The impact of hydrogen peroxide supply on LPMO activity and overall saccharification efficiency of a commercial cellulase cocktail.

Background: The 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 HO, rather than O, 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 HO.

Results: The controlled supply of anaerobic hydrolysis reactions with HO and sub-stoichiometric amounts of reductant increased apparent LPMO activity by almost two orders of magnitude compared to standard aerobic reactions utilizing O 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 HO feeding rates (in the range of 90-600 µM h). Tight control of LPMO reactions by controlled supply of HO under anaerobic conditions was possible.

Conclusion: We report saccharification rates and yields for a model substrate (Avicel) and industrial lignocellulosic substrates that, at low HO 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, HO, provides an alternative to the currently available processes, which likely is cheaper and more easy to control, while giving higher product yields.