The synergy between LPMOs and cellulases in enzymatic saccharification of cellulose is both enzyme- and substrate-dependent

Tokin, Radina; Ipsen, Johan Ø.; Westh, Peter; Johansen, Katja Salomon

doi:10.1007/s10529-020-02922-0

Cited by 71 publications

(68 citation statements)

References 31 publications

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“…The mechanism for the impeding effect has never been investigated, but different interpretations have been put forward. Current suggestions that could explain the impeding effect are: enzyme-enzyme competition for sites (17), enzymes blocking the activity of each other, oxidative inactivation (21,39), and inability or reduced ability of the cellobiohydrolases to attack C1-oxidized cellulose chain ends (22,23). In this present study, we investigated the mechanism behind this undesirable effect.…”

Section: Discussionmentioning

confidence: 91%

“…While the boosting effect of LPMOs is well documented on cellulase cocktails, pronounced impeding effects have been reported between C1-oxidizing LPMOs and reducing endcellobiohydrolases from Trichoderma spp. (17,(21)(22)(23). This may be of high industrial relevance as GH7 cellobiohydrolases are often the most prevalent members of fungal cellulolytic cocktails (24).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to the common perception of synergistic effects between LPMOs and glycoside hydrolases, a few studies have reported pronounced impeding effects of C1-oxidizing LPMOs on the activity of GH7 (reducing end) cellobiohydrolases from Trichoderma spp. (17,(21)(22)(23). This may have important implications, as GH7 cellobiohydrolases make up a significant part of industrial cellulolytic cocktails (24).…”

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confidence: 99%

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A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases

Keller

Badino

Røjel

et al. 2021

Journal of Biological Chemistry

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Lytic polysaccharide monooxygenases (LPMOs) are known to act synergistically with glycoside hydrolases in industrial cellulolytic cocktails. However, a few studies have reported severe impeding effects of C1-oxidizing LPMOs on the activity of reducing-end cellobiohydrolases. The mechanism for this effect remains unknown, but it may have important implications as reducing-end cellobiohydrolases make up a significant part of such cocktails. To elucidate whether the impeding effect is general for different reducing-end cellobiohydrolases and study the underlying mechanism, we conducted a comparative biochemical investigation of the cooperation between a C1-oxidizing LPMO from Thielavia terrestris and three reducing-end cellobiohydrolases; Trichoderma reesei ( Tr Cel7A), T. terrestris ( Tt Cel7A), and Myceliophthora heterothallica ( Mh Cel7A). The enzymes were heterologously expressed in the same organism and thoroughly characterized biochemically. The data showed distinct differences in synergistic effects between the LPMO and the cellobiohydrolases; Tr Cel7A was severely impeded, Tt Cel7A was moderately impeded, while Mh Cel7A was slightly boosted by the LPMO. We investigated effects of C1-oxidations on cellulose chains on the activity of the cellobiohydrolases and found reduced activity against oxidized cellulose in steady-state and pre-steady-state experiments. The oxidations led to reduced maximal velocity of the cellobiohydrolases and reduced rates of substrate complexation. The extent of these effects differed for the cellobiohydrolases and scaled with the extent of the impeding effect observed in the synergy experiments. Based on these results, we suggest that C1-oxidized chain ends are poor attack sites for reducing-end cellobiohydrolases. The severity of the impeding effects varied considerably among the cellobiohydrolases, which may be relevant to consider for optimization of industrial cocktails.

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Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases

Keller

Badino

Røjel

et al. 2021

Journal of Biological Chemistry

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“…Recent insights concerning the role of H 2 O 2 and enzyme inactivation suggest that so far, we have not harnessed the full potential of LPMOs. Furthermore, despite much research on LPMOs in the past decade, exactly how these enzymes co-operate with classical cellulases remains largely unknown (see [343] for a recent study). Finally, recent work suggests that LPMOs could play a role in removing (traces of) recalcitrant hemicellulose, which may promote cellulolytic processes [68,150].…”

Section: Discussionmentioning

confidence: 99%

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

Østby

Hansen

Horn

et al. 2020

Journal of Industrial Microbiology and Biotechnology

136

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Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies and different enzyme process setups and present an in-depth discussion of the various enzyme types that are currently in use. We pay ample attention to the role of the recently discovered lytic polysaccharide monooxygenases (LPMOs), which have led to renewed interest in the role of redox enzyme systems in lignocellulose processing. Better understanding of the interplay between the various enzyme types, as they may occur in a commercial enzyme cocktail, is likely key to further process improvements.

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“…The same inhibitory effect of LPMOs has been observed on rice straw, while addition of AfCel6A almost did not impact the release of reducing sugars (Figure 8c). The divergent results among the three agricultural residues pointed to the substrate-dependence and substrate specificity of AfCel6A and AfAA9_B synergism with cellulases [78]. Compared to Af-EGL7 alone, the association between Af-EGL7 and AfCel6A increased the amount of reducing sugars released from the three biomasses: ~ 163%, ~ 118%, and ~ 88% for SEB (Figure 9a), corncob (Figure 9b), and rice straw (Figure 9c), respectively, after 48 h. The combination of Af-EGL7 and AfAA9_B also improved hydrolysis of SEB and corncob, but it had no effect on rice straw degradation.…”

Section: Synergistic Action On Cellulose Hydrolysismentioning

confidence: 96%

LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Bost Enzymatic Saccharification Activity of Cellulase Cocktail

Dinamarco¹,

Bernardi²,

Gerolamo³

et al. 2020

Preprint

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Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Because breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from A. fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (kcat of 147.9 s-1 and 0.64 s-1, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. Association of AfCel6A and AfAA9_B inhibits the activity of AfCel6A, an outcome that needs to be further investigated. However, addition of AfCel6A or AfAA9_B boosts the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase Af-EGL7. The supplementation of an enzymatic cocktail with AfCel6A or AfAA9_B boosted the yield of fermentable sugars from complex substrates, especially sugarcane exploded bagasse, by up to 95%. The synergism between the cellulase cocktail and AfAA9_B is enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass.

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The synergy between LPMOs and cellulases in enzymatic saccharification of cellulose is both enzyme- and substrate-dependent

Cited by 71 publications

References 31 publications

A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases

A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Bost Enzymatic Saccharification Activity of Cellulase Cocktail

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