Natural photoredox catalysts promote light-driven lytic polysaccharide monooxygenase reactions and enzymatic turnover of biomass.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of crystalline polysaccharides such as cellulose and chitin and are important for biomass conversion in the biosphere as well as in biorefineries. The target polysaccharides of LPMOs naturally occur in copolymeric structures such as plant cell walls and insect cuticles that are rich in phenolic compounds, which contribute rigidity and stiffness to these materials. Since these phenolics may be photoactive and since LPMO action depends on reducing equivalents, we hypothesized that LPMOs may enable light-driven biomass conversion. Here, we show that redox compounds naturally present in shed insect exoskeletons enable harvesting of light energy to drive LPMO reactions and thus biomass conversion. The primary underlying mechanism is that irradiation of exoskeletons with visible light leads to the generation of H2O2, which fuels LPMO peroxygenase reactions. Experiments with a cellulose model substrate show that the impact of light depends on both light and exoskeleton dosage and that light-driven LPMO activity is inhibited by a competing H2O2-consuming enzyme. Degradation experiments with the chitin-rich exoskeletons themselves show that solubilization of chitin by a chitin-active LPMO is promoted by light. The fact that LPMO reactions, and likely reactions catalyzed by other biomass-converting redox enzymes, are fueled by light-driven abiotic reactions in nature provides an enzyme-based explanation for the known impact of visible light on biomass conversion.

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.

Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase.

Lytic Polysaccharide Monooxygenases (LPMOs) are copper-dependent enzymes that play a pivotal role in the enzymatic conversion of the most recalcitrant polysaccharides, such as cellulose and chitin. Hence, protein engineering is highly required to enhance their catalytic efficiencies. To this effect, we optimized the protein sequence encoding for an LPMO from Bacillus amyloliquefaciens (BaLPMO10A) using the sequence consensus method. Enzyme activity was determined using the chromogenic substrate 2,6-Dimethoxyphenol (2,6-DMP). Compared with the wild type (WT), the variants exhibit up to a 93.7% increase in activity against 2,6-DMP. We also showed that BaLPMO10A can hydrolyze p-nitrophenyl-ß-D-cellobioside (PNPC), carboxymethylcellulose (CMC), and phosphoric acid-swollen cellulose (PASC). In addition to this, we investigated the degradation potential of BaLPMO10A against various substrates such as PASC, filter paper (FP), and Avicel, in synergy with the commercial cellulase, and it showed up to 2.7-, 2.0- and 1.9-fold increases in production with the substrates PASC, FP, and Avicel, respectively, compared to cellulase alone. Moreover, we examined the thermostability of BaLPMO10A. The mutants exhibited enhanced thermostability with an apparent melting temperature increase of up to 7.5 °C compared to the WT. The engineered BaLPMO10A with higher activity and thermal stability provides a better tool for cellulose depolymerization.

The Role of the Residue at Position 2 in the Catalytic Activity of AA9 Lytic Polysaccharide Monooxygenases.

AA9 lytic polysaccharide monooxygenases (LPMOs) are copper-dependent metalloenzymes that play a major role in cellulose degradation and plant infection. Understanding the AA9 LPMO mechanism would facilitate the improvement of plant pathogen control and the industrial application of LPMOs. Herein, via point mutation, we investigated the role of glycine 2 residue in cellulose degradation by Thermoascus aurantiacus AA9 LPMOs (TaAA9). A computational simulation showed that increasing the steric properties of this residue by replacing glycine with threonine or tyrosine altered the H-bonding network of the copper center and copper coordination geometry, decreased the surface charge of the catalytic center, weakened the TaAA9-substrate interaction, and enhanced TaAA9-product binding. Compared with wild-type TaAA9, G2T-TaAA9 and G2Y-TaAA9 variants showed attenuated copper affinity, reduced oxidative product diversity and decreased substrate Avicel binding, as determined using ITC, MALDI-TOF/TOF MS and cellulose binding analyses, respectively. Consistently, the enzymatic activity and synergy with cellulase of the G2T-TaAA9 and G2Y-TaAA9 variants were lower than those of TaAA9. Hence, the investigated residue crucially affects the catalytic activity of AA9 LPMOs, and we propose that the electropositivity of copper may correlate with AA9 LPMO activity. Thus, the relationship among the amino acid at position 2, surface charge and catalytic activity may facilitate an understanding of the proteins in AA9 LPMOs.

Comparison of Six Lytic Polysaccharide Monooxygenases from Thermothielavioides terrestris Shows That Functional Variation Underlies the Multiplicity of LPMO Genes in Filamentous Fungi.

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. IMPORTANCE The discovery of LPMOs in 2010 has revolutionized the industrial biotechnology field, mainly by increasing the efficiency of cellulolytic enzyme cocktails. Nonetheless, the biological purpose of the multiplicity of LPMO-encoding genes in filamentous fungi has remained an open question. Here, we address this point by showing that six AA9 LPMOs from a single fungal strain have various substrate preferences and activities on tested cellulosic and hemicellulosic substrates, including several native xylan substrates. Importantly, several of these activities could only be detected when using copolymeric substrates that likely resemble plant cell walls more than single fractionated polysaccharides do. Our results suggest that LPMOs have evolved to contribute to the degradation of different complex structures in plant cell walls where different biomass polymers are closely associated. This knowledge together with the elucidated novel xylanolytic activities could aid in further optimization of enzymatic cocktails for efficient degradation of lignocellulosic substrates and more.

On the expansion of biological functions of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) constitute an enigmatic class of enzymes, the discovery of which has opened up a new arena of riveting research. LPMOs can oxidatively cleave the glycosidic bonds found in carbohydrate polymers enabling the depolymerisation of recalcitrant biomasses, such as cellulose or chitin. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. In the present review, we propose a historical perspective of LPMO research providing a succinct overview of the major achievements of LPMO research over the past decade. This journey through LPMOs landscape leads us to dive into the emerging biological functions of LPMOs and LPMO-like proteins. We notably highlight roles in fungal and oomycete plant pathogenesis (e.g. potato late blight), but also in mutualistic/commensalism symbiosis (e.g. ectomycorrhizae). We further present the potential importance of LPMOs in other microbial pathogenesis including diseases caused by bacteria (e.g. pneumonia), fungi (e.g. human meningitis), oomycetes and viruses (e.g. entomopox), as well as in (micro)organism development (including several plant pests). Our assessment of the literature leads to the formulation of outstanding questions, promising for the coming years exciting research and discoveries on these moonlighting proteins.

Chitin Biodegradation by Lytic Polysaccharide Monooxygenases from Streptomyces coelicolor In Vitro and In Vivo.

Lytic polysaccharide monooxygenases (LPMOs) have the potential to improve recalcitrant polysaccharide hydrolysis by the oxidizing cleavage of glycosidic bond. Streptomyces species are major chitin decomposers in soil ecological environments and encode multiple lpmo genes. In this study, we demonstrated that transcription of the lpmo gene, Sclpmo10G, in the Streptomyces coelicolor A3(2) (ScA3(2)) strain is strongly induced by chitin. The ScLPMO10G protein was further expressed in Escherichia coli and characterized in vitro. The ScLPMO10G protein showed oxidation activity towards chitin. Chitinase synergy experiments demonstrated that the addition of ScLPMO10G resulted in a substantial in vitro increase in the reducing sugar levels. Moreover, in vivo the LPMO-overexpressing strain ScΔLPMO10G(+) showed stronger chitin-degrading ability than the wild-type, leading to a 2.97-fold increase in reducing sugar level following chitin degradation. The total chitinase activity of ScΔLPMO10G(+) was 1.5-fold higher than that of ScA3(2). In summary, ScLPMO10G may play a role in chitin biodegradation in S. coelicolor, which could have potential applications in biorefineries.

Quantifying Oxidation of Cellulose-Associated Glucuronoxylan by Two Lytic Polysaccharide Monooxygenases from Neurospora crassa.

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) ß-(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 cellulose-beechwood 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 xylan-active 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 xylan-active 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.

Functional characterization of cellulose-degrading AA9 lytic polysaccharide monooxygenases and their potential exploitation.

Cellulose-degrading auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are known to be widely distributed among filamentous fungi and participate in the degradation of lignocellulose via the oxidative cleavage of celluloses, cello-oligosaccharides, or hemicelluloses. AA9 LPMOs have been reported to have extensive interactions with not only cellulases but also oxidases. The addition of AA9 LPMOs can greatly reduce the amount of cellulase needed for saccharification and increase the yield of glucose. The discovery of AA9 LPMOs has greatly changed our understanding of how fungi degrade cellulose. In this review, apart from summarizing the recent discoveries related to their catalytic reaction, functional diversity, and practical applications, the stability, expression system, and protein engineering of AA9 LPMOs are reviewed for the first time. This review may provide a reference value to further broaden the substrate range of AA9 LPMOs, expand the scope of their practical applications, and realize their customization for industrial utilization.Key Points• The stability and expression system of AA9 LPMOs are reviewed for the first time.• The protein engineering of AA9 LPMOs is systematically summarized for the first time.• The latest research results on the catalytic mechanism of AA9 LPMOs are summarized.• The application of AA9 LPMOs and their relationship with other enzymes are reviewed.

Sequence and Structural Analysis of AA9 and AA10 LPMOs: An Insight into the Basis of Substrate Specificity and Regioselectivity.

Lytic polysaccharide monooxygenases (LPMOs) are key enzymes in both the natural carbon cycle and the biorefinery industry. Understanding the molecular basis of LPMOs acting on polysaccharide substrates is helpful for improving industrial cellulase cocktails. Here we analyzed the sequences, structures, and substrate binding modes of LPMOs to uncover the factors that influence substrate specificity and regioselectivity. Our results showed that the different compositions of a motif located on L2 affect the electrostatic potentials of substrate binding surfaces, which in turn affect substrate specificities of AA10 LPMOs. A conserved Asn at a distance of 7 Å from the active center Cu might, together with the conserved Ser immediately before the second catalytic His, determine the localization of LPMOs on substrate, and thus contribute to C4-oxidizing regioselectivity. The findings in this work provide an insight into the molecular basis of substrate specificity and regioselectivity of LPMOs.

The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases.

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases,CfLPMO10 andTbLPMO10 fromCellulomonas fimiandThermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducingCtCBM3a, from theClostridium thermocellumcellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact ofCtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM fromCfLPMO10 or the introduction of a family 10 CBM fromCellvibrio japonicusLPMO10B intoTbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.

A family of AA9 lytic polysaccharide monooxygenases in Aspergillus nidulans is differentially regulated by multiple substrates and at least one is active on cellulose and xyloglucan.

Fungal genomes contain multiple genes encoding AA9 lytic polysaccharide monooxygenases (LPMOs), a recently discovered class of enzymes known to be active on cellulose and expressed when grown on biomass. Because of extensive genetic and biochemical data already available, Aspergillus nidulans offers an excellent model system to study the need for multiple AA9 LPMOs and their activity during oxidative degradation of biomass. We provide the first report on regulation of the entire family of AA9 LPMOs in A. nidulans over a range of polysaccharides including xylan, xyloglucan, pectin, glucan, and cellulose. We have successfully cloned and expressed AN3046, an AA9 LPMO in A. nidulans that is active on cellulose. Additionally, we performed mass spectral analyses that show the enzyme is active on the hemicellulose xyloglucan. The AN3046 LPMO showed synergy with other hydrolases in degrading sorghum stover. Our data showing activity of the overexpressed LPMO on cellulose and xyloglucan provides further evidence for the breadth of substrates acted on by AA9 LPMOs.

Microbial decomposition of keratin in nature-a new hypothesis of industrial relevance.

Discovery of keratin-degrading enzymes from fungi and bacteria has primarily focused on finding one protease with efficient keratinase activity. Recently, an investigation was conducted of all keratinases secreted from a fungus known to grow on keratinaceous materials, such as feather, horn, and hooves. The study demonstrated that a minimum of three keratinases is needed to break down keratin, an endo-acting, an exo-acting, and an oligopeptide-acting keratinase. Further, several studies have documented that disruption of sulfur bridges of the keratin structure acts synergistically with the keratinases to loosen the molecular structure, thus giving the enzymes access to their substrate, the protein structure. With such complexity, it is relevant to compare microbial keratin decomposition with the microbial decomposition of well-studied polymers such as cellulose and chitin. Interestingly, it was recently shown that the specialized enzymes, lytic polysaccharide monoxygenases (LPMOs), shown to be important for breaking the recalcitrance of cellulose and chitin, are also found in keratin-degrading fungi. A holistic view of the complex molecular self-assembling structure of keratin and knowledge about enzymatic and boosting factors needed for keratin breakdown have been used to formulate a hypothesis for mode of action of the LPMOs in keratin decomposition and for a model for degradation of keratin in nature. Testing such hypotheses and models still needs to be done. Even now, the hypothesis can serve as an inspiration for designing industrial processes for keratin decomposition for conversion of unexploited waste streams, chicken feather, and pig bristles into bioaccessible animal feed.

Diversely regio-oxidative degradation of konjac glucomannan by lytic polysaccharide monooxygenase AA10 and generating antibacterial hydrolysate.

Konjac glucomannan (KGM) hydrolysate exhibit various biological activities and health-promoting effects. Lytic polysaccharide monooxygenases (LPMOs) play an important role on enzymatic degradation of recalcitrant polysaccharides to obtain fermentable sugars. It is generally accepted that LPMOs exhibits high substrate specificity and oxidation regioselectivity. Here, a bacteria-derived SmAA10A, with chitin-active with strict C1 oxidation, was used to catalyse KGM degradation. Through ethanol precipitation, two hydrolysed KGM components (4 kDa (KGM-1) and 5 kDa (KGM-2)) were obtained that exhibited antibacterial activity against Staphylococcus aureus. In natural KGM, KGM-1, and KGM-2, the molar ratios of mannose to glucose were 1:2.19, 1:3.05, and 1:2.87, respectively, indicating that SmAA10A preferentially degrades mannose in KGM. Fourier-transform infrared spectroscopy and scanning electron microscopy imaging revealed the breakage of glycosylic bonds during enzymatic catalysis. The regioselectivity of SmAA10A for KGM degradation was determined based on the fragmentation behaviour of the KGM-1 and KGM-2 oligosaccharides and their NaBD4-reduced forms. SmAA10A exhibited diverse oxidation degradation of KGM and generated single C1-, single C4-, and C1/C4-double oxidised oligosaccharide forms. This study provides an alternative method for obtaining KGM degradation components with antibacterial functions and expands the substrate specificity and oxidation regioselectivity of bacterial LPMOs.

Defective copper-cobalt binuclear Prussian blue analogue nanozymes with high specificity as lytic polysaccharide monooxygenase-mimic via axial ligation of histidine.

Degradation of polysaccharides based on lytic polysaccharide monooxygenases (LPMOs) has received considerably interest in the environment and energy fields since 2010. With the rapid development of nanozymes in various fields, it is highly desirable but challenging to develop LPMO-like nanozymes with high specificity and satisfied activity. Here, a defective copper-cobalt binuclear Prussian blue analogue (CuCoPBA) nanozyme was developed via a facile and ingenious methodology based on single histidine (His). For the first time, His-CuCoPBA nanozyme was found to exhibit LPMO-like activity with H2O2 as a cosubstrate at room temperature and neutral pH, which can efficiently catalyze the degradation of galactomannans selectively. Significantly, the high degradation activity at pH 10 expands the application of Fenton-like nanozymes in alkaline condition. Singlet oxygen (1O2), as a main reactive intermediate, plays a crucial role in the galactomannan degradation catalyzed by His-CuCoPBA nanozyme. Both control experimental and density functional theory (DFT) results indicate Cu-NxHis contributes to the efficiently and selectively catalytic activity of His-CuCoPBA nanozymes by emulating the binding and catalytic sites of LPMOs. The present work not only represents a fundamental breakthrough toward degradation of polysaccharide based on nanozyme, but also contributes to understanding the catalytic mechanism of natural Cu-dependent LPMOs.

Advances in the understanding and exploitation of carbohydrate-active enzymes.

Carbohydrate-active enzymes (CAZymes) are responsible for the biosynthesis, modification and degradation of all glycans in Nature. Advances in genomic and metagenomic methodologies, in conjunction with lower cost gene synthesis, have provided access to a steady stream of new CAZymes with both well-established and novel mechanisms. At the same time, increasing access to cryo-EM has resulted in exciting new structures, particularly of transmembrane glycosyltransferases of various sorts. This improved understanding has resulted in widespread progress in applications of CAZymes across diverse fields, including therapeutics, organ transplantation, foods, and biofuels. Herein, we highlight a few of the many important advances that have recently been made in the understanding and applications of CAZymes.

Structural dissection of two redox proteins from the shipworm symbiont Teredinibacter turnerae.

The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria.

Enzymatic hydrolysis cocktail optimization for the intensification of sugar extraction from sugarcane bagasse.

Lignocellulosic biomasses have a very important role as a raw material to produce biofuels and biochemicals. However, a sustainable, efficient, and economically competitive process for the release of sugars from such materials has still not been achieved. In this work, the optimization of the enzymatic hydrolysis cocktail was evaluated as an approach to maximize sugar extraction from mildly pretreated sugarcane bagasse. Different additives and enzymes, including hydrogen peroxide (H2O2), laccase, hemicellulase and the surfactants Tween 80 and PEG4000 were added to a cellulolytic cocktail with the aim of improving biomass hydrolysis. An increase of 39 % and 46 % of glucose and xylose concentrations, respectively, compared to the control (when only the cellulolytic cocktail (20 or 35 FPU g-1 dry mass), was obtained when H2O2 (0.24 mM) was added at the beginning of the hydrolysis. On the other hand, the addition of hemicellulase (81-162 µL g-1 DM) increased the production of glucose up to 38 % and xylose up to 50 %. The findings of this study reveal that it is possible to increase the extraction of sugars from mildly pretreated lignocellulosic biomass by using an appropriate enzymatic cocktail supplemented with additives. This opens up new opportunities for the development of a more sustainable, efficient, and economically competitive process for biomass fractionation.

Structural and binding studies of a new chitin-active AA10 lytic polysaccharide monooxygenase from the marine bacterium Vibrio campbellii.

Vibrio spp. play a crucial role in the global recycling of the highly abundant recalcitrant biopolymer chitin in marine ecosystems through their ability to secrete chitin-degrading enzymes to efficiently hydrolyse chitinous materials and use them as their major carbon source. In this study, the first crystal structures of a complete four-domain chitin-active AA10 lytic polysaccharide monooxygenase from the chitinolytic bacterium Vibrio campbellii type strain ATCC BAA-1116 are reported. The crystal structures of apo and copper-bound VhLPMO10A were resolved as homodimers with four distinct domains: an N-terminal AA10 catalytic (CatD) domain connected to a GlcNAc-binding (GbpA_2) domain, followed by a module X domain and a C-terminal carbohydrate-binding module (CBM73). Size-exclusion chromatography and small-angle X-ray scattering analysis confirmed that VhLPMO10A exists as a monomer in solution. The active site of VhLPMO10A is located on the surface of the CatD domain, with three conserved residues (His1, His98 and Phe170) forming the copper(II)-binding site. Metal-binding studies using synchrotron X-ray absorption spectroscopy and X-ray fluorescence, together with electron paramagnetic resonance spectroscopy, gave consistently strong copper(II) signals in the protein samples, confirming that VhLPMO10A is a copper-dependent enzyme. ITC binding data showed that VhLPMO10A could bind various divalent cations but bound most strongly to copper(II) ions, with a Kd of 0.1 ± 0.01 µM. In contrast, a Kd of 1.9 nM was estimated for copper(I) ions from redox-potential measurements. The presence of ascorbic acid is essential for H2O2 production in the reaction catalysed by VhLPMO10A. MALDI-TOF MS identified VhLPMO10A as a C1-specific LPMO, generating oxidized chitooligosaccharide products with different degrees of polymerization (DP2ox-DP8ox). This new member of the chitin-active AA10 LPMOs could serve as a powerful biocatalyst in biofuel production from chitin biomass.

Unravelling the secrets of multi-domain lytic polysaccharide monooxygenases (LPMOs).

A new chitin-active AA10 lytic polysaccharide monooxygenase from the marine bacterium Vibrio campbellii is described in the paper by Zhou et al. [(2023), Acta Cryst. D79, 479-497].