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 pI 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-leucine and N-methyl-DL-valine to lesser extents. The apparent Km values of sarcosine, N-methyl-DL-alanine, N-methyl-L-leucine and N-methyl-DL-valine were 12.2, 6.8, 106 and 173 mM, respectively. The enzyme was inactivated by N-bromosuccinimide, Zn2+, Fe3+ and Hg2+, but not by ethylenediaminetetraacetate, p-chloromercuribenzoate, monoiodoacetate or p-toluenesulfonyl-chloride.