Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) play an important role in the conversion of recalcitrant polysaccharides, but their mode of action has remained largely enigmatic. It is generally believed that catalysis by LPMOs requires molecular oxygen and a reductant that delivers two electrons per catalytic cycle. Using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show here that HO, rather than O, is the preferred co-substrate of LPMOs. By controlling HO supply, stable reaction kinetics are achieved, the LPMOs work in the absence of O, and the reductant is consumed in priming rather than in stoichiometric amounts. The use of HO by a monocopper enzyme that is otherwise cofactor-free offers new perspectives regarding the mode of action of copper enzymes. Furthermore, these findings have implications for the enzymatic conversion of biomass in Nature and in industrial biorefining.
BackgroundThe discovery of enzymes named lytic polysaccharide monooxygenases (LPMOs) has had a major impact on the efficiency of current commercial cellulase cocktails for saccharification of lignocellulosic biomass. However, the notion that LPMOs use molecular oxygen as a co-substrate and require two externally delivered electrons per catalytic cycle poses a challenge in the development of efficient large-scale industrial processes. Building on the recent discovery that H2O2, rather than O2, is the co-substrate of LPMOs, we show here how cellulose degradation by the LPMO-containing commercial cellulase cocktail Cellic® CTec2 can be controlled and boosted by supplying the reaction with H2O2.ResultsThe controlled supply of anaerobic hydrolysis reactions with H2O2 and sub-stoichiometric amounts of reductant increased apparent LPMO activity by almost two orders of magnitude compared to standard aerobic reactions utilizing O2 and stoichiometric amounts of reductant. Improved LPMO activity was correlated with enhanced saccharification rates and yields for a model cellulosic substrate (Avicel) as well as industrial lignocellulosic substrates (sulfite-pulped Norway spruce and steam-exploded birch), although the magnitude of the effects was substrate dependent. Improvements in lignocellulose conversions were achieved at low H2O2 feeding rates (in the range of 90–600 µM h−1). Tight control of LPMO reactions by controlled supply of H2O2 under anaerobic conditions was possible.ConclusionWe report saccharification rates and yields for a model substrate (Avicel) and industrial lignocellulosic substrates that, at low H2O2 feeding rates, are higher than those seen under standard aerobic conditions. In an industrial setting, controlling and supplying molecular oxygen and stoichiometric amounts of reductant are challenging. The present report shows that the use of small amounts of a liquid bulk chemical, H2O2, provides an alternative to the currently available processes, which likely is cheaper and more easy to control, while giving higher product yields.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1199-4) contains supplementary material, which is available to authorized users.
The discovery of lytic polysaccharide monooxygenases (LPMOs) has revolutionized enzymatic processing of polysaccharides, in particular, recalcitrant insoluble polysaccharides, such as cellulose. These monocopper enzymes display intriguing and unprecedented catalytic chemistry, which make them highly valuable in industrial bioprocessing, but also generate considerable challenges in terms of scientific understanding and optimal implementation. One issue of particular interest is the fact that both molecular oxygen and hydrogen peroxide can drive LPMO reactions. Here, we review recent insights into the catalytic mechanism of LPMOs derived from structural, spectroscopic, and functional studies. We then turn to the question of how one can optimally harness the potential of LPMOs in biomass processing, given the current knowledge of their catalytic mechanism. Finally, we review recent, more applied studies that have addressed the importance of LPMOs in enzymatic conversion of lignocellulosic biomass and discuss how the impact of these powerful enzymes could be improved.
The catalytically crucial N-terminal histidine (His1) of fungal lytic polysaccharide monooxygenases (LPMOs) is post-translationally modified to carry a methylation. The functional role of this methylation remains unknown. We have carried out an in-depth functional comparison of two variants of a family AA9 LPMO from Thermoascus aurantiacus (TaLPMO9A), one with, and one without the methylation on His1. Various activity assays showed that the two enzyme variants are identical in terms of substrate preferences, cleavage specificities and the ability to activate molecular oxygen. During the course of this work, new functional features of TaLPMO9A were discovered, in particular the ability to cleave xyloglucan, and these features were identical for both variants. Using a variety of techniques, we further found that methylation has minimal effects on the pK of His1, the affinity for copper and the redox potential of bound copper. The two LPMOs did, however, show clear differences in their resistance against oxidative damage. Studies with added hydrogen peroxide confirmed recent claims that low concentrations of H O boost LPMO activity, whereas excess H O leads to LPMO inactivation. The methylated variant of TaLPMO9A, produced in Aspergillus oryzae, was more resistant to excess H O and showed better process performance when using conditions that promote generation of reactive-oxygen species. LPMOs need to protect themselves from reactive oxygen species generated in their active sites and this study shows that methylation of the fully conserved N-terminal histidine provides such protection.
BackgroundRecent advances in the development of enzyme cocktails for degradation of lignocellulosic biomass, especially the discovery of lytic polysaccharide monooxygenases (LPMOs), have opened new perspectives for process design and optimization. Softwood biomass is an abundant resource in many parts of the world, including Scandinavia, but efficient pretreatment and subsequent enzymatic hydrolysis of softwoods are challenging. Sulfite pulping-based pretreatments, such as in the BALI™ process, yield substrates that are relatively easy to degrade. We have assessed how process conditions affect the efficiency of modern cellulase preparations in processing of such substrates.ResultsWe show that efficient degradation of sulfite-pulped softwoods with modern, LPMO-containing cellulase preparations requires the use of conditions that promote LPMO activity, notably the presence of molecular oxygen and sufficient reducing power. Under LPMO activity-promoting conditions, glucan conversion after 48-h incubation with Cellic® CTec3 reached 73.7 and 84.3% for Norway spruce and loblolly pine, respectively, at an enzyme loading of 8 mg/g of glucan. The presence of free sulfite ions had a negative effect on hydrolysis efficiency. Lignosulfonates, produced from lignin during sulfite pretreatment, showed a potential to activate LPMOs. Spiking of Celluclast®, a cellulase cocktail with low LPMO activity, with monocomponent cellulases or an LPMO showed that the addition of the LPMO was clearly more beneficial than the addition of any classical cellulase. Addition of the LPMO in reactions with spruce increased the saccharification yield from approximately 60% to the levels obtained with Cellic® CTec3.ConclusionsIn this study, we have demonstrated the importance of LPMOs for efficient enzymatic degradation of sulfite-pulped softwood. We have also shown that to exploit the full potential of LPMO-rich cellulase preparations, conditions promoting LPMO activity, in particular the presence of oxygen and reducing equivalents are necessary, as is removal of residual sulfite from the pretreatment step. The use of lignosulfonates as reductants may reduce the costs related to the addition of small molecule reductants in sulfite pretreatment-based biorefineries.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0862-5) contains supplementary material, which is available to authorized users.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.