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

Abstract

BackgroundThe 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.ResultsIn this study, we combined enzymatic assays and real-time imaging using atomic force microscopy (AFM) to study the molecular interactions of an LPMO [TrAA9A, formerly known as TrCel61A) from Trichoderma reesei] and a cellobiohydrolase I (TlCel7A from T. longibrachiatum) with bacterial microcrystalline cellulose (BMCC) as a substrate. Cellulose conversion by TlCel7A alone was enhanced from 46 to 54% by the addition of TrAA9A. Conversion by a mixture of TlCel7A, endoglucanase, and β-glucosidase was increased from 79 to 87% using pretreated BMCC with TrAA9A for 72 h. AFM imaging demonstrated that individual TrAA9A 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 TrAA9A and TlCel7A were added together compared to TrAA9A alone; TlCel7A alone caused no separation.ConclusionsTrAA9A 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.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1023-1) contains supplementary material, which is available to authorized users.

Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes

“…The synergistic activities of conventional endoand exocellobiohydrolases are well known. More recently, LPMO has been shown to boost the performance of these hydrolases (42,43). Although our study selected for eukaryotic mRNA, genes most closely related to bacteria were occasionally identified.…”

Polysaccharide oxidation by lytic polysaccharide monooxygenase is enhanced by engineered cellobiose dehydrogenase

“…Lytic polysaccharide monooxygenases (LPMOs, EC: 1.14.99.53-56, CAZy IDs: AA9-11, AA13-16) are copper-containing enzymes found in chitin-, starch-, and plant cell wall-degrading organisms [1][2][3][4][5]. LPMOs use an unprecedented oxidative reaction mechanism to cleave and locally decrystallize these biopolymers, which enhances the activity of associated hydrolases [1,2,[6][7][8]. The reaction cycle starts with the reduction of LPMO's mononuclear type-II copper by external electron donors.…”

Enzyme biotechnology in degradation and modification of plant cell wall polymers

“…High‐throughput sequencing combined with bioinformatic sequence mining tools, heterologous expression and novel substrates and screening methods have been developed for CAZymes (Cragg et al , Deng et al , Vidal‐Melgosa et al ). One of the recent findings in lignocellulose saccharification was the discovery of LPMOs ability to remarkably enhance hydrolysis of lignocelluloses, apparently by improving the accessibility of the polysaccharides to GHs (Harris et al , Cannella et al , Eibinger et al , Song et al ). A similar role in lignocellulose hydrolysis has been proposed for swollenins and other expansin‐like proteins (Gourlay et al ).…”

“…Combination of LPMO, endoglucanase and xylanase in treatment of pulp has also been reported to enhance nanofibrillation of pulp (Hu et al 2018). Interestingly, the ability of LPMO to split cellulose nanocrystals was published recently (Song et al 2018). The combination of production of nanocellulose with biofuel production using enzymatic hydrolysis and fractionation of pulp has also been proposed (Zhu et al 2011, Yarbrough et al 2017.…”