Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs)

Kont, Riin; Bissaro, Bastien; Eijsink, Vincent G. H.; Väljamaë, Priit

doi:10.1038/s41467-020-19561-8

Cited by 80 publications

(93 citation statements)

References 64 publications

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“…Apparent LPMO catalytic rates observed in cellulose degradation reactions are average rates and may thus reflect an average of a mixture of inactive and active LPMOs. Indeed, apparent reaction rates of LPMOs deduced from cellulose degradation reactions similar to those described here [ 17 ], were orders of magnitude lower than the maximum catalytic rates found for pure LPMOs in proper enzyme kinetics studies [ 18 , 19 , 25 ]. This supports the notion that in these H 2 O 2 -limited reactions, only a fraction of the LPMOs is needed to utilize the available co-substrate.…”

Section: Resultsmentioning

confidence: 48%

“…The oxidative action of LPMOs requires the presence of either O 2 and stoichiometric amounts of reducing agent or lignin [ 4 , 5 ] or H 2 O 2 and priming amounts of reducing agent [ 6 , 15 , 16 ]. While the action of LPMOs, as shown by the formation of oxidized sugars and glucose by LPMO-containing cellulase cocktails, can be harnessed in bioreactors using both the O 2 -dependent [ 3 , 14 ] and the H 2 O 2 -dependent [ 17 ] reaction mechanisms, the H 2 O 2 -dependent reaction mechanism has been shown to be more effective, both at laboratory scale [ 17 – 19 ] and recently also at demonstration scale [ 20 ] for lignin-poor biomass. The use of added H 2 O 2 enables LPMO activity without the need for gas–liquid mass transfer of oxygen.…”

Section: Introductionmentioning

confidence: 99%

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

Kadić

Várnai

Eijsink

et al. 2021

Biotechnol Biofuels

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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.

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Section: Resultsmentioning

confidence: 48%

Section: Introductionmentioning

confidence: 99%

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

Kadić

Várnai

Eijsink

et al. 2021

Biotechnol Biofuels

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“…In search of an explanation for this paradoxical finding, it is important to consider the limitations of the HRP/Amplex Red assay [ 29 ]. The assay is based on single electron oxidation of Amplex Red by HRP in the presence of H 2 O 2 as a co-substrate [ 30 ].…”

Section: Resultsmentioning

confidence: 99%

“…On another note, this study presents an illustration of the complex nature and limitations of the HRP/Amplex Red assay (see also [ 29 ]). We demonstrate that apparent H 2 O 2 production rates obtained by this method using low concentrations of reductants (a rather common approach employed in many LPMO studies) may not necessarily describe trends that exist in reactions on cellulose at standard aerobic conditions (i.e., at ≥ 1 mM reductant concentration).…”

Section: Discussionmentioning

confidence: 99%

Unraveling the roles of the reductant and free copper ions in LPMO kinetics

Stepnov

Forsberg

Sørlie

et al. 2021

Biotechnol Biofuels

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Background Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative depolymerization of industrially relevant crystalline polysaccharides, such as cellulose, in a reaction that depends on an electron donor and O2 or H2O2. While it is well known that LPMOs can utilize a wide variety of electron donors, the variation in reported efficiencies of various LPMO-reductant combinations remains largely unexplained. Results In this study, we describe a novel two-domain cellulose-active family AA10 LPMO from a marine actinomycete, which we have used to look more closely at the effects of the reductant and copper ions on the LPMO reaction. Our results show that ascorbate-driven LPMO reactions are extremely sensitive to very low amounts (micromolar concentrations) of free copper because reduction of free Cu(II) ions by ascorbic acid leads to formation of H2O2, which speeds up the LPMO reaction. In contrast, the use of gallic acid yields steady reactions that are almost insensitive to the presence of free copper ions. Various experiments, including dose–response studies with the enzyme, showed that under typically used reaction conditions, the rate of the reaction is limited by LPMO-independent formation of H2O2 resulting from oxidation of the reductant. Conclusion The strong impact of low amounts of free copper on LPMO reactions with ascorbic acid and O2, i.e. the most commonly used conditions when assessing LPMO activity, likely explains reported variations in LPMO rates. The observed differences between ascorbic acid and gallic acid show a way of making LPMO reactions less copper-dependent and illustrate that reductant effects on LPMO action need to be interpreted with great caution. In clean reactions, with minimized generation of H2O2, the (O2-driven) LPMO reaction is exceedingly slow, compared to the much faster peroxygenase reaction that occurs when adding H2O2.

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“… a The redox potentials for the Cu(II)/Cu(I) redox couples are indicated in the column headers. The signals obtained in the Amplex Red signal were corrected for the effect of ascorbic acid 28 and the rates were corrected for the rate in reactions with only ascorbic acid. …”

Section: Resultsmentioning

confidence: 99%

Kinetic Characterization of a Putatively Chitin-Active LPMO Reveals a Preference for Soluble Substrates and Absence of Monooxygenase Activity

et al. 2021

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Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are recognized as important contributors to aerobic enzymatic degradation of recalcitrant polysaccharides such as chitin and cellulose. LPMOs are remarkably abundant in nature, with some fungal species possessing more than 50 LPMO genes, and the biological implications of this diversity remain enigmatic. For example, chitin-active LPMOs have been encountered in biological niches where chitin conversion does not seem to take place. We have carried out an in-depth kinetic characterization of a putatively chitin-active LPMO from Aspergillus fumigatus ( Af AA11B), which, as we show here, has multiple unusual properties, such as a low redox potential and high oxidase activity. Furthermore, Af AA11B is hardly active on chitin, while being very active on soluble oligomers of N -acetylglucosamine. In the presence of chitotetraose, the enzyme can withstand considerable amounts of H 2 O 2 , which it uses to efficiently and stoichiometrically convert this substrate. The unique properties of Af AA11B allowed experiments showing that it is a strict peroxygenase and does not catalyze a monooxygenase reaction. This study shows that nature uses LPMOs for breaking glycosidic bonds in non-polymeric substrates in reactions that depend on H 2 O 2 . The quest for the true substrates of these enzymes, possibly carbohydrates in the cell wall of the fungus or its competitors, will be of major interest.

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Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs)

Cited by 80 publications

References 64 publications

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

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

Unraveling the roles of the reductant and free copper ions in LPMO kinetics

Kinetic Characterization of a Putatively Chitin-Active LPMO Reveals a Preference for Soluble Substrates and Absence of Monooxygenase Activity

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