| 1. |
- Costa, Thales H.F., et al.
(författare)
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Demonstration-scale enzymatic saccharification of sulfite-pulped spruce with addition of hydrogen peroxide for LPMO activation
- 2020
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Ingår i: Biofuels, Bioproducts and Biorefining. - : Wiley. - 1932-104X .- 1932-1031. ; 14:4, s. 734-745
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Tidskriftsartikel (refereegranskat)abstract
- The saccharification of lignocellulosic materials like Norway spruce is challenging due to the recalcitrant nature of the biomass, and it requires optimized and efficient pretreatment and enzymatic hydrolysis processes to make it industrially feasible. In this study, we report successful enzymatic saccharification of sulfite-pulped spruce (Borregaard's BALI™ process) at demonstration scale, achieved through the controlled delivery of hydrogen peroxide (H2O2) for the activation of lytic polysaccharide monooxygenases (LPMOs) present in the cellulolytic enzyme preparation. We achieved 85% saccharification yield in 4 days using industrially relevant conditions – that is, an enzyme dose of 4% (w/w dry matter of substrate) of the commercial cellulase cocktail Cellic CTec3 and a substrate loading of 12% (w/w). Addition of H2O2 and the resulting controlled and high LPMO activity had a positive effect on the rate of saccharification and the final sugar titer. Clearly, the high LPMO activity was dependent on feeding the reactors with the LPMO co-substrate H2O2, as in situ generation of H2O2 from molecular oxygen was limited. These demonstration-scale experiments provide a solid basis for the use of H2O2 to improve enzymatic saccharification of lignocellulosic biomass at large industrial scale.
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| 2. |
- Hegnar, Olav A., et al.
(författare)
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Quantifying Oxidation of Cellulose-Associated Glucuronoxylan by Two Lytic Polysaccharide Monooxygenases from Neurospora crassa
- 2021
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Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 87:24
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Tidskriftsartikel (refereegranskat)abstract
- Family AA9 lytic polysaccharide monooxygenases (LPMOs) are abundant in fungi, where they catalyze oxidative depolymerization of recalcitrant plant biomass. These AA9 LPMOs cleave cellulose and some also act on hemicelluloses, primarily other (substituted) beta-(1 -> 4)-glucans. Oxidative cleavage of xylan has been shown for only a few AA9 LPMOs, and it remains unclear whether this activity is a minor side reaction or primary function. Here, we show that Neurospora crassa LPMO9F (NcLPMO9F) and the phylogenetically related, hitherto uncharacterized NcLPMO9L from N. crassa are active on both cellulose and cellulose-associated glucuronoxylan but not on glucuronoxylan alone. A newly developed method for simultaneous quantification of xylan-derived and cellulose-derived oxidized products showed that NcLPMO9F preferentially cleaves xylan when acting on a cellulosebeechwood glucuronoxylan mixture, yielding about three times more xylan-derived than cellulose-derived oxidized products. Interestingly, under similar conditions, NcLPMO9L and the previously characterized McLPMO9H, from Malbranchea cinnamomea, showed different xylan-to-cellulose preferences, giving oxidized product ratios of about 0.5:1 and 1:1, respectively, indicative of functional variation among xylanactive LPMOs. Phylogenetic and structural analysis of xylan-active AA9 LPMOs led to the identification of characteristic structural features, including unique features that do not occur in phylogenetically remote AA9 LPMOs, such as four AA9 LPMOs whose lack of activity toward glucuronoxylan was demonstrated in the present study. Taken together, the results provide a path toward discovery of additional xylanactive LPMOs and show that the huge family of AA9 LPMOs has members that preferentially act on xylan. These findings shed new light on the biological role and industrial potential of these fascinating enzymes. IMPORTANCE Plant cell wall polysaccharides are highly resilient to depolymerization by hydrolytic enzymes, partly due to cellulose chains being tightly packed in microfibrils that are covered by hemicelluloses. Lytic polysaccharide monooxygenases (LPMOs) seem well suited to attack these resilient copolymeric structures, but the occurrence and importance of hemicellulolytic activity among LPMOs remain unclear. Here, we show that certain AA9 LPMOs preferentially cleave xylan when acting on a cellulose-glucuronoxylan mixture, and that this ability is the result of protein evolution that has resulted in a clade of AA9 LPMOs with specific structural features. Our findings strengthen the notion that the vast arsenal of AA9 LPMOs in certain fungal species provides functional versatility and that AA9 LPMOs may have evolved to promote oxidative depolymerization of a wide variety of recalcitrant, copolymeric plant polysaccharide structures. These findings have implications for understanding the biological roles and industrial potential of LPMOs.
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| 3. |
- Hegnar, Olav, et al.
(författare)
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Challenges and opportunities in mimicking non-enzymatic brown-rot decay mechanisms for pretreatment of Norway spruce
- 2019
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Ingår i: Wood Science and Technology. - : Springer Science and Business Media LLC. - 0043-7719 .- 1432-5225. ; 53:2, s. 291-311
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Tidskriftsartikel (refereegranskat)abstract
- The recalcitrance bottleneck of lignocellulosic materials presents a major challenge for biorefineries, including second-generation biofuel production. Because of their abundance in the northern hemisphere, softwoods, such as Norway spruce, are of major interest as a potential feedstock for biorefineries. In nature, softwoods are primarily degraded by basidiomycetous fungi causing brown rot. These fungi employ a non-enzymatic oxidative system to depolymerize wood cell wall components prior to depolymerization by a limited set of hydrolytic and oxidative enzymes. Here, it is shown that Norway spruce pretreated with two species of brown-rot fungi yielded more than 250% increase in glucose release when treated with a commercial enzyme cocktail and that there is a good correlation between mass loss and the degree of digestibility. A series of experiments was performed aimed at mimicking the brown-rot pretreatment, using a modified version of the Fenton reaction. A small increase in digestibility after pretreatment was shown where the aim was to generate reactive oxygen species within the wood cell wall matrix. Further experiments were performed to assess the possibility of performing pretreatment and saccharification in a single system, and the results indicated the need for a complete separation of oxidative pretreatment and saccharification. A more severe pretreatment was also completed, which interestingly did not yield a more digestible material. It was concluded that a biomimicking approach to pretreatment of softwoods using brown-rot fungal mechanisms is possible, but that there are additional factors of the system that need to be known and optimized before serious advances can be made to compete with already existing pretreatment methods.
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| 4. |
- Hüttner, Silvia, 1984, et al.
(författare)
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Specific xylan activity revealed for AA9 Lytic Polysaccharide Monooxygenases of the thermophilic fungus Malbranchea cinnamomea by functional characterization
- 2019
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Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 85:23
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Tidskriftsartikel (refereegranskat)abstract
- The thermophilic biomass-degrader Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight ( M. cinnamomea AA9s) Mc AA9s, namely, Mc AA9A, Mc AA9B, Mc AA9F, and Mc AA9H, to gain a deeper understanding about their roles in the fungus. The characterized Mc AA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the Mc AA9s, Mc AA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, Mc AA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. Mc AA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers’ physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall.
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| 5. |
- Kadić, Adnan, et al.
(författare)
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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
- 2021
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 14:1
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Tidskriftsartikel (refereegranskat)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.
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| 6. |
- Penttilä, Paavo A., et al.
(författare)
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Xylan as limiting factor in enzymatic hydrolysis of nanocellulose
- 2013
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Ingår i: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 129, s. 135-141
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Tidskriftsartikel (refereegranskat)abstract
- The role of xylan as a limiting factor in the enzymatic hydrolysis of cellulose was studied by hydrolysing nanocellulose samples prepared by mechanical fibrillation of birch pulp with varying xylan content. Analyzing the nanocelluloses and their hydrolysis residues with dynamic FT-IR spectroscopy revealed that a certain fraction of xylan remained tightly attached to cellulose fibrils despite partial hydrolysis of xylan with xylanase prior to pulp fibrillation and that this fraction remained in the structure during the hydrolysis of nanocellulose with cellulase mixture as well. Thus, a loosely bound fraction of xylan was predicted to have been more likely removed by purified xylanase. The presence of loosely bound xylan seemed to limit the hydrolysis of crystalline cellulose, indicated by an increase in cellulose crystallinity and by preserved crystal width measured with wide-angle X-ray scattering. Removing loosely bound xylan led to a proportional hydrolysis of xylan and cellulose with the cellulase mixture.
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| 7. |
- Tõlgo, Monika, 1994, et al.
(författare)
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Comparison of Six Lytic Polysaccharide Monooxygenases from Thermothielavioides terrestris Shows That Functional Variation Underlies the Multiplicity of LPMO Genes in Filamentous Fungi
- 2022
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 1098-5336 .- 0099-2240. ; 88:6
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Tidskriftsartikel (refereegranskat)abstract
- Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that oxidatively degrade various polysaccharides. Genes encoding LPMOs in the AA9 family are abundant in filamentous fungi while their multiplicity remains elusive. We describe a detailed functional characterization of six AA9 LPMOs from the ascomycetous fungus Thermothielavioides terrestris LPH172 (syn. Thielavia terrestris). These six LPMOs were shown to be upregulated during growth on different lignocellulosic substrates in our previous study. Here, we produced them heterologously in Pichia pastoris and tested their activity on various model and native plant cell wall substrates. All six T. terrestris AA9 (TtAA9) LPMOs produced hydrogen peroxide in the absence of polysaccharide substrate and displayed peroxidase-like activity on a model substrate, yet only five of them were active on selected cellulosic substrates. TtLPMO9A and TtLPMO9E were also active on birch acetylated glucuronoxylan, but only when the xylan was combined with phosphoric acid-swollen cellulose (PASC). Another of the six AA9s, TtLPMO9G, was active on spruce arabinoglucuronoxylan mixed with PASC. TtLPMO9A, TtLPMO9E, TtLPMO9G, and TtLPMO9T could degrade tamarind xyloglucan and, with the exception of TtLPMO9T, beechwood xylan when combined with PASC. Interestingly, none of the tested enzymes were active on wheat arabinoxylan, konjac glucomannan, acetylated spruce galactoglucomannan, or cellopentaose. Overall, these functional analyses support the hypothesis that the multiplicity of the fungal LPMO genes assessed in this study relates to the complex and recalcitrant structure of lignocellulosic biomass. Our study also highlights the importance of using native substrates in functional characterization of LPMOs, as we were able to demonstrate distinct, previously unreported xylan-degrading activities of AA9 LPMOs using such substrates.
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