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Influence of cellobiose oxidase on peroxidases from Phanerochaete chrysosporium.

ABSTRACT: Reduction of H2O2-oxidized manganese peroxidase (MnP), lignin peroxidase and, to some extent, horseradish peroxidase, was studied in the presence of cellobiose oxidase (CbO) and cellobiose. It was found that the reversion rates for MnP compound II and lignin peroxidase compound II back to native enzymes increased significantly in the presence of CbO and cellobiose. However, the reduction of cytochrome c by CbO plus cellobiose was 40 times faster than the reduction of MnP compound II. Also, the lag phase before reversion to the native states decreased for all three peroxidases in the presence of CbO and cellobiose. Active CbO did not repress formation of compounds I or II of the peroxidases, and Mn2+/veratryl alcohol reduced compound II of the peroxidases much more rapidly than did active CbO. This indicates that, in the presence of Mn2+ or veratryl alcohol, MnP and lignin peroxidase can complete their catalytic cycles and function normally without interference from CbO. Without the presence of peroxidase substrates, active CbO reduced compound II of the above peroxidases.

SUBMITTER: Ander P

PROVIDER: S-EPMC1134378 | biostudies-other | 1993 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundCellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp).ResultsPcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-β-D-glucosaminidase in the supernatant.ConclusionsHeterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

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Influence of cellobiose oxidase on peroxidases from Phanerochaete chrysosporium.

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