ and Hg2+, but not by ethylenediaminetetraacetate, p-chloromercuribenzoate monoiodoacetate or p-toluenesulfonyl-. © 1987, The Pharmaceutical Society of Japan. All rights reserved.