Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus

Abstract

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. IMPORTANCE 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.

Characterization of a New Glyoxal Oxidase from the Thermophilic Fungus Myceliophthora thermophila M77: Hydrogen Peroxide Production Retained in 5-Hydroxymethylfurfural Oxidation

“…GLOx couples the two-electron oxidation of aldehydes to carboxylic acids to the reduction of O 2 to H 2 O 2 [21,24,25], α-Hydroxy carbonyl and α-diol substrates can also be oxidized in two steps to carboxylic acids. Previous reports on the enzymatic activity of GLOxes from Phanerochaete chrysosporium [26] and Pycnoporus cinnabarinus [27] indicate that glyoxal and methylglyoxal are the best substrates for the enzymes. Both aldehydes are recognized as products of glucose degradation through a combination of retroaldol condensation and auto-oxidation , and are found in ligninolytic cultures [28,29].…”

“…This copper ion is coordinated by two histidine residues and a tyrosine, which is cross-linked to a cysteine residue by an unusual thioester bond. These residues are conserved in GLOxes, CgrAlcOx, and galactose-6-oxidases [27,31,32].…”

Evaluation of 18 isolates of basidiomycetes for Lignocellulose degrading enzymes

“…While laccase requires oxygen for its activity, MnP and LiP require hydrogen peroxide. Auxiliary enzymes such as aryl alcohol oxidase and glyoxal oxidase produce the hydrogen peroxide required for their activity (Daou et al, 2016). Since many basidiomycetes are known to degrade both cellulose and lignin efficiently (Zhu et al, 2016), they may be used as sources of enzymes for both biological pre-treatment and saccharification.…”

A Lytic Polysaccharide Monooxygenase from a White-Rot Fungus Drives the Degradation of Lignin by a Versatile Peroxidase

“…First, white-rot fungi have versatile extracellular H 2 O 2 -generating enzymes that participate in the lignin degradation reaction of class II peroxidase, such as aryl-alcohol oxidase (AAO) and glyoxal oxidase (GLOX) (23). The expression of H 2 O 2 -generating enzymes depends on the fungal isolate, wood substrate species, and physiological factors, and different H 2 O 2 -generating enzymes play roles in different stages of fungal decay (40,41).…”

“…It is well known that lignin-degrading peroxidases use H 2 O 2 as a cosubstrate to mineralize the lignin. Multiple extracellular enzymes, such as glyoxal oxidases (GLOX), aryl-alcohol oxidases (AAO), and copper radical oxidases (23)(24)(25), generate H 2 O 2 for lignin degradation. Some studies showed that multiple copies of LPMOs are coexpressed with peroxidases during white-rot fungal decay (21).…”