Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus.
The genome of the white rot fungus Pycnoporus cinnabarinus includes 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 from Phanerochaete chrysosporium The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2 and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced in Aspergillus niger strain D15#26 (pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for PciGLOX1 and PciGLOX2, 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 two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but PciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde.



This study addresses the poorly understood role of how fungal peroxidases obtain an in situ supply 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 H2O2 in 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 by Pycnoporus cinnabarinus illustrates the alternative mechanisms existing in a single fungus, together with the utilization of these enzymes to prepare platform chemicals for industry.
Aix Marseille Université, INRA, BBF (Biodiversité et Biotechnologie Fongiques), Marseille, France.
Marianne
M
Daou
Aix Marseille Université, INRA, BBF (Biodiversité et Biotechnologie Fongiques), Marseille, France.
François
F
Piumi
USDA, Forest Products Laboratory, Madison, Wisconsin, USA.
Daniel
D
Cullen
Aix Marseille Université, INRA, BBF (Biodiversité et Biotechnologie Fongiques), Marseille, France.
Eric
E
Record
Aix Marseille Université, INRA, BBF (Biodiversité et Biotechnologie Fongiques), Marseille, France craig.faulds@univ-amu.fr.
Craig B
CB
Faulds
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D030781:Organisms, Genetically Modified / Q000378:metabolism; D010084:Oxidation-Reduction; D010802:Phylogeny; D055494:Pycnoporus / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D013379:Substrate Specificity
D005656:Fungal Proteins; D000429:Alcohol Oxidoreductases; C052371:glyoxal oxidase