Photodegradation in terrestrial ecosystems.

The first step in carbon (C) turnover, where senesced plant biomass is converted through various pathways into compounds that are released to the atmosphere or incorporated into the soil, is termed litter decomposition. This review is focused on recent advances of how solar radiation can affect this important process in terrestrial ecosystems. We explore the photochemical degradation of plant litter and its consequences for biotic decomposition and C cycling. The ubiquitous presence of lignin in plant tissues poses an important challenge for enzymatic litter decomposition due to its biological recalcitrance, creating a substantial bottleneck for decomposer organisms. The recognition that lignin is also photolabile and can be rapidly altered by natural doses of sunlight to increase access to cell wall carbohydrates and even bolster the activity of cell wall degrading enzymes highlights a novel role for lignin in modulating rates of litter decomposition. Lignin represents a key functional connector between photochemistry and biochemistry with important consequences for our understanding of how sunlight exposure may affect litter decomposition in a wide range of terrestrial ecosystems. A mechanistic understanding of how sunlight controls litter decomposition and C turnover can help inform management and other decisions related to mitigating human impact on the planet.

A modular enzyme with combined hemicellulose-removing and LPMO activity increases cellulose accessibility in softwood.

Because of the association with other complex polysaccharides, extracting and utilizing cellulose from lignocellulosic materials requires the combined action of a broad range of carbohydrate-active enzymes, including multiple glycoside hydrolases (GHs) and lytic polysaccharide monooxygenases (LPMOs). The interplay between these enzymes and the way in which Nature orchestrates their co-existence and combined action are topics of great scientific and industrial interest. To gain more insight into these issues, we have studied the lignocellulose-degrading abilities of an enzyme from Caldibacillus cellulovorans (CcLPMO10-Man5), comprising an LPMO domain, a GH5 mannanase domain and two family 3 carbohydrate-binding modules (CBM3). Using a natural softwood substrate, we show that this enzyme promotes cellulase activity, i.e., saccharification of cellulose, both by removing mannan covering the cellulose and by oxidatively breaking up the cellulose structure. Synergy with CcLPMO10-Man5 was most pronounced for two tested cellobiohydrolases, whereas effects were smaller for a tested endoglucanase, which is in line with the notion that cellobiohydrolases and LPMOs attack the same crystalline regions of the cellulose, whereas endoglucanases attack semi-crystalline and amorphous regions. Importantly, the LPMO domain of CcLPMO10-Man5 is incapable of accessing the softwood cellulose in absence of the mannanase domain. Considering that LPMOs not bound to a substrate are sensitive to autocatalytic inactivation, this intramolecular synergy provides a perfect rationale for the evolution of modular enzymes such as CcLPMO10-Man5. The intramolecular coupling of the LPMO with a mannanase and two CBMs ensures that the LPMO is directed to areas where mannans are removed and cellulose thus becomes available.

Heterologous Expression and Biochemical Characterization of a Novel Lytic Polysaccharide Monooxygenase from Chitinilyticum aquatile CSC-1.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the oxidative cleavage of recalcitrant polysaccharides. There are limited reports on LPMOs capable of concurrently catalyzing the oxidative cleavage of both cellulose and chitin. In this study, we identified and cloned a novel LPMO from the newly isolated bacterium Chitinilyticum aquatile CSC-1, designated as CaLPMO10. When using 2, 6-dimethylphenol (2, 6-DMP) as the substrate, CaLPMO10 exhibited optimal activity at 50 °C and pH 8, demonstrating good temperature stability at 30 °C. Even after a 6 h incubation at pH 8 and 30 °C, CaLPMO10 retained approximately 83.03 ± 1.25% residual enzyme activity. Most metal ions were found to enhance the enzyme activity of CaLPMO10, with ascorbic acid identified as the optimal reducing agent. Mass spectrometry analysis indicated that CaLPMO10 displayed oxidative activity towards both chitin and cellulose, identifying it as a C1/C4-oxidized LPMO. CaLPMO10 shows promise as a key enzyme for the efficient utilization of biomass resources in future applications.

Comparison of simultaneous saccharification and fermentation with LPMO-supported hybrid hydrolysis and fermentation.

Enzymatic saccharification is used to convert polysaccharides in lignocellulosic biomass to sugars which are then converted to ethanol or other bio-based fermentation products. The efficacy of commercial cellulase preparations can potentially increase if lytic polysaccharide monooxygenase (LPMO) is included. However, as LPMO requires both a reductant and an oxidant, such as molecular oxygen, a reevaluation of process configurations and conditions is warranted. Saccharification and fermentation of pretreated softwood was investigated in demonstration-scale experiments with 10 m3 bioreactors using an LPMO-containing cellulase preparation, a xylose-utilizing yeast, and either simultaneous saccharification and fermentation (SSF) or hybrid hydrolysis and fermentation (HHF) with a 24-hour or 48-hour initial phase and with 0.15 vvm aeration before addition of the yeast. The conditions used for HHF, especially with 48 h initial phase, resulted in better glucan conversion, but in poorer ethanol productivity and in poorer initial ethanol yield on consumed sugars than the SSF. In the SSF, hexose sugars such as glucose and mannose were consumed faster than xylose, but, in the end of the fermentation >90% of the xylose had been consumed. Chemical analysis of inhibitory pretreatment by-products indicated that the concentrations of heteroaromatic aldehydes (such as furfural), aromatic aldehydes, and an aromatic ketone decreased as a consequence of the aeration. This was attributed mainly to evaporation caused by the gas flow. The results indicate that further research is needed to fully exploit the advantages of LPMO without compromising fermentation conditions.

Characterization of a novel AA16 lytic polysaccharide monooxygenase from Thermothelomyces thermophilus and comparison of biochemical properties with an LPMO from AA9 family.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which are categorized in the CAZy database under auxiliary activities families AA9-11, 13, 14-17. Secreted by various microorganisms, they play a crucial role in carbon recycling, particularly in fungal saprotrophs. LPMOs oxidize polysaccharides through monooxygenase/peroxygenase activities and exhibit peroxidase and oxidase activities, with variations among different families. AA16, a newly identified LPMO family, is noteworthy due to limited studies on its members, thus rendering the characterization of AA16 enzymes vital for addressing controversies around their functions. This study focused on heterologous expression and biochemical study of an AA16 LPMO from Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila), namely MtLPMO16A. Substrate specificity evaluation of MtLPMO16A showed oxidative cleavage of hemicellulosic substrates and no activity on cellulose, accompanied by a strong oxidase activity. A comparative analysis with an LPMO from AA9 family explored correlations between these families, while MtLPMO16A was shown to boost the activity of some AA9 family LPMOs. The results offer new insights into the AA16 family's action mode and microbial hemicellulose decomposition mechanisms in nature.

Oxidized reducing ends in celluloses: Quantitative profiling relative to molar mass distribution by fluorescence labeling.

Fluorescence labeling with N-(1-naphthyl)ethylenediamine is highly effective for quantifying oxidized reducing end groups (REGs) in cellulosic materials. When combined with size exclusion chromatography in DMAc/LiCl, along with fluorescence / multiple-angle laser light scattering / refractive index detection, a detailed profile of C1-oxidized REGs relative to the molecular weight distribution of the cellulosic material can be obtained. In this work, the derivatization process was extensively optimized, to be carried out heterogeneously in the solvent N-methyl-2-pyrrolidone. Furthermore, we show that to achieve high selectivity for carboxyl groups at the C1 position, keto and aldehyde groups need to be selectively reduced (e.g., by NaBH4), and carboxyl groups other than at C1 need to be blocked (e.g., by methylation with (trimethylsilyl)diazomethane) prior to fluorescence labeling of carboxyl groups at C1 position. Finally, we demonstrate the practical value of the analytical method by measuring the content of the C1-oxidized REGs in cellulose samples after chemical (by Pinnick oxidation) or enzymatic (by treatment with C1-oxidizing LPMO enzymes) oxidation of various pulp samples.

Action of AA9 lytic polysaccharide monooxygenase enzymes on different cellulose allomorphs.

Lytic polysaccharide monooxygenase (LPMO)-catalyzed oxidative processes play a major role in natural biomass conversion. Despite their oxidative cleavage at the surface of polysaccharides, understanding of their mode of action, and the impact of structural patterns of the cellulose fiber on LPMO activity is still not fully understood. In this work, we investigated the action of two different LPMOs from Podospora anserina on celluloses showing different structural patterns. For this purpose, we prepared cellulose II and cellulose III allomorphs from cellulose I cotton linters, as well as amorphous cellulose. LPMO action was monitored in terms of surface morphology, molar mass changes and monosaccharide profile. Both PaLPMO9E and PaLPMO9H were active on the different cellulose allomorphs (I, II and III), and on amorphous cellulose (PASC) whereas they displayed a different behavior, with a higher molar mass decrease observed for cellulose I. Overall, the pretreatment with LPMO enzymes clearly increased the accessibility of all types of cellulose, which was quantified by the higher carboxylate content after carboxymethylation reaction on LPMO-pretreated celluloses. This work gives more insight into the action of LPMOs as a tool for deconstructing lignocellulosic biomass to obtain new bio-based building blocks.

Genome and secretome insights: unravelling the lignocellulolytic potential of Myceliophthora verrucosa for enhanced hydrolysis of lignocellulosic biomass.

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.

Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities.

BACKGROUND: The polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear. RESULTS: An LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid). CONCLUSIONS: This study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

A novel starch-active lytic polysaccharide monooxygenase discovered with bioinformatics screening and its application in textile desizing.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative cleavage of different types of polysaccharides have potential to be used in various industries. However, AA13 family LPMOs which specifically catalyze starch substrates have relatively less members than AA9 and AA10 families to limit their application range. Amylase has been used in enzymatic desizing treatment of cotton fabric for semicentury which urgently need for new assistant enzymes to improve reaction efficiency and reduce cost so as to promote their application in the textile industry. RESULTS: A total of 380 unannotated new genes which probably encode AA13 family LPMOs were discovered by the Hidden Markov model scanning in this study. Ten of them have been successfully heterologous overexpressed. AlLPMO13 with the highest activity has been purified and determined its optimum pH and temperature as pH 5.0 and 50 °C. It also showed various oxidative activities on different substrates (modified corn starch > amylose > amylopectin > corn starch). The results of enzymatic textile desizing application showed that the best combination of amylase (5 g/L), AlLPMO13 (5 mg/L), and H2O2 (3 g/L) made the desizing level and the capillary effects increased by 3 grades and more than 20%, respectively, compared with the results treated by only amylase. CONCLUSION: The Hidden Markov model constructed basing on 34 AA13 family LPMOs was proved to be a valid bioinformatics tool for discovering novel starch-active LPMOs. The novel enzyme AlLPMO13 has strong development potential in the enzymatic textile industry both concerning on economy and on application effect.

Heterologously Expressed Cellobiose Dehydrogenase Acts as Efficient Electron-Donor of Lytic Polysaccharide Monooxygenase for Cellulose Degradation in Trichoderma reesei.

The conversion of lignocellulosic biomass to second-generation biofuels through enzymes is achieved at a high cost. Filamentous fungi through a combination of oxidative enzymes can easily disintegrate the glycosidic bonds of cellulose. The combination of cellobiose dehydrogenase (CDH) with lytic polysaccharide monooxygenases (LPMOs) enhances cellulose degradation in many folds. CDH increases cellulose deconstruction via coupling the oxidation of cellobiose to the reductive activation of LPMOs by catalyzing the addition of oxygen to C-H bonds of the glycosidic linkages. Fungal LPMOs show different regio-selectivity (C1 or C4) and result in oxidized products through modifications at reducing as well as nonreducing ends of the respective glucan chain. T. reesei LPMOs have shown great potential for oxidative cleavage of cellobiose at C1 and C4 glucan bonds, therefore, the incorporation of heterologous CDH further increases its potential for biofuel production for industrial purposes at a reduced cost. We introduced CDH of Phanerochaete chrysosporium (PcCDH) in Trichoderma reesei (which originally lacked CDH). We purified CDH through affinity chromatography and analyzed its enzymatic activity, electron-donating ability to LPMO, and the synergistic effect of LPMO and CDH on cellulose deconstruction. The optimum temperature of the recombinant PcCDH was found to be 45 °C and the optimum pH of PcCDH was observed as 4.5. PcCDH has high cello-oligosaccharide kcat, Km, and kcat/Km values. The synergistic effect of LPMO and cellulase significantly improved the degradation efficiency of phosphoric acid swollen cellulose (PASC) when CDH was used as the electron donor. We also found that LPMO undergoes auto-oxidative inactivation, and when PcCDH is used an electron donor has the function of a C1-type LPMO electron donor without additional substrate increments. This work provides novel insights into finding stable electron donors for LPMOs and paves the way forward in discovering efficient CDHs for enhanced cellulose degradation.

Influences of the Carbohydrate-Binding Module on a Fungal Starch-Active Lytic Polysaccharide Monooxygenase.

Noncatalytic carbohydrate-binding modules (CBMs) play important roles in the function of lytic polysaccharide monooxygenases (LPMOs) but have not been well demonstrated for starch-active AA13 LPMO. In this study, four new CBMs were investigated systematically for their influence on MtLPMO toward starch in terms of substrate binding, H2O2 production activity, oxidative product yields, and the degradation effect with α-amylase and glucoamylase toward different starch substrates. Among the four MtLPMO-CBM chimeras, MtLPMO-CnCBM harboring the CBM fromColletotrichum nymphaeae showed the highest substrate binding toward different types of starch compared to MtLPMO without CBM. MtLPMO-PvCBM harboring the CBM from Pseudogymnoascus verrucosus and MtLPMO-CnCBM showed dramatically enhanced H2O2 production activity of 4.6-fold and 3.6-fold, respectively, than MtLPMO without CBM. More importantly, MtLPMO-CBM generated more oxidative products from starch polysaccharides degradation than MtLPMO alone, with 6.0-fold and 4.6-fold enhancement obtained from the oxidation of amylopectin and corn starch with MtLPMO-CnCBM, and a 5.2-fold improvement obtained with MtLPMO-AcCBM for amylose. MtLPMO-AcCBM significantly boosted the yields of reducing sugar with α-amylase upon degrading amylopectin and corn starch. These findings demonstrate that CBMs greatly influence the performance of starch-active AA13 LPMOs due to their enhanced binding and H2O2 production activity.

LPMO-supported saccharification of biomass: effects of continuous aeration of reaction mixtures with variable fractions of water-insoluble solids and cellulolytic enzymes.

BACKGROUND: High substrate concentrations and high sugar yields are important aspects of enzymatic saccharification of lignocellulosic substrates. The benefit of supporting the catalytic action of lytic polysaccharide monooxygenase (LPMO) through continuous aeration of slurries of pretreated softwood was weighed against problems associated with increasing substrate content (quantitated as WIS, water-insoluble solids, in the range 12.5-17.5%), and was compared to the beneficial effect on the saccharification reaction achieved by increasing the enzyme preparation (Cellic CTec3) loadings. Aerated reactions were compared to reactions supplied with N2 to assess the contribution of LPMO to the saccharification reactions. Analysis using 13C NMR spectroscopy, XRD, Simons' staining, BET analysis, and SEM analysis was used to gain further insights into the effects of the cellulolytic enzymes on the substrate under different reaction conditions. RESULTS: Although glucose production after 72 h was higher at 17.5% WIS than at 12.5% WIS, glucan conversion decreased with 24% (air) and 17% (N2). Compared to reactions with N2, the average increases in glucose production for aerated reactions were 91% (12.5% WIS), 70% (15.0% WIS), and 67% (17.5% WIS). Improvements in glucan conversion through aeration were larger (55-86%) than the negative effects of increasing WIS content. For reactions with 12.5% WIS, increased enzyme dosage with 50% improved glucan conversion with 25-30% for air and N2, whereas improvements with double enzyme dosage were 30% (N2) and 39% (air). Structural analyses of the solid fractions revealed that the enzymatic reaction, particularly with aeration, created increased surface area (BET analysis), increased disorder (SEM analysis), decreased crystallinity (XRD), and increased dye adsorption based on the cellulose content (Simons' staining). CONCLUSIONS: The gains in glucan conversion with aeration were larger than the decreases observed due to increased substrate content, resulting in higher glucan conversion when using aeration at the highest WIS value than when using N2 at the lowest WIS value. The increase in glucan conversion with double enzyme preparation dosage was smaller than the increase achieved with aeration. The results demonstrate the potential in using proper aeration to exploit the inherent capacity of LPMO in enzymatic saccharification of lignocellulosic substrates and provide detailed information about the characteristics of the substrate after interaction with cellulolytic enzymes.

Lytic Polysaccharide Monooxygenase Activity of Tma12 Is Critical for Its Toxicity to Whitefly.

Lytic polysaccharide monooxygenases (LPMOs) are powerful redox enzymes that transform complex carbohydrates through oxidation and make them suitable for saccharification by canonical hydrolases. Due to this property, LPMOs are considered to be a valuable component of enzymatic consortia for industrial biorefineries. Tma12 is a fern entomotoxic protein that kills whitefly and has structural similarities with chitinolytic LPMO. However, its enzymatic activity is poorly understood. Studying the role of the LPMO-like activity in the insecticidal function of Tma12 can be of considerable importance. Our results show that Tma12 preferentially binds and digests ß-chitin. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis shows that the digestion of chitin produces chitin oligosaccharides of various lengths (DP2-DP7). The Michaelis constant (km) and catalytic constant (kcat) for hydrocoerulignone are 0.022 mM and 0.044 s-1, respectively. The attenuation of catalytic activity through diethylpyrocarbonate modification abolishes the insecticidal activity of the protein. Our findings reveal that (a) Tma12 is an active LPMO and (b) LPMO activity is indispensable for its function as a bioinsecticide.

Lytic polysaccharide monooxygenase activity increases productive binding capacity of cellobiohydrolases on cellulose.

Cellobiohydrolases are crucial for cellulose breakdown, but their efficiency on crystalline cellulose is hampered by limited access to single chain ends to initiate hydrolysis. As a result, they depend on enzymes like lytic polysaccharide monooxygenases (LPMOs), which directly target the crystalline cellulose surface. This study investigated how LPMO pretreatment affected the productive binding capacity of a Trichoderma longibrachiatum cellobiohydrolase, TlCBHI, on crystalline cellulose by applying an amperometric cellobiose dehydrogenase biosensor. After the 24-hour of LPMO pretreatment, the productive binding capacity of TlCBHI significantly increased in all reactions. However, with a shorter 5-hour LPMO pretreatment, minimal to no effect on productive binding capacity was observed. Of note, all LPMO reactions were inactivated around this time point. This delayed LPMO effect suggests that the improved binding capacity for cellulases does not directly result from cellulose chain cleavage by LPMOs but rather from the cellulose decrystallization following the oxidative cleavage.

The effect of linker conformation on performance and stability of a two-domain lytic polysaccharide monooxygenase.

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the WT enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using small-angle X-ray scattering, NMR, molecular dynamics simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the WT and a serine-rich variant, which have the same linker length, demonstrated better performance compared with those with either a shorter linker or a longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, whereas the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass-processing enzymes is more complex than mere structural tethering.

Revisiting the AA14 family of lytic polysaccharide monooxygenases and their catalytic activity.

Lytic polysaccharide monooxygenases (LPMOs) belonging to the AA14 family are believed to contribute to the enzymatic degradation of lignocellulosic biomass by specifically acting on xylan in recalcitrant cellulose-xylan complexes. Functional characterization of an AA14 LPMO from Trichoderma reesei, TrAA14A, and a re-evaluation of the properties of the previously described AA14 from Pycnoporus coccineus, PcoAA14A, showed that these proteins have oxidase and peroxidase activities that are common for LPMOs. However, we were not able to detect activity on cellulose-associated xylan or any other tested polysaccharide substrate, meaning that the substrate of these enzymes remains unknown. Next to raising questions regarding the true nature of AA14 LPMOs, the present data illustrate possible pitfalls in the functional characterization of these intriguing enzymes.

Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification.

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.

Synergetic effect of lytic polysaccharide monooxygenase from Thermobifida fusca on saccharification of agrowastes.

Saccharification is one of the most noteworthy processes in biomass-based biorefineries. In particular, the lytic polysaccharide monooxygenase has recently emerged as an oxidative cleavage-recalcitrant polysaccharide; however, there is insufficient information regarding its application to actual biomass. Accordingly, this study focused optimizing the recombinant expression level of a bacterial lytic polysaccharide monooxygenase from Thermobifida fusca (TfLPMO), which was characterized as a cellulolytic enzyme. Finally, the synergistic effect of the lytic polysaccharide monooxygenase and a commercial cellulase cocktail on the saccharification of agrowaste was investigated. TfLPMO functioned on various cellulosic and hemicellulosic substrates, and the combination of TfLPMO with cellulase exhibited a synergistic effect on the saccharification of agrowastes, resulting in a 19.2% and 14.1% increase in reducing sugars from rice straw and corncob, respectively. The results discussed herein can lead to an in-depth understanding of enzymatic saccharification and suggest viable options for valorizing agrowastes as renewable feedstocks in biorefineries.

Reductants fuel lytic polysaccharide monooxygenase activity in a pH-dependent manner.

Polysaccharide-degrading mono-copper lytic polysaccharide monooxygenases (LPMOs) are efficient peroxygenases that require electron donors (reductants) to remain in the active Cu(I) form and to generate the H2 O2 co-substrate from molecular oxygen. Here, we show how commonly used reductants affect LPMO catalysis in a pH-dependent manner. Between pH 6.0 and 8.0, reactions with ascorbic acid show little pH dependency, whereas reactions with gallic acid become much faster at increased pH. These dependencies correlate with the reductant ionization state, which affects its ability to react with molecular oxygen and generate H2 O2 . The correlation does not apply to l-cysteine because, as shown by stopped-flow kinetics, increased H2 O2 production at higher pH is counteracted by increased binding of l-cysteine to the copper active site. The findings highlight the importance of the choice of reductant and pH in LPMO reactions.