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

Abstract

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 °C with the secretome was as efficient as saccharification with Cellic CTec2 at 50 °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 °C was clearly most efficient. At 60 °C, enzyme inactivation became apparent for both enzyme blends, likely due to detrimental redox processes. Addition of H 2 O 2 -generating glucose oxidase to reactions with Cellic CTec2 at 50 °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.

Insights into the H2O2-Driven Lytic Polysaccharide Monooxygenase Activity on Efficient Cellulose Degradation in the White Rot Fungus Irpex lacteus

“…Considering the high catalytic efficiency of LPMO in the presence of H 2 O 2 , the H 2 O 2 -driven LPMO catalysis from natural microorganisms had gradually attracted academic and industrial attention in recent years. 14,18,37,38 Similar to previous reports, the H 2 O 2 -driven LPMO catalysis in I. lacteus was more efficient than the O 2 -driven LPMO catalysis for cellulose depolymerization, showing orders of magnitude improvement in the native and oxidized cello-oligosaccharide production. Nonetheless, it should not be ignored that excess H 2 O 2 could cause oxidative damage to LPMO activity and result in a significant reduction in the native and oxidized cellooligosaccharide production.…”

In situ H2O2 Generation by Choline Oxidase and Its Application in Amino Polysaccharide Degradation by Coupling to Lytic Polysaccharide Monooxygenase

“…As discussed above, system efficiency, i. e., reaction kinetics, LPMO stability, and total yield of oxidized products, can be manipulated by varying various reaction parameters. For applications in biomass processing, further, application‐specific optimization will be needed, since LPMO action and optimal supply rates of H 2 O 2 depend on the nature of the substrate, the substrate concentration, the presence of other redox‐active compounds, and the interplay with other degradative enzymes (e. g., chitinases or cellulases) [26] …”

“…For applications in biomass processing, further, application-specific optimization will be needed, since LPMO action and optimal supply rates of H 2 O 2 depend on the nature of the substrate, the substrate concentration, the presence of other redox-active compounds, and the interplay with other degradative enzymes (e. g., chitinases or cellulases). [26] Interestingly, solvent engineering plays an important role in current attempts to develop greener routes for biomass processing. [17b,27] For example, deep eutectic solvents (DES) have been used for extracting and partially deacetylating chitin.…”

Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase

“…The nature of this intermediate has not been experimentally determined but has been explored in multiple theoretical calculations, which propose it to be either a triplet state ( S = 1) Cu II –oxyl, [Cu–O] + or a singlet state ( S = 0) Cu III –hydroxide, [Cu–OH] 2+ (or possibly free hydroxyl radicals, following the enzyme-catalyzed homolytic fission of the O–O bond in H 2 O 2 ). − As with all oxygenase and peroxygenase enzymes, however, these intermediates can cause damage to the enzyme through oxidative cleavage of bonds on adjacent amino acids, , an action which compromises enzyme efficiency. − Thus, an outstanding question about the mode of action of LPMOs is how does the enzyme mitigate/prevent oxidative damage to preserve the integrity of key active site residues? In this regard, it is known that the type of substrate, , the concentration of peroxide, the type of reducing agent, the degree of glycosylation and methylation of His1 of LPMOs are all factors in the degree of substrate versus enzyme oxidation. Nonetheless, little is known about the fate of reactive intermediates at a mechanistic level, save for proposals from recent QM/MM calculations which suggest that the formation of a copper­(II)–histidyl radical complex occurs during uncoupled turnover of AA9 LPMOs with peroxide …”

“…15−17 Thus, an outstanding question about the mode of action of LPMOs is how does the enzyme mitigate/prevent oxidative damage to preserve the integrity of key active site residues? In this regard, it is known that the type of substrate, 18,19 the concentration of peroxide, 17 the type of reducing agent, 20 the degree of glycosylation 21 ation of His1 22 of LPMOs are all factors in the degree of substrate versus enzyme oxidation. Nonetheless, little is known about the fate of reactive intermediates at a mechanistic level, save for proposals from recent QM/MM calculations which suggest that the formation of a copper(II)−histidyl radical complex occurs during uncoupled turnover of AA9 LPMOs with peroxide.…”