GLYOXAL OXIDASE OF PHANEROCHAETE-CHRYSOSPORIUM - ITS CHARACTERIZATION AND ACTIVATION BY LIGNIN PEROXIDASE

Glyoxal oxidase (GLOX) is an extracellular H2O2-generating enzyme produced by ligninolytic cultures of Phanerochete chrysosporium. The production, purification, and partial characterization of GLOX from agitated cultures are described here. High-oxygen levels are critical for GLOX production as for lignin peroxidase. GLOX purified by anion-exchange chromatography appears homogeneous by NaDod-SO4/PAGE (molecular mass = 68 kDa). However, analysis by isoelectric focusing indicates two major bands (pI 4.7 and 4.9) that stain as glycoproteins as well as for H2O2-producing activity in the presence of methylglyoxal. Purified GLOX shows a marked stimulation in activity when incubated with Cu2+; full activation takes more than 1 hr with 1 mM CuSO4 at pH 6. The steady-state kinetic parameters for the GLOX oxidation of methylglyoxal, glyceraldehyde, dihydroxyacetone, glycolaldehyde, acetaldehyde, glyoxal, glyoxylic acid, and formaldehyde, were determined by using a lignin peroxidase coupled-assay at pH 4.5. Of these substrates, the best is the extracellular metabolite methylglyoxal with a K(m) of 0.64 mM and an apparent rate of catalysis, k(cat), of 198 s-1 under air-saturated conditions. The K(m) for oxygen is greater than the concentration of oxygen possible at ambient pressure - i.e., >1.3 mM at 25°C. Importantly, oxygen-uptake experiments show that purified GLOX is inactive unless coupled to the peroxidase reaction. With this coupled reaction, for each mol of methylglyoxal, veratryl alcohol (a lignin peroxidase substrate), and oxygen consumed, 1 mol each of pyruvate and veratraldehyde is produced. The importance of these results is discussed in relation to the physiology of lignin biodegradation and possible extracellular regulatory mechanisms for the control of oxidase and peroxidase activities.