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The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay.

ABSTRACT: Background:Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H2O2 is a more efficient cosubstrate for the enzyme than O2, which could greatly affect the utilization of LPMOs in industrial settings. Results:We probe the reactivity of LPMO9C from the cellulose-degrading fungus Neurospora crassa with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and H2O2 as a cosubstrate. The measurements were also followed by continuous electrochemical H2O2 detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of H2O2 and for the reduction of LPMO's active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate. Conclusions:We found for all systems that the supply of H2O2 limited LPMO's cellulose depolymerization activity, which supports the function of H2O2 as the relevant cosubstrate. The turbidimetric assay allowed rapid determination of LPMO activity on a cellulosic substrate without the need for time-consuming and instrumentally elaborate analysis methods.

SUBMITTER: Filandr F

PROVIDER: S-EPMC7057652 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay.

Filandr Frantisek F Man Petr P Halada Petr P Chang Hucheng H Ludwig Roland R Kracher Daniel D

Biotechnology for biofuels 20200305

<h4>Background</h4>Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H2O2 is a more efficient cosubstrate for the enzyme than O2, which could greatly affect the utilization of LPMOs in industrial settin ...[more]

PMID: 32158501

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Project description:Background:Cellulose-active lytic polysaccharide monooxygenases (LPMOs) secreted by filamentous fungi play a key role in the degradation of recalcitrant lignocellulosic biomass. They can occur as multidomain proteins fused to a carbohydrate-binding module (CBM). From a biotech perspective, LPMOs are promising innovative tools for producing nanocelluloses and biofuels, but their direct action on cellulosic substrates is not fully understood. Results:In this study, we probed the role of the CBM from family 1 (CBM1) appended to the LPMO9H from Podospora anserina (PaLPMO9H) using model cellulosic substrates. Deletion of the CBM1 weakened the binding to cellulose nanofibrils, amorphous and crystalline cellulose. Although the release of soluble sugars from cellulose was drastically reduced under standard conditions, the truncated LPMO retained some activity on soluble oligosaccharides. The cellulolytic action of the truncated LPMO was demonstrated using synergy experiments with a cellobiohydrolase (CBH). The truncated LPMO was still able to improve the efficiency of the CBH on cellulose nanofibrils in the same range as the full-length LPMO. Increasing the substrate concentration enhanced the performance of PaLPMO9H without CBM in terms of product release. Interestingly, removing the CBM also altered the regioselectivity of PaLPMO9H, significantly increasing cleavage at the C1 position. Analysis of the insoluble fraction of cellulosic substrates evaluated by optical and atomic force microscopy confirmed that the CBM1 module was not strictly required to promote disruption of the cellulose network. Conclusions:Absence of the CBM1 does not preclude the activity of the LPMO on cellulose but its presence has an important role in driving the enzyme to the substrate and releasing more soluble sugars (both oxidized and non-oxidized), thus facilitating the detection of LPMO activity at low substrate concentration. These results provide insights into the mechanism of action of fungal LPMOs on cellulose to produce nanocelluloses and biofuels.

Dataset Information

The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay.

Publications

The H<sub>2</sub>O<sub>2</sub>-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay.

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