Purification and characterization of an L-amino-acid oxidase from Chlamydomonas reinhardtii.

An L-amino-acid oxidase (EC 1.4.3.1) that catalyzes the oxidative deamination of twelve L-amino acids has been purified 21-fold and with 14% yield to electrophoretic homogeneity from Chlamydomonas reinhardtii cells by ammonium-sulfate fractionation, gel filtration through Sephacryl and Superose, anion-exchange chromatography and preparative electrophoresis in polyacrylamide gels. The native enzyme is a protein of 470 kDa and consists of eight identical or similarsized subunits of 60 kDa each. Optimum pH and temperature were 8.2 and 55° C, respectively, with a Q10 (45-55° C) of 1.7 and an activation energy of 45 kJ · mol(-1). Its absorption spectrum showed, in the visible region, maxima at 360 and 444 nm, characteristic of a flavoprotein with a calculated flavin content of 7.7 mol FAD per mol of native enzyme. Apparent K m values of the twelve L-amino acids which can act as substrates of L-amino-acid oxidase ranged between 31 µM for phenylalanine and 176 µM for methionine. The effect of several specific group reagents, chelating agents and bivalent cations on enzyme activity has also been studied.