Real-time imaging reveals that lytic polysaccharide monooxygenase promotes cellulase activity by increasing cellulose accessibility.

Background: The high cost of enzymes is one of the key technical barriers that must be overcome to realize the economical production of biofuels and biomaterials from biomass. Supplementation of enzyme cocktails with lytic polysaccharide monooxygenase (LPMO) can increase the efficiency of these cellulase mixtures for biomass conversion. The previous studies have revealed that LPMOs cleave polysaccharide chains by oxidization of the C1 and/or C4 carbons of the monomeric units. However, how LPMOs enhance enzymatic degradation of lignocellulose is still poorly understood.

Results: In this study, we combined enzymatic assays and real-time imaging using atomic force microscopy (AFM) to study the molecular interactions of an LPMO [AA9A, formerly known as Cel61A) from ] and a cellobiohydrolase I (Cel7A from ) with bacterial microcrystalline cellulose (BMCC) as a substrate. Cellulose conversion by Cel7A alone was enhanced from 46 to 54% by the addition of AA9A. Conversion by a mixture of Cel7A, endoglucanase, and β-glucosidase was increased from 79 to 87% using pretreated BMCC with AA9A for 72 h. AFM imaging demonstrated that individual AA9A molecules exhibited intermittent random movement along, across, and penetrating into the ribbon-like microfibril structure of BMCC, which was concomitant with the release of a small amount of oxidized sugars and the splitting of large cellulose ribbons into fibrils with smaller diameters. The dividing effect of the cellulose microfibril occurred more rapidly when AA9A and Cel7A were added together compared to AA9A alone; Cel7A alone caused no separation.

Conclusions: AA9A increases the accessible surface area of BMCC by separating large cellulose ribbons, and thereby enhances cellulose hydrolysis yield. By providing the first direct observation of LPMO action on a cellulosic substrate, this study sheds new light on the mechanisms by which LPMO enhances biomass conversion.