Single-molecule study of oxidative enzymatic deconstruction of cellulose

Abstract

LPMO (lytic polysaccharide monooxygenase) represents a unique paradigm of cellulosic biomass degradation by an oxidative mechanism. Understanding the role of LPMO in deconstructing crystalline cellulose is fundamental to the enzyme’s biological function and will help to specify the use of LPMO in biorefinery applications. Here we show with real-time atomic force microscopy that C1 and C4 oxidizing types of LPMO from Neurospora crassa (NcLPMO9F, NcLPMO9C) bind to nanocrystalline cellulose with high preference for the very same substrate surfaces that are also used by a processive cellulase (Trichoderma reesei CBH I) to move along during hydrolytic cellulose degradation. The bound LPMOs, however, are immobile during their adsorbed residence time ( ~ 1.0 min for NcLPMO9F) on cellulose. Treatment with LPMO resulted in fibrillation of crystalline cellulose and strongly ( ≥ 2-fold) enhanced the cellulase adsorption. It also increased enzyme turnover on the cellulose surface, thus boosting the hydrolytic conversion.

Structural Features on the Substrate-Binding Surface of Fungal Lytic Polysaccharide Monooxygenases Determine Their Oxidative Regioselectivity

“…Of all lignocellulolytic enzymes present in the T. reesei cocktail, cellobiohydrolase I (CHBI, Cel7A) has been suggested to benefit most from LPMO treatment, but whether the oxidative regioselectivity of the LPMO plays a role as well, is still unclear. On the other hand, it was recently shown by Eibinger et al that LPMOs stay adsorbed to the substrate much longer than the processively active CBHI, allowing up to 6 catalytic events per retention time . As these two enzymes are suggested to have overlapping specificity for crystalline regions, it seems likely that the boosting effect of LPMOs could be further enhanced by optimizing the frequency of adsorption and desorption by carefully balancing out the oxidative and binding capacity.…”

Differential activity of lytic polysaccharide monooxygenases on celluloses of different crystallinity. Effectiveness in the sustainable production of cellulose nanofibrils

“…Cellulolytic hydrolytic enzymes can be classified according to their modes of action as endoglucanase, exoglucanase, or β-glucosidase (Lombard et al, 2014). In addition, other complementary enzymes like LPMOs and expansins seem to trigger and/or improve the cooperative action of cellulases in cellulosic degradation (Bunterngsook, Mhuantong, Champreda, Thamchaipenet, & Eurwilaichitr, 2014;Eibinger, Sattelkow, Ganner, Plank, & Nidetzky, 2017a).…”

“…Maximum synergism effect was achieved in a range between 0.5-2 µM Cel9B, which produced 3-fold higher amount of reducing sugars. Although stimulation of cellulase activity has been reported for several combinations of cellulases and LPMOs, important differences have been described in terms of substrate, dose, time of incubation and type of cellulase (endo or exo hydrolysis) (Eibinger, Sattelkow, Ganner, Plank, & Nidetzky, 2017b;Moser et al, 2008;Vermaas, Crowley, Beckham, & Payne, 2015). Hu et al (2014) Nanocellulose from flax pulp NFC is usually manufactured from paper pulp by a mechanical treatment preceded by a chemical pretreatment, including acid or TEMPO-mediated oxidation (Fillat et al, 2018;Saito, Nishiyama, Putaux, Vignon, & Isogai, 2006).…”

Enhancing Enzymatic Properties of Endoglucanase I Enzyme from Trichoderma Reesei via Swapping from Cellobiohydrolase I Enzyme

“…Lignocellulose is considered the most abundant and sustainable energy resource in the world [1,2]. Due to the growing demand on green alternative energy sources, a substantial global effort is being exerted to redesign the global carbon cycle [3] in an effort to provide sustainabile fuels from plant-based cellulosic materials [4]. Plant-based cellulosic materials as a biofuel resource offer significant advantages over fossil fuel sources, e.g., decreasing global emission, providing a long-term rural development [5,6], the particular challenges still have to be surmounted in the deconstruction processes of cellulose to cellulosic biomass.…”