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[72] Lactate oxygenase of Mycobacterium phlei

1975; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(75)41074-6

1557-7988

Shigeki Takemori, Masayuki Katagiri,

Synthesis and Catalytic Reactions

This chapter discusses the assay method, purification procedure, and properties of lactate oxygenase of Mycobacterium phlei. Lactate oxygenase, which also has been termed “lactate oxidative decarboxylase,” is a flavoprotein with FMN as the prosthetic group and catalyzes the incorporation of one atom of oxygen into substrate, while the other is reduced to water. The activity of the enzyme is assayed at 25° by measuring the consumption of molecular oxygen dissolved in the reaction medium. A manometric technique may also be used. Mixtures are placed in the reaction vessel that contain 72 μmoles of the buffer, 270 μmoles of lactate, as well as an appropriate amount of the enzyme in a total volume of 3.6 ml. The reactions are initiated by adding enzyme. The DEAE-Sephadex step in the purification procedurecan be satisfactorily omitted, and the enzyme is directly crystallized from the preparation obtained with ammonium sulfate fractionation. All operations are carried out at room temperature. The enzyme has a typical flavoprotein absorption spectrum with maxima at 280, 375, and 454 nm with shoulders at 430 and 480 nm. The enzyme is reversibly resolved into FMN and apoenzyme moieties by the acid ammonium sulfate treatment. The molecular weight values of the enzyme determined by various methods are summarized.