In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose

Abstract

Background Biochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation. Results Here, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation–reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt. Conclusion Our results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation–reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics.

The impact of reductants on the catalytic efficiency of a lytic polysaccharide monooxygenase and the special role of dehydroascorbic acid

“…The progress curve shows that product formation diminished after a fast and linear initial phase. This is indicative of enzyme inactivation, which is to be expected in experiments with high amounts of added H 2 O 2 [24,56,57].…”

H2O2 in Liquid Fractions of Hydrothermally Pretreated Biomasses: Implications of Lytic Polysaccharide Monooxygenases

“…Although H 2 O 2 is used efficiently in polysaccharide peroxygenase reaction, a reductant peroxidase side reaction may lead to the irreversible inactivation of LPMOs by H 2 O 2. ,,,, Indeed, our recent results demonstrated that cellulose-free Tr AA9A can perform an average of 138 ascorbic acid peroxidase reactions before complete inactivation of the enzyme . Therefore, a fine-tuned control over the levels of H 2 O 2 is a prerequisite for the maintenance of LPMO stability.…”

“…14,35,50 A study by Kadic et al demonstrated that the optimal H 2 O 2 supply rate for anaerobic degradation of cellulose (Avicel at 50 g L −1 ) by the enzyme cocktail (Cellic CTec3) was around 50 μM h −1 , whereas supply rates of 100 μM h −1 and above were deleterious for the LPMO activity. 50 In aerobic conditions, H 2 O 2 is also formed in reactions between O 2 and compounds in pretreated biomass. 38 While O 2 diffusion may be potentially rate-limiting under high dry matter conditions of lignocellulose saccharification, the experiments used here for the measurement of the rates of H 2 O 2 production were set up so that the O 2 diffusion was not rate-limiting (Figure S5).…”

“…These results suggest that the originally described monooxygenase activity may, at least in part, stem from the reaction of LPMO with H 2 O 2 that has been produced in a nonenzymatic reaction of O 2 with reductants or in the uncoupled reaction of reduced LPMO with O 2 . , Many compounds including lignocellulosic biomass-derived phenolic compounds ,,− as well as some redox-active enzymes ,− are able to deliver electrons to LPMOs. While the monooxygenase reaction assumes the stoichiometric delivery of two electrons per glyosidic bond cleavage, , the peroxygenase reaction implies only the initial “priming” reduction of LPMO to its catalytically active Cu­(I) form. ,, However, uncoupled side reactions involving the re-oxidation of polysaccharide-free LPMO by H 2 O 2 /O 2 in a peroxidase/oxidase-like reaction will increase the turnover of the reductant. ,, Most importantly, the re-oxidation of LPMO by H 2 O 2 may lead to an oxidative damage and irreversible inactivation of an enzyme. ,,,, …”

Kinetics of H2O2-driven catalysis by a lytic polysaccharide monooxygenase from the fungus Trichoderma reesei

“…Thus, the values of the kinetic parameters and their dependency on cellulose concentration suggest that the flow of H 2 O 2 through the cellulolytic peroxygenase reaction is strongly favored over side reactions. However, because the contact times required to achieve target conversion are usually in the range of 72 to 96 h ( 55 ), the stability of LPMOs is a major issue in the application of these enzymes in lignocellulose conversion ( 24 , 56 ). Our results suggest that LPMOs and their variants with low efficiency of the reductant peroxidase reaction and low probability of enzyme inactivation should be more stable.…”