Abstract
BackgroundCellulose-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.ResultsIn 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.ConclusionsAbsence 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.
Highlights
Cellulose-active lytic polysaccharide monooxygenases (LPMOs) secreted by filamentous fungi play a key role in the degradation of recalcitrant lignocellulosic biomass
Production of PaLPMO9H with and without CBM from family 1 (CBM1) To gain insight into the contribution of the CBM1 to the catalytic function of AA9 LPMOs, we selected PaLPMO9H based on the previous biochemical analyses [18, 19, 25]
Absence of CBM1 alters LPMO cellulolytic activity at low substrate concentration The action of LPMO full-length (LPMO-FL) was first evaluated on three different types of cellulose, i.e., phosphoric acid-swollen cellulose (PASC), nanofibrillated cellulose (NFC), and bacterial microcrystalline cellulose (BMCC) (Fig. 2a)
Summary
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). Huge effort is being invested in the development of biofuels made from cellulosic biomass feedstocks, known as second-generation biofuels [2]. In parallel, nanomaterials such as nanofibers and nanocrystals are being isolated from wood and agricultural resources by mechanical and/or chemical treatments, offering unique properties with a wide range of applications (paper, pharmaceutical, cosmetics and food industries) [3,4,5]. All characterized LPMOs that belong to the AA9 family are able to oxidatively cleave cellulose [15,16,17,18], and recent studies have focused on their use to defibrillate cellulose and facilitate the production of nanocelluloses [19,20,21]
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