Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus
ABSTRACTThe genome of the white rot fungusPycnoporus cinnabarinusincludes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated fromPhanerochaete chrysosporium. The glyoxal oxidase ofP. chrysosporiumis physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases ofP. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced inAspergillus nigerstrain D15#26 (pyrGnegative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein forPciGLOX1 andPciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The twoP. cinnabarinusenzymes generally exhibited similar substrate preferences, butPciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde.IMPORTANCEThis study addresses the poorly understood role of how fungal peroxidases obtain anin situsupply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H2O2in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these enzymes utilize fungal metabolites as the substrates. Two glyoxal oxidases have been isolated and characterized to date, and the differentiation of the substrate specificity of the two enzymes produced byPycnoporus cinnabarinusillustrates the alternative mechanisms existing in a single fungus, together with the utilization of these enzymes to prepare platform chemicals for industry.