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 similar-sized subunits of 60 kDa each. Optimum pH and temperature were 8.2 and 55-degrees-C, respectively, with a Q10 (45-55-degrees-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-mu-M for phenylalanine and 176-mu-M for methionine. The effect of several specific group reagents, chelating agents and bivalent cations on enzyme activity has also been studied.