Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H2O2-Producing Glucose Oxidase

Understanding and improving the efficiency of enzymatic saccharification of lignocellulosic biomass will promote the use of this renewable material. Here, we have studied several process parameters (reaction temperature, type of enzyme blend, type of substrate, type of reductant, and in situ supply of hydrogen peroxide) to better understand how saccharification could be optimized, focusing on the role of lytic polysaccharide monooxygenases (LPMOs). Comparison of a simple, LPMO-rich cellulolytic secretome from the thermophilic fungus Thermoascus aurantiacus with the commercial cellulase preparation Cellic CTec2 showed that saccharification of (lignin-poor) sulfite-pulped spruce at 60 degrees C with the secretome was as efficient as saccharification with Cellic CTec2 at 50 degrees C. Quantification of LPMO products showed that while LPMO activity contributed to saccharification efficiency, high levels of LPMO activity were not necessarily beneficial. Reactions with steam-exploded birch, rich in redox-active lignin, highlighted a strong impact of the feedstock on enzyme performance. In this case, the reaction with Cellic CTec2 at 50 degrees C was clearly most efficient. At 60 degrees C, enzyme inactivation became apparent for both enzyme blends, likely due to detrimental redox processes. Addition of H2O2-generating glucose oxidase to reactions with Cellic CTec2 at 50 degrees C led to strongly increased LPMO activity and, only for reactions with the lignin-poor substrate, improved saccharification yields. These results underpin the potential of the T. aurantiacus secretome for hydrolysis of lignin-poor substrates, and the usefulness of glucose oxidase for optimizing their saccharification. They also show that the efficiency of LPMO-containing cellulase preparations is highly dependent on the nature of the reductant and the substrate.

Automated summary

Understanding and improving the efficiency of enzymatic saccharification of lignocellulosic biomass is crucial for utilizing renewable materials. This study focuses on the role of lytic polysaccharide monooxygenases (LPMOs) and investigates various process parameters. The results demonstrate the potential of LPMO-rich cellulolytic secretome and the usefulness of glucose oxidase for optimizing saccharification.