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Stimulation of Lignocellulosic Biomass Hydrolysis by Proteins of Glycoside Hydrolase Family 61: Structure and Function of a Large, Enigmatic Family
Abstract
Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.
... Activation of C-H and cleavage of glycosidic bonds in these crystalline polysaccharides necessitate powerful redox chemistry to surmount an activation energy barrier of approximately 95 kcal/mol [4,5]. LPMOs are currently classified in eight Auxiliary Activity families (AA9-11, [13][14][15][16][17] in the Carbohydrate Active enZyme database [6]. LPMOs are monocopper enzymes in which copper is coordinated by a conserved histidine brace [7]. ...
... Engineering LPMOs through point mutations, coupled with computational and structural studies, provide invaluable data, however mutations to the second coordination sphere remain relatively unexplored. It is expected that these substitutions will alter to some extent enzyme activity and/or enzyme binding to its solid substrate [11][12][13][14][15]. ...
... Wang et al. 's molecular dynamics simulations indicated that, in the presence of an electron donor (specifically ascorbic acid) and substrate, His1 was the residue most proximal to the electron donor interaction [39]. Although the significance of His1 is unquestionable, direct substitution attempts in the past led to the abrogation of LPMO activity as demonstrated by Harris et al. [14]. In our rationale approach to probe the vicinity of His1 depicted in Fig. 4, exploration based on the AlphaFold model of TthLPMO9G, seven amino acids were discerned within a 5 Å radius of His1. ...
Background
The field of enzymology has been profoundly transformed by the discovery of lytic polysaccharide monooxygenases (LPMOs). LPMOs hold a unique role in the natural breakdown of recalcitrant polymers like cellulose and chitin. They are characterized by a “histidine brace” in their active site, known to operate via an O2/H2O2 mechanism and require an electron source for catalytic activity. Although significant research has been conducted in the field, the relationship between these enzymes, their electron donors, and H2O2 production remains complex and multifaceted.
Results
This study examines TthLPMO9G activity, focusing on its interactions with various electron donors, H2O2, and cellulose substrate interactions. Moreover, the introduction of catalase effectively eliminates H2O2 interference, enabling an accurate evaluation of each donor’s efficacy based on electron delivery to the LPMO active site. The introduction of catalase enhances TthLPMO9G’s catalytic efficiency, leading to increased cellulose oxidation. The current study provides deeper insights into specific point mutations, illuminating the crucial role of the second coordination sphere histidine at position 140. Significantly, the H140A mutation not only impacted the enzyme’s ability to oxidize cellulose, but also altered its interaction with H2O2. This change was manifested in the observed decrease in both oxidase and peroxidase activities. Furthermore, the S28A substitution, selected for potential engagement within the His1–electron donor–cellulose interaction triad, displayed electron donor-dependent alterations in cellulose product patterns.
Conclusion
The interaction of an LPMO with H2O2, electron donors, and cellulose substrate, alongside the impact of catalase, offers deep insights into the intricate interactions occurring at the molecular level within the enzyme. Through rational alterations and substitutions that affect both the first and second coordination spheres of the active site, this study illuminates the enzyme’s function. These insights enhance our understanding of the enzyme’s mechanisms, providing valuable guidance for future research and potential applications in enzymology and biochemistry.
... As previously outlined, the biomass degradation has been traditionally explained in terms of synergistic action of glycosyl hydrolases, but the discovery of LPMOs in recent past has radically changed our understanding (Harris et al. 2010). They include AA9 (active on cellulose polymer), AA10 (acting on either cellulose or chitin), AA11 (attacking chitin) and AA13 (hydrolyzing starch) (Haris et al. 2010;Forsberg et al. 2014;Hemsworth et al. 2014;Lo Leggio et al. 2015). ...
... enzyme was pre-incubated with Cu 2+ and Mn 2+ . Additionally, the high LPMO mediated catalysis using a buffer containing Mn 2+ has also been reported by Harris et al. (2010). The CDH enzyme activity was potentially influenced by Ca 2+ (163.60%), ...
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.
... 7 Consequently, the discovery of a new class of enzymes that boosts the degradation of polysaccharides has attracted considerable attention. [7][8][9][10][11][12] This enzyme class is now termed lytic polysaccharide monooxygenase (LPMO) and is currently grouped into eight families: AA9 to AA11 and AA13 to AA17. 8,9,[13][14][15][16][17][18][19] The LPMOs differ in substrate-specicity 8,14-16,19-25 but they generally catalyze the oxidation of the glycosidic C-H bonds in the polysaccharide, leading ultimately to a cleavage of the glycosidic bond. ...
... [7][8][9][10][11][12] This enzyme class is now termed lytic polysaccharide monooxygenase (LPMO) and is currently grouped into eight families: AA9 to AA11 and AA13 to AA17. 8,9,[13][14][15][16][17][18][19] The LPMOs differ in substrate-specicity 8,14-16,19-25 but they generally catalyze the oxidation of the glycosidic C-H bonds in the polysaccharide, leading ultimately to a cleavage of the glycosidic bond. 10,11,26 This cleavage causes the boosting effect and is administered by a copper-containing active site with Cu coordinated by two histidines 7,[16][17][18][19]27 that has been coined the histidine brace. ...
Lytic polysaccharide monooxygenase (LPMO) is a new class of oxidoreductases that boosts polysaccharide degradation employing a copper active site. This boost may facilitate the cost-efficient production of biofuels and high-value chemicals from polysaccharides such as lignocellulose. Unfortunately, self-oxidation of the active site inactivates LPMOs. Other oxidoreductases employ hole-hopping mechanisms as protection against oxidative damage, but little is generally known about the details of these mechanisms. Herein, we employ highly accurate theoretical models based on density functional theory (DFT) molecular mechanics (MM) hybrids to understand the initial steps in LPMOs' protective measures against self-oxidation; we identify several intermediates recently proposed from experiment, and quantify which are important for protective hole-hopping pathways. Investigations on two different LPMOs show consistently that a tyrosine residue close to copper is crucial for protection: this explains recent experiments, showing that LPMOs without this tyrosine are more susceptible to self-oxidation.
... PMOs are also known as lytic polysaccharide monooxygenases (LPMOs) and they work in synergy with cellobiose hydrolases and other cellulolytic enzymes to significantly enhance the enzymatic degradation of cellulose [2,4,8,9,10,11]. Indeed, several studies have reported that the addition of a small amount of PMO (1-5%) to the typical hydrolytic cellulase mixture greatly improved cellulolytic activity, resulting in a 3-5 times more efficient conversion of cellulose to fermentable sugars [12]. ...
... In the CAZy database, PMOs are classified as auxiliary activity (AA) enzymes. Currently, PMOs are divided into six families based on their amino acid sequence similarities and substrates [13]: (i) cellulose-active fungal PMOs [8,11,12,14] (AA9, formerly classified in the glycoside hydrolase family GH61, and AA16); (ii) chitin-and/ or cellulose-active bacterial PMOs [3,4,5] (CBM33 or AA10); (iii) chitin-active fungal PMOs (AA11) [3,4,5]; (iv) starch-specific fungal PMOs (AA13) [6,7]; (v) xylan-active fungal PMOs (AA14), which act on xylan coating cellulose fibers [15]; (vi) cellulose and chitinactive PMOs (AA15) of animal origin [16]. ...
... Previously, it has been shown that BaLPMO10A has C1 oxidation activity against chitin but no activity against cellulose [30]. Earlier studies have shown that some LPMOs can hydrolyze cellulose or carboxymethylcellulose (CMC) [18,31], albeit with low activity. To test whether BaLPMO10A has hydrolysis activity, CMC was used as a substrate and mixed with WT BaLPMO10A (Figure 2A). ...
... It has been indicated that the LPMO reaction is crucial to enhancing the degradation of microcrystalline cellulosic substrates. A study reported the enhanced depolymerization of a pre-treated corn stover where the addition of LPMOs to the cellulase mixture led to a reduction in half of the enzyme load required to reach 90% of the hydrolysis process of cellulose [31]. Moreover, another more recent report showed that the addition of LPMOs to the cellulase resulted in an up to 50% increase in the saccharification process efficiency of the microcrystalline cellulose [37]. ...
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-β-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.
... In contrast to cellulolytic hydrolases, cellulose-active LPMOs are mono-copper enzymes with a relatively flat active site that uses O 2 and/or H 2 O 2 as co-substrates. LPMOs cleave their substrates by oxidizing C1 or C4 carbons, or both [23][24][25][26]. As the Cu(II) core must be reduced to Cu(I), reaction conditions for LPMOs must be tailored carefully with respect to reductant, co-substrate, and potential auto-inactivation processes [27][28][29]. ...
... Time-course reactions were performed in triplicates for each time point (0, 2,4,8,24,48, and 72 h), in 2 mL screw-cap tubes with a total reaction weight of 1.8 g. The loaded substrate (2% w/w of dry mass) was suspended in 0.15 M acetate buffer (pH 5). ...
Background
To realize the full potential of softwood-based forest biorefineries, the bottlenecks of enzymatic saccharification of softwood need to be better understood. Here, we investigated the potential of lytic polysaccharide monooxygenases (LPMO9s) in softwood saccharification. Norway spruce was steam-pretreated at three different severities, leading to varying hemicellulose retention, lignin condensation, and cellulose ultrastructure. Hydrolyzability of the three substrates was assessed after pretreatment and after an additional knife-milling step, comparing the efficiency of cellulolytic Celluclast + Novozym 188 and LPMO-containing Cellic CTec2 cocktails. The role of Thermoascus aurantiacus TaLPMO9 in saccharification was assessed through time-course analysis of sugar release and accumulation of oxidized sugars, as well as wide-angle X-ray scattering analysis of cellulose ultrastructural changes.
Results
Glucose yield was 6% (w/w) with the mildest pretreatment (steam pretreatment at 210 °C without catalyst) and 66% (w/w) with the harshest (steam pretreatment at 210 °C with 3%(w/w) SO2) when using Celluclast + Novozym 188. Surprisingly, the yield was lower with all substrates when Cellic CTec2 was used. Therefore, the conditions for optimal LPMO activity were tested and it was found that enough O2 was present over the headspace and that the reducing power of the lignin of all three substrates was sufficient for the LPMOs in Cellic CTec2 to be active. Supplementation of Celluclast + Novozym 188 with TaLPMO9 increased the conversion of glucan by 1.6-fold and xylan by 1.5-fold, which was evident primarily in the later stages of saccharification (24–72 h). Improved glucan conversion could be explained by drastically reduced cellulose crystallinity of spruce substrates upon TaLPMO9 supplementation.
Conclusion
Our study demonstrated that LPMO addition to hydrolytic enzymes improves the release of glucose and xylose from steam-pretreated softwood substrates. Furthermore, softwood lignin provides enough reducing power for LPMOs, irrespective of pretreatment severity. These results provided new insights into the potential role of LPMOs in saccharification of industrially relevant softwood substrates.
... This enzymatic depolymerisation of cellulose was largely considered to be catalysed by glycoside hydrolases (GHs), collectively termed cellulases, which catalyse glycoside bond cleavage through the activation of water as a nucleophile [5]. About 12 years ago, however, a previously less well-understood enzyme class was discovered to play a key role in this process -lytic polysaccharide monooxygenases (LPMOs) [6][7][8]. ...
... LPMOs have been demonstrated to significantly improve the efficiency of cellulose (and other polysaccharide) degradation by GHs [6][7][8][9][10][11]. They catalyse the addition of a single atom of oxygen at either the C1 or C4 position of the sugar ring within polysaccharide chains, thereby destabilising the Schematic drawings illustrating the differences when (A) O 2 is used as the LPMO cosubstrate as opposed to (B) H 2 O 2 . ...
The plant cell wall is rich in carbohydrates and many fungi and bacteria have evolved to take advantage of this carbon source. These carbohydrates are largely locked away in polysaccharides and so these organisms deploy a range of enzymes that can liberate individual sugars from these challenging substrates. Glycoside hydrolases (GHs) are the enzymes that are largely responsible for bringing about this sugar release; however, 12 years ago, a family of enzymes known as lytic polysaccharide monooxygenases (LPMOs) were also shown to be of key importance in this process. LPMOs are copper-dependent oxidative enzymes that can introduce chain breaks within polysaccharide chains. Initial work demonstrated that they could activate O2 to attack the substrate through a reaction that most likely required multiple electrons to be delivered to the enzyme. More recently, it has emerged that LPMO kinetics are significantly improved if H2O2 is supplied to the enzyme as a cosubstrate instead of O2. Only a single electron is required to activate an LPMO and H2O2 cosubstrate and the enzyme has been shown to catalyse multiple turnovers following the initial one-electron reduction of the copper, which is not possible if O2 is used. This has led to further studies of the roles of the electron donor in LPMO biochemistry, and this review aims to highlight recent findings in this area and consider how ongoing research could impact our understanding of the interplay between redox processes in nature.
... However, the poor efficiency of enzymatic hydrolysis of some recalcitrant lignocellulosic materials remains a key limiting step in their processing [19]. In recent years, great attention has been given to the auxiliary enzyme family 9 (AA9, formerly GH61) due to its booster activity in cellulose enzymatic degradation [20,21]. ...
... In the present study, we explored the feasibility of the production of two AA9 LPMO proteins by tobacco plastid transformation, since, previously, this approach enabled very encouraging results in terms of protein yield, hydrolytic activity, and stability of recombinant cellulolytic enzymes [14]. TrAA9B from T. reesei was chosen as this filamentous fungus is one of the best-studied organisms used in biomass conversion; in fact, it produces a multitude of (hemi)cellulases and is the best known and commercially used cellulolytic system [20]; TaAA9B from T. aurantiacus, instead, was selected for its putative thermophilic properties. Neither TrAA9B nor TaAA9B were previously expressed in planta. ...
Plant biomass is the most abundant renewable resource in nature. In a circular economy perspective, the implementation of its bioconversion into fermentable sugars is of great relevance. Lytic Polysaccharide MonoOxygenases (LPMOs) are accessory enzymes able to break recalcitrant polysaccharides, boosting biomass conversion and subsequently reducing costs. Among them, auxiliary activity of family 9 (AA9) acts on cellulose in synergism with traditional cellulolytic enzymes. Here, we report for the first time, the production of the AA9 LPMOs from the mesophilic Trichoderma reesei (TrAA9B) and the thermophilic Thermoascus aurantiacus (TaAA9B) microorganisms in tobacco by plastid transformation with the aim to test this technology as cheap and sustainable manufacture platform. In order to optimize recombinant protein accumulation, two different N-terminal regulatory sequences were used: 5′ untranslated region (5′-UTR) from T7g10 gene (DC41 and DC51 plants), and 5′ translation control region (5′-TCR), containing the 5′-UTR and the first 14 amino acids (Downstream Box, DB) of the plastid atpB gene (DC40 and DC50 plants). Protein yields ranged between 0.5 and 5% of total soluble proteins (TSP). The phenotype was unaltered in all transplastomic plants, except for the DC50 line accumulating AA9 LPMO at the highest level, that showed retarded growth and a mild pale green phenotype. Oxidase activity was spectrophotometrically assayed and resulted higher for the recombinant proteins without the N-terminal fusion (DC41 and DC51), with a 3.9- and 3.4-fold increase compared to the fused proteins.
... There are many methods to increase the activity of cellulases, and the cellulases auxiliary protein is one of the resolvents. Cellulases auxiliary proteins are mainly lytic polysaccharide monooxygenase (LPMO), cellobiose dehydrogenase (CDH), expansin, and swollenin (SWOI), which can effectively promote the hydrolysis of cellulose, and some of them have a certain cellulose hydrolysis ability (Chen et al., 2010;Harris et al., 2010;Wang and Lu, 2016;Chase et al., 2020). ...
Cellobiose dehydrogenase (CDH) is one of the cellulase auxiliary proteins, which is widely used in the field of biomass degradation. However, how to efficiently and cheaply apply it in industrial production still needs further research. Aspergillus niger C112 is a significant producer of cellulase and has a relatively complete lignocellulose degradation system, but its CDH activity was only 3.92 U. To obtain a recombinant strain of A. niger C112 with high cellulases activity, the CDH from the readily available white-rot fungus Grifola frondose had been heterologously expressed in A. niger C112, under the control of the gpdA promoter. After cultivation in the medium with alkali-pretreated poplar fiber as substrate, the enzyme activity of recombinant CDH reached 36.63 U/L. Compared with the original A. niger C112, the recombinant A. niger transformed with Grifola frondosa CDH showed stronger lignocellulase activity, the activities of cellulases, β-1, 4-glucosidase and manganese peroxidase increased by 28.57, 35.07 and 121.69%, respectively. The result showed that the expression of the gcdh gene in A. niger C112 could improve the activity of some lignocellulose degrading enzymes. This work provides a theoretical basis for the further application of gcdh gene in improving biomass conversion efficiency.
... This modification destabilizes the 1,4 glycosidic linkage and thereby brings about oxidative polysaccharide cleavage (Vaaje-Kolstad et al., 2010;Quinlan et al., 2011;Phillips et al., 2011;Bissaro et al., 2017), and can boost the ability of other canonical glycoside hydrolases to further degrade the substrate. This boosting action leads to significant increases in the level of glucose that can be obtained from lignocellulosic biomass for processing into bioethanol (Vaaje-Kolstad et al., 2010;Cannella et al., 2012;Harris et al., 2010;Lo Leggio et al., 2015;Sabbadin, Hemsworth et al., 2018), and more recently, new biological roles for LPMOs have also emerged, with these enzymes being implicated in virulence (Askarian et al., 2021(Askarian et al., , 2023Sabbadin et al., 2021), copper homeostasis (Garcia-Santamarina et al., 2020) and cell-wall remodelling (Zhong et al., 2022). ...
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.
... The GH class contains the endoand exo-acting enzymes targeting cellulose and hemicelluloses. The AA class contains laccases (AA1) and class II peroxidases (AA2) involved in lignin degradation, and lytic polysaccharide monooxygenases (LPMOs, AA9) proposed to play an important role in the early phase of wood degradation (5)(6)(7). Reactive oxygen species (ROS), particularly hydrogen peroxide (H 2 O 2 ), play a crucial role in the early phase of wood decay (8)(9)(10). H 2 O 2 is generated by glucose-methanol-choline oxidoreductases (AA3) and copper radical oxidases (AA5) that target carbohydrate or aliphatic/aromatic alcohols (11,12). ...
White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H 2 O 2 ), to degrade lignocellulose in wood. H 2 O 2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO 2 ) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H 2 ¹⁸ O 2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.
IMPORTANCE
The study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H 2 O 2 ) on secreted proteins remains unclear. This study aims at evaluating the effect of H 2 O 2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H 2 O 2 . The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.
... Coniferous substrates have more lignin than broad-leaved tree substrates, which makes the role of glycoside hydrolase in lignocellulose degradation limited (Zhang et al., 2006). However, the synergy of lytic polysaccharide monooxygenase (LPMO) and glycoside hydrolase (GHs) can improve lignocellulose degradation (Harris et al., 2010;Li et al., 2022). The study identified a higher number of genes related to lignocellulose degradation in conifers, such as AA9 (LPMO), GH30 (glucuronoarabinoxylan endo-β-1,4-xylanase) and GH31 (βglucosidase), GH47 (α-mannosidase), GH7 (cellobiohydrolase), and GH71 (glucanase), which may be attributed to the high lignin content present in these trees. ...
Pleurotus placentodes (PPL) and Pleurotus cystidiosus (PCY) are economically valuable species. PPL grows on conifers, while PCY grows on broad-leaved trees. To reveal the genetic mechanism behind PPL’s adaptability to conifers, we performed de novo genome sequencing and comparative analysis of PPL and PCY. We determined the size of the genomes for PPL and PCY to be 36.12 and 42.74 Mb, respectively, and found that they contain 10,851 and 15,673 protein-coding genes, accounting for 59.34% and 53.70% of their respective genome sizes. Evolution analysis showed PPL was closely related to P. ostreatus with the divergence time of 62.7 MYA, while PCY was distantly related to other Pleurotus species with the divergence time of 111.7 MYA. Comparative analysis of carbohydrate-active enzymes (CAZYmes) in PPL and PCY showed that the increase number of CAZYmes related to pectin and cellulose degradation (e.g., AA9, PL1) in PPL may be important for the degradation and colonization of conifers. In addition, geraniol degradation and peroxisome pathways identified by comparative genomes should be another factors for PPL’s tolerance to conifer substrate. Our research provides valuable genomes for Pleurotus species and sheds light on the genetic mechanism of PPL’s conifer adaptability, which could aid in breeding new Pleurotus varieties for coniferous utilization.
... Its complete hydrolysis requires thermo-chemical pre-treatment to loosen up its structure, followed by the action of specialized enzymes which can specifically hydrolyze the β-linkages between glucose moieties [6,7]. Classical enzymes such as cellulases, hemicellulases, and pectinases hydrolyze the plant cell wall components along with auxiliary activity enzymes, which act synergistically with the classical enzymes to loosen up the lignocellulosic structure, increasing the accessibility of hydrolyzing enzymes [8,9]. These enzymes are categorized as Carbohydrate Active Enzymes (CAZymes). ...
Background
Penicillium funiculosum NCIM1228 is a filamentous fungus that was identified in our laboratory to have high cellulolytic activity. Analysis of its secretome suggested that it responds to different carbon substrates by secreting specific enzymes capable of digesting those substrates. This phenomenon indicated the presence of a regulatory system guiding the expression of these hydrolyzing enzymes. Since transcription factors (TFs) are the key players in regulating the expression of enzymes, this study aimed first to identify the complete repertoire of Carbohydrate Active Enzymes (CAZymes) and TFs coded in its genome. The regulation of CAZymes was then analysed by studying the expression pattern of these CAZymes and TFs in different carbon substrates—Avicel (cellulosic substrate), wheat bran (WB; hemicellulosic substrate), Avicel + wheat bran, pre-treated wheat straw (a potential substrate for lignocellulosic ethanol), and glucose (control).
Results
The P. funiculosum NCIM1228 genome was sequenced, and 10,739 genes were identified in its genome. These genes included a total of 298 CAZymes and 451 TF coding genes. A distinct expression pattern of the CAZymes was observed in different carbon substrates tested. Core cellulose hydrolyzing enzymes were highly expressed in the presence of Avicel, while pre-treated wheat straw and Avicel + wheat bran induced a mixture of CAZymes because of their heterogeneous nature. Wheat bran mainly induced hemicellulases, and the least number of CAZymes were expressed in glucose. TFs also exhibited distinct expression patterns in each of the carbon substrates. Though most of these TFs have not been functionally characterized before, homologs of NosA, Fcr1, and ATF21, which have been known to be involved in fruiting body development, protein secretion and stress response, were identified.
Conclusions
Overall, the P. funiculosum NCIM1228 genome was sequenced, and the CAZymes and TFs present in its genome were annotated. The expression of the CAZymes and TFs in response to various polymeric sugars present in the lignocellulosic biomass was identified. This work thus provides a comprehensive mapping of transcription factors (TFs) involved in regulating the production of biomass hydrolyzing enzymes.
... [1,2] For this reason, the application of powerful oxidoreductases for efficient polysaccharide depolymerization processes becomes increasingly prominent. Especially lytic polysaccharide monooxygenases (LPMOs) [3,4] are active on a wide variety of carbohydrate polymers as substrates, support the action of hydrolytic enzymes, and degrade recalcitrant plant polymers. LPMOs enhance the degradation of cellulose and other polysaccharides by the regioselective hydroxylation of carbon atoms and thereby induced cleavage of the glycosidic bond. ...
The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD‐containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure‐based engineering. The electron transfer kinetics of wild‐type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped‐flow spectrophotometry and structural effects were studied by small‐angle X‐ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway – its mutation reducing the interdomain electron transfer 10‐fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.
... Significant inspiration for the development of efficient biocatalytic methods to overcome these challenges comes from the microbial world: Diverse bacteria and fungi have evolved specialized hydrolytic and oxidative enzymatic systems to target individual biomass polymers for nutrient acquisition [13][14][15][16][17][18][19][20]. Lytic polysaccharide monooxygenases (LPMOs) have attracted attention for biomass deconstruction and biofuels production, due to their ability to potentiate the activity of cellulases and thereby enhance lignocellulose saccharification [21][22][23][24]. More recently, LPMOs have been harnessed for nanocellulose production [25][26][27][28][29][30][31] and the surgical introduction of carboxylate groups as reactive handles on cellulose and chitin surfaces [32,33]. ...
Background
Microbial lytic polysaccharide monooxygenases (LPMOs) cleave diverse biomass polysaccharides, including cellulose and hemicelluloses, by initial oxidation at C1 or C4 of glycan chains. Within the Carbohydrate-Active Enzymes (CAZy) classification, Auxiliary Activity Family 9 (AA9) comprises the first and largest group of fungal LPMOs, which are often also found in tandem with non-catalytic carbohydrate-binding modules (CBMs). LPMOs originally attracted attention for their ability to potentiate complete biomass deconstruction to monosaccharides. More recently, LPMOs have been applied for selective surface modification of insoluble cellulose and chitin.
Results
To further explore the catalytic diversity of AA9 LPMOs, over 17,000 sequences were extracted from public databases, filtered, and used to construct a sequence similarity network (SSN) comprising 33 phylogenetically supported clusters. From these, 32 targets were produced successfully in the industrial filamentous fungus Aspergillus niger , 25 of which produced detectable LPMO activity. Detailed biochemical characterization of the eight most highly produced targets revealed individual C1, C4, and mixed C1/C4 regiospecificities of cellulose surface oxidation, different redox co-substrate preferences, and CBM targeting effects. Specifically, the presence of a CBM correlated with increased formation of soluble oxidized products and a more localized pattern of surface oxidation, as indicated by carbonyl-specific fluorescent labeling. On the other hand, LPMOs without native CBMs were associated with minimal release of soluble products and comparatively dispersed oxidation pattern.
Conclusions
This work provides insight into the structural and functional diversity of LPMOs, and highlights the need for further detailed characterization of individual enzymes to identify those best suited for cellulose saccharification versus surface functionalization toward biomaterials applications.
... Another protein with weak endoglucanase activity (EGIV) is classified into the glycoside hydrolases family (GH61) [33]. Further structural studies show that CBM33 and GH61 are far from other sibling families, but both have conservative metal binding sites [34]. ...
Every year, seafood waste produced globally contains about 10 million tons of wasted crab, shrimp and lobster shells, which are rich in chitin resources. The exploitation and utilization of chitin resources are of great significance to environmental protection, economic development and sustainable development. Lytic polysaccharide monooxygenases (LPMOs) can catalyze polysaccharides by oxidative breakage of glycosidic bonds and have catalytic activity for chitin and cellulose, so they play an important role in the transformation of refractory polysaccharides into biomass. Although there have been many studies related to LPMOs, the research related to lytic chitin monooxygenases (LCMs) is still very limited. The specific catalytic mechanism of LCMs has not been fully elucidated, which poses a challenge to their application in industrial biomass conversion. This review introduces the present situation of resource development and utilization in chitin, the origin and classification of different LCMs families, the structural characteristics of LCMs and the relationship between structure and function. The research results related to activity detection, screening, preparation and transformation of LCMs were summarized and discussed. Finally, the synergistic effect of LCMs and chitin enzyme on biomass degradation was reviewed, and the existing problems and future research directions were pointed out. This is the first review focusing on Chitin-Active LPMOs in recent years, intending to provide a reference for applying chitin degradation enzymes system in the industry.
... In this process, the glycosidic bonds of cellulose are broken down by cellobiohydrolases into cellobiose, which is the substrate of cellobiose dehydrogenase (CDH) [3,4]. CDH is an auxiliary enzyme to lytic polysaccharide monooxygenase (LPMO) [5,6], which enhances the degradation of cellulose but also other polysaccharides by an oxidative mechanism cleaving glycosidic bonds and thereby forming new chain end for processive hydrolases [7]. The improved degradation efficiency caused by this enzymatic system shows the importance of these two carbohydrate-active enzymes in fungal cellulose degradation [8]. ...
The interdomain electron transfer between the catalytic flavodehydrogenase domain and the electron transferring cytochrome domain of cellobiose dehydrogenase plays an essential role in biocatalysis, biosensors, and biofuel cells as well as for its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of cellobiose dehydrogenase (CDH), which is hypothesised to limit interdomain electron transfer in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase of CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multi-state modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multi-state modelling, and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the interdomain electron transfer governed by the movement of the CDH cytochrome domain.
... Lignocellulose is the most abundant renewable resource in nature, which provides a sustainable substitute for fossil fuels, and has a wide application potential in the field of biorefinery. The discovery of lytic polysaccharide monooxygenases (LPMO) was of great significance for the degradation of polysaccharides in lignocellulose [1,2]. LPMO can oxidize and break the glycosidic bonds of polysaccharides such as cellulose, chitin, and starch, thereby reducing the resistance of polysaccharide conversion and promoting the hydrolysis of polysaccharide substrates by glycoside hydrolases [3]. ...
Lytic polysaccharide monooxygenase (LPMO) could oxidize and cleavage the glycosidic bonds of polysaccharides in lignocellulose, thereby promoting the hydrolysis of polysaccharide substrates by glycoside hydrolases and significantly improving the saccharification efficiency of lignocellulose. Brown-rot fungi are typical degraders of lignocellulose and contain multiple LPMO genes of the AA14 family and AA9 family, however, the AA14 LPMO from brown-rot fungi was rarely reported. Herein, the transcriptomic analysis of Serpula lacrymans incubated in the presence of pine exhibited that an AA14 LPMO (SlLPMO14A) was significantly upregulated and there were redox interactions between LPMOs and other enzymes (AA3, AA6, and hemicellulose degrading enzyme), indicating that SlLPMO14A may be involved in the degradation of polysaccharides. Enzymatic profiling of SlLPMO14A showed the optimal pH of 8.0 and temperature of 50 °C and it had higher reaction activity in the presence of 40% glycerol and acetonitrile. SlLPMO14A could significantly improve the saccharification of pine and xylan-coated cellulose substrate to release glucose and xylose by cellulase and xylanase by disturbing the surface structure of lignocellulose based on environmental scanning electron microscope and atomic force microscopy analysis.
... LPMOs can effectively disrupt and loosen the intrinsic structure of crystalline polysaccharides, providing more accessible sites for hydrolases [10]. Therefore, the synergistic action of LPMO and cellulase can accelerate the decomposition of cellulose and make the conversion of plant biomass more economical [11][12][13][14][15]. ...
Lytic polysaccharide monooxygenases (LPMOs) can oxidatively cleave the glycosidic bonds of crystalline polysaccharides, providing more accessible sites for polysaccharide hydrolases and promoting efficient conversion of biomass. In order to promote industrial applications of LPMOs, the stability of an LPMO of Myceliophthora thermophila C1 (MtC1LPMO) was improved by adding disulfide bonds in this study. Firstly, the structural changes of wild-type (WT) MtC1LPMO at different temperatures were explored using molecular dynamics simulations, and eight mutants were selected by combining the predicted results from Disulfide by Design (DBD), Multi agent stability prediction upon point mutations (Maestro) and Bridge disulfide (BridgeD) websites. Then, the enzymatic properties of the different mutants were determined after their expression and purification, and the mutant S174C/A93C with the highest thermal stability was obtained. The specific activities of unheated S174C/A93C and WT were 160.6 ± 1.7 U/g and 174.8 ± 7.5 U/g, respectively, while those of S174C/A93C and WT treated at 70 °C for 4 h were 77.7 ± 3.4 U/g and 46.1 ± 0.4 U/g, respectively. The transition midpoint temperature of S174C/A93C was 2.7 °C higher than that of WT. The conversion efficiency of S174C/A93C for both microcrystalline cellulose and corn straw was about 1.5 times higher than that of WT. Finally, molecular dynamics simulations revealed that the introduction of disulfide bonds increased the β-sheet content of the H1-E34 region, thus improving the rigidity of the protein. Therefore, the overall structural stability of S174C/A93C was improved, which in turn improved its thermal stability.
... In our study we have uncovered a fundamental role of the CnCel1 protein in stress adaption and virulence of the human fungal pathogen C. neoformans. The CnCel1 protein phylogenetically groups with AA9 cellulose-active LPMOs, sharing the aromatic amino acids known to be involved in AA9 LPMO substrate recognition, the copper binding histidines, as well as the secondary sphere coordinating tyrosine [60,61]. Consequentially, the expression of an N-terminal histidine to alanine CnCel1 variant (cel1Δ C H17A ), does not complement the stress phenotypes of the cel1Δ mutant strain, demonstrating that an intact active site is essential for CnCel1 ...
Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment.
... Interestingly, some of the LPMOs performing best in industrially relevant setups [95] are promiscuous LPMOs; TaLPMO9A from Thermoascus aurantiacus is active on cellulose and xyloglucan [96], while TtLPMO9E from T. terrestris is active on cellulose and xylan [77]. While contributions of cellulose-active LPMOs to biorefining by way of addition to commercial cellulase cocktails is well established [20,[97][98][99], the potential and effective contribution of hemicellulolytic LPMOs to lignocellulose bioprocessing remain relatively underexplored. ...
Lignocellulosic biomass is the most abundant source of carbon-based material on a global basis, serving as a raw material for cellulosic fibers, hemicellulosic polymers, platform sugars, and lignin resins or monomers. In nature, the various components of lignocellulose (primarily cellulose, hemicellulose, and lignin) are decomposed by saprophytic fungi and bacteria utilizing specialized enzymes. Enzymes are specific catalysts and can, in many cases, be produced on-site at lignocellulose biorefineries. In addition to reducing the use of often less environmentally friendly chemical processes, the application of such enzymes in lignocellulose processing to obtain a range of specialty products can maximize the use of the feedstock and valorize many of the traditionally underutilized components of lignocellulose, while increasing the economic viability of the biorefinery. While cellulose has a rich history of use in the pulp and paper industries, the hemicellulosic fraction of lignocellulose remains relatively underutilized in modern biorefineries, among other reasons due to the heterogeneous chemical structure of hemicellulose polysaccharides, the composition of which varies significantly according to the feedstock and the choice of pretreatment method and extraction solvent. This paper reviews the potential of hemicellulose in lignocellulose processing with focus on what can be achieved using enzymatic means. In particular, we discuss the various enzyme activities required for complete depolymerization of the primary hemicellulose types found in plant cell walls and for the upgrading of hemicellulosic polymers, oligosaccharides, and pentose sugars derived from hemicellulose depolymerization into a broad spectrum of value-added products.
... The other two loops, called LS and LC, are found in AA9 and AA13 LPMOs, and LC in most AA9 members harbors Tyr residue(s) [57]. The involvement of AA9 surface aromatic residues in substrate binding was confirmed by mutagenesis studies [96] and the removal of a helix insert in AA9 L2, that contains the conserved Tyr can shift the C1/C4 regioselectivity to an exclusive C1 oxidation, suggesting that aromatic residues in L2 play a long range role in driving oxidation at the C4 position [65,97]. Interestingly, the presence of an insertion in the L2 of one chitin/cellulose-active AA10 LPMO (lacking in a purely chitin-active one) has been instead correlated to cellulose selectivity [65,98]. ...
Molecular modeling techniques have become indispensable in many fields of molecular sciences in which the details related to mechanisms and reactivity need to be studied at an atomistic level. This review article provides a collection of computational modeling works on a topic of enormous interest and urgent relevance: the properties of metalloenzymes involved in the degradation and valorization of natural biopolymers and synthetic plastics on the basis of both circular biofuel production and bioremediation strategies. In particular, we will focus on lytic polysaccharide monooxygenase, laccases, and various heme peroxidases involved in the processing of polysaccharides, lignins, rubbers, and some synthetic polymers. Special attention will be dedicated to the interaction between these enzymes and their substrate studied at different levels of theory, starting from classical molecular docking and molecular dynamics techniques up to techniques based on quantum chemistry.
... Secretome analysis has revealed the presence of proteins that participate in cellulose degradation by a previously unknown oxidative mechanism [144,145]. Table 2, also see Table 1). ...
Discussion on lignocellulolytic enzymes in this chapter is mainly concerned with enzymes produced by filamentous fungi, which display a broad array of hydrolytic enzymes including cellulases, hemicellulases, and a suite of oxidative enzymes serving diverse functions in lignocellulose degradation. The carbohydrate-active enzymes (CAZymes) are classified into families of Glycoside Hydrolases (GHs), Glycosyl Transferases (GTs), Polysaccharide Lyases (PLs), Carbohydrate Esterases (CEs), and Auxiliary Activities (AAs) based on similarities in their amino-acid sequence. Some of their key properties, genetic modification, and production strategies are discussed here and elsewhere in the book. Among the challenges in working toward economically sustainable enzymatic hydrolysis of lignocellulose for cost-effective production of biofuels and other products is lowering the price of the enzymes and reducing the enzyme load required for hydrolysis. The latter is currently being addressed, for example, by attempts to improve the thermotolerance and catalytic activity of the key enzymes such as CBHI (Cel7A). A more recent line of research is exploring novel enzymes and nonhydrolytic proteins assisting in biomass degradation and designing substrate-tailored enzyme mixtures exposed by secretome displays and genome sequence analysis.
... While the first crystallographic studies of proteins known today as LPMOs showed the presence of a single metal-binding site [3,6], the nature of this metal ion remained unclear [2,22], until it was established in 2011 [23,24] that LPMOs are monocopper enzymes. The copper is bound by a highly conserved arrangement of two histidine residues referred to as the histidine-brace [24][25][26][27]. ...
The discovery of oxidative cleavage of glycosidic bonds by enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) has profoundly changed our current understanding of enzymatic processes underlying the conversion of polysaccharides in the biosphere. LPMOs are truly unique enzymes, harboring a single copper atom in a solvent-exposed active site, allowing them to oxidize C-H bonds at the C1 and/or C4 carbon of glycosidic linkages found in recalcitrant, often crystalline polysaccharides such as cellulose and chitin. To catalyze this challenging reaction, LPMOs harness and control a powerful oxidative reaction that involves Fenton-like chemistry. In this essay, we first draw a brief portrait of the LPMO field, notably explaining the shift from the monooxygenase paradigm (i.e., using O2 as cosubstrate) to that of a peroxygenase (i.e., using H2O2). Then, we briefly review current understanding of how LPMOs generate and control a hydroxyl radical (HO•) generated through Cu(I)-catalyzed H2O2 homolysis, and how this radical is used to create the proposed Cu(II)-oxyl species, abstracting hydrogen atom of the C-H bond. We also point at the complexity of analyzing redox reactions involving reactive oxygen species and address potential deficiencies in the interpretation of existing LPMO data. Being the first copper enzymes shown to enable site-specific Fenton-like chemistry, and maybe not the only ones, LPMOs may serve as a blueprint for future research on monocopper peroxygenases.
... They are classified into auxiliary activity (AA) protein families in Carbohydrate Active enZyme (CAZy) database (http://www.cazy.org/Auxiliary-Activities.html), in which AA9 family represents the fungal cellulose oxidizing LPMOs. The oxidative action of LPMOs was discovered approximately ten years ago in connection with their remarkable ability to improve saccharification of recalcitrant lignocellulosic materials (Harris et al., 2010;Müller et al., 2015). In contrast to endoglucanases, the AA9 family LPMOs can cleave internal linkages in crystalline celluloses, thus facilitating the degradation of most recalcitrant celluloses. ...
Cellulose oxidation and enzymatic treatments can be employed to reduce the energy consumption in production of cellulose nanofibrils (CNFs). Lytic polysaccharide monooxygenases (LPMOs) offer new biocatalytic tools for oxidative pretreatment of cellulosic fibres in this process. In this work the capability of LPMO enzymes to enhance fibrillation of bleached softwood kraft fibres was studied using two LPMO enzymes, i.e. C1/C4-oxidising AA9A from Trichoderma reesei (Tr AA9A) and C1-oxidising AA9E from Podospora anserina (Pa AA9E). The enzymatic treatments were carried out after mechanical pre-refining (grinding) of the pulp, which resulted in clear reduction of the intrinsic viscosity of the refined fibres compared to the previously published work, presumably due to better access of the enzymes on the fibre polysaccharides. The Tr AA9A treatments were carried out using different enzyme dosages (0.25–10 mg/g dry fibre) and fixed reaction time (24 h), resulting in fibres characterized by increased aldehyde content, which is in line with the C1/C4 oxidising activity of this enzyme. Comparison of the Tr AA9A to Pa AA9E with fixed enzyme dosage (2 mg/g dry fibre) and reaction time (24 h), showed that no remarkable increase in total charge of the cellulosic fibres could be obtained with either of the LPMO enzymes in these conditions. All the LPMO pretreatments improved fibrillation of mechanically refined fibres in microfluidization, seen as clearly lower residual fibre content and higher proportion nanosized material. In comparison of the Pa AA9E to Tr AA9A, clearly faster fibrillation was achieved with the Pa AA9E pretreatment. The mechanical and oxygen barrier properties of CNF films prepared from LPMO pretreated fibres were very similar to the reference CNF films while water vapour transmission rate was somewhat higher.
... Lytic polysaccharide monooxygenases LPMOs dramatically improve cellulase performance (Harris et al., 2010). These enzymes are classified into four families of the AA class. ...
... The reaction involves a divalent copper ion within the active site, in addition to molecular oxygen/hydrogen peroxide and externally provided electrons to potentiate LPMOs activity [8,9]. The oxidative cleavage of glycosidic bonds in crystalline polysaccharides by LPMOs improves the accessibility of substrates for glycoside hydrolases, thereby enhancing the overall efficiency of recalcitrant polysaccharides using complex enzymatic systems [7,[10][11][12]. 2 of 12 LPMOs are now classified as auxiliary activities (AA) and grouped into eight families (AA9-11, AA13-17) in the CAZy database (http://www.cazy.org/Auxiliary-Activities. html/ (accessed on 4 March 2022)) [7,[13][14][15][16][17][18]. LPMOs are widespread and highly abundant in the fungal and bacterial genomes, and more than 20 lpmo genes have been identified in the genomes of some ascomycetous and basidiomycetous fungi [19]. ...
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.
... Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that catalyze oxidative depolymerization of complex carbohydrate substrates, such as chitin or cellulose, in the presence of electron donors (Forsberg et al., 2011;Phillips, Beeson, Cate, & Marletta, 2011;Quinlan et al., 2011;Vaaje-Kolstad et al., 2010). LPMOs are known for a unique ability to act directly on crystalline surfaces of their substrates rather than on isolated and amorphous polysaccharide chains, thus exposing these substrates to the action of canonical hydrolytic enzymes (Harris et al., 2010;Vaaje-Kolstad et al., 2010;Vaaje-Kolstad, Horn, van Aalten, Synstad, & Eijsink, 2005). The contribution of LPMOs to the efficiency of enzymatic biomass conversion has attracted significant attention from industry. ...
Lytic polysaccharide monooxygenases (LPMOs) are unique redox enzymes capable of disrupting the crystalline surfaces of industry-relevant recalcitrant polysaccharides, such as chitin and cellulose. Historically, LPMOs were thought to be slow enzymes relying on O2 as the co-substrate, but it is now clear that these enzymes prefer H2O2, allowing for fast depolymerization of polysaccharides through a peroxygenase reaction. Thus, quantifying H2O2 in LPMO reaction set-ups is of a great interest. The horseradish peroxidase (HRP)/Amplex Red (AR) assay is one of the most popular and accessible tools for measuring hydrogen peroxide. This assay has been used in various types of biological and biochemical studies, including LPMO research, but suffers from pitfalls that need to be accounted for. In this Chapter, we discuss this method and its use for assessing the often rate-limiting in situ formation of H2O2 in LPMO reactions. We show that, after accounting for multiple potential side reactions, quantitative data on H2O2 production obtained with the HRP/Amplex Red assay provide useful clues for understanding the catalytic activity of LPMOs, including the impact of reductants and transition metal ions.
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.
Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes that oxidatively cleave the strong C-H bonds in recalcitrant polysaccharide substrates, thereby playing a crucial role in biomass degradation. Recently, LPMOs have also...
Lytic polysaccharide monooxygenases (LPMOs) have an essential role in global carbon cycle, industrial biomass processing and microbial pathogenicity by catalysing the oxidative cleavage of recalcitrant polysaccharides. Despite initially being considered monooxygenases, experimental and theoretical studies show that LPMOs are essentially peroxygenases, using a single copper ion and H2O2 for C-H bond oxygenation. Here, we examine LPMO catalysis, emphasizing key studies that have shaped our comprehension of their function, and address side and competing reactions that have partially obscured our understanding. Then, we compare this novel copper-peroxygenase reaction with reactions catalysed by haem iron enzymes, highlighting the different chemistries at play. We conclude by addressing some open questions surrounding LPMO catalysis, including the importance of peroxygenase and monooxygenase reactions in biological contexts, how LPMOs modulate copper site reactivity and potential protective mechanisms against oxidative damage.
Cellulose degradation is a critical process in soil ecosystems, playing a vital role in nutrient cycling and organic matter decomposition. Enzymatic degradation of cellulosic biomass is the most sustainable and green method of producing liquid biofuel. It has gained intensive research interest with future perspective as the majority of terrestrial lignocellulose biomass has a great potential to be used as a source of bioenergy. However, the recalcitrant nature of lignocellulose limits its use as a source of energy. Noteworthy enough, enzymatic conversion of cellulose biomass could be a leading future technology. Fungal enzymes play a central role in cellulose degradation. Our understanding of fungal cellulases has substantially redirected in the past few years with the discovery of a new class of enzymes and Cellulosome. Efforts have been made from time to time to develop an economically viable method of cellulose degradation. This review provides insights into the current state of knowledge regarding cellulose degradation in soil and identifies areas where further research is needed.
New materials’ synthesis and utilization have shown many critical challenges in healthcare and other industrial sectors as most of these materials are directly or indirectly developed from fossil fuel resources. Environmental regulations and sustainability concepts have promoted the use of natural compounds with unique structures and properties that can be biodegradable, biocompatible, and eco-friendly. In this context, nanocellulose (NC) utility in different sectors and industries is reported due to their unique properties including biocompatibility and antimicrobial characteristics. The bacterial nanocellulose (BNC)-based materials have been synthesized by bacterial cells and extracted from plant waste materials including pineapple plant waste biomass. These materials have been utilized in the form of nanofibers and nanocrystals. These materials are found to have excellent surface properties, low density, and good transparency, and are rich in hydroxyl groups for their modifications to other useful products. These materials are well utilized in different sectors including biomedical or health care centres, nanocomposite materials, supercapacitors, and polymer matrix production. This review explores different approaches for NC production from pineapple waste residues using biotechnological interventions, approaches for their modification, and wider applications in different sectors. Recent technological developments in NC production by enzymatic treatment are critically discussed. The utilization of pineapple waste-derived NC from a bioeconomic perspective is summarized in the paper. The chemical composition and properties of nanocellulose extracted from pineapple waste may have unique characteristics compared to other sources. Pineapple waste for nanocellulose production aligns with the principles of sustainability, waste reduction, and innovation, making it a promising and novel approach in the field of nanocellulose materials.
Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H2O2 splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.
Lytic polysaccharide monooxygenases (LPMOs) are taxonomically widespread copper-enzymes boosting biopolymers conversion (e.g. cellulose, chitin) in Nature. White-rot Polyporales, which are major fungal wood decayers, may possess up to 60 LPMO-encoding genes belonging to the auxiliary activities family 9 (AA9). Yet, the functional relevance of such multiplicity remains to be uncovered. Previous comparative transcriptomic studies of six Polyporales fungi grown on cellulosic substrates had shown the overexpression of numerous AA9-encoding genes, including some holding a C-terminal domain of unknown function (“X282”). Here, after carrying out structural predictions and phylogenetic analyses, we selected and characterized six AA9-X282s with different C-term modularities and atypical features hitherto unreported. Unexpectedly, after screening a large array of conditions, these AA9-X282s showed only weak binding properties to cellulose, and low to no cellulolytic oxidative activity. Strikingly, proteomic analysis revealed the presence of multiple phosphorylated residues at the surface of these AA9-X282s, including a conserved residue next to the copper site. Further analyses focusing on a 9 residues glycine-rich C-term extension suggested that it could hold phosphate-binding properties. Our results question the involvement of these AA9 proteins in the degradation of plant cell wall and open new avenues as to the divergence of function of some AA9 members.
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.
Polysaccharide monooxygenases (PMOs) and Glycoside Hydrolases (GH) play a crucial role in breaking down chitin and cellulose. Research on PMOs has mainly focused on their role in the production of bioethanol from biomass. Still, the potential of PMOs as virulence factors in microorganisms is not been fully studied. Some pathogenic bacteria secrete PMOs, which have been identified as virulence factors. The ability of Vibrio species to metabolize chitin is well-known, but the question of which Vibrio species contains the PMO gene remains unanswered. Materials and methods: Black tiger shrimp Penaeus monodon was collected from a culture pond in Long An, Vietnam. Thiosulfate-citrate-bile salts-sucrose and CHROMagar Vibrio agar were used to isolate, Vibrio bacteria from the shrimp samples. Mixed bases primers were designed to detect PMOs gene from Vibrio strains. Results: Total of 35 different strains of bacteria were isolated from the guts and pancreas of the shrimp sample. Based on biochemical tests, there were a total of 20 different Vibrio strains identified among 35 isolated strains. The PCR results showed that six isolates could have the PMOs gene. The partial 16S rDNA fragments of six isolate Vibrio strains were sequenced. Comparing 16S rDNA sequences with sequences available in NCBI databases revealed that all the sequences are Vibrio spp. 16S rDNA sequences.
Spindles are intracellular crystals of the fusolin protein that enhance the oral virulence of insect poxviruses by disruption of the larval chitinous peritrophic matrix. The enigmatic fusolin protein is classified as a lytic polysaccharide monooxygenase (LPMO) by sequence and structure. Although circumstantial evidence points towards a role for fusolin in chitin degradation, no biochemical data exist to verify this claim. In the present study we demonstrate that fusolin released from over 40-year-old spindles, stored for 10 years at 4 °C, are chitin-degrading LPMOs. Not only were the fusolins active after long-term storage, but, in its crystalline form, fusolin also withstood high temperature and oxidative stress, highlighting an extreme stability that is beneficial to viral persistence and desirable for potential biotechnology applications.
The lytic polysaccharide monooxygenases (LPMOs) comprise a super-family of copper enzymes that boost the depolymerisation of polysaccharides by oxidatively disrupting the glycosidic bonds connecting the sugar units. Industrial use of LPMOs for cellulose depolymerisation has already begun but is still far from reaching its full potential. One issue is that the LPMOs self-oxidise and thereby deactivate. The mechanism of this self-oxidation is unknown, but histidine residues coordinating to the copper atom are the most susceptible. An unusual methyl modification of the NE2 atom in one of the coordinating histidine residues has been proposed to have a protective role. Furthermore, substrate binding is also known to reduce oxidative damage. We here for the first time investigate the mechanism of histidine oxidation with combined quantum and molecular mechanical (QM/MM) calculations, with outset in intermediates previously shown to form from a reaction with peroxide and a reduced LPMO. We show that an intermediate with a [Cu–O] + moiety is sufficiently potent to oxidise the nearest C–H bond on both histidine residues, but methylation of the NE2 atom of His-1 increases the reaction barrier of this reaction. The substrate further increases the activation barrier. We also investigate a [Cu–OH] 2 + intermediate with a deprotonated tyrosine radical. This intermediate was previously proposed to have a protective role, and we also find it to have higher barriers than the corresponding a [Cu–O] + intermediate.
Graphical abstract
With the increasing awareness and concern about the dependency on fossil resources and the depletion of crude oil reserves, experts from industrial biotechnology, renewable resources, green chemistry, and biorefineries are stimulating the transition from the fossil-based to the bio-based economy. This text confronts scientific and economic challenges and strategies for making this crucial transition.
Renewable Resources for Biorefineries is the work of a strongly interdisciplinary authorship, offering perspectives from biology, chemistry, biochemical engineering, materials science, and industry. This unique approach provides an opportunity for a much broader coverage of biomass and valorisation than has been attempted in previous titles. This book also represents the fundamentally important technical and policy aspects of a bio-based economy, to ground this important science in a realistic and viable economic framework. Chapters in this book cover a diverse range of topics, including: advanced generation bioenergy sectors; biobased polymers and materials; chemical platform molecules; industrial crops and biorefineries; financing and policy for change; and valorisation of biomass waste streams.
This is an ideal book for upper level undergraduate and postgraduate students taking modules on Renewable resources, green chemistry, sustainable development, environmental science, agricultural science and environmental technology. It will also benefit industry professionals and product developers who are looking to improve economic and environmental ways to utilise renewable resources in current and future biorefineries.
Since the 1980s, plastic waste in the environment has been accumulating, and little is known about fungi biodegradation, especially in dry environments. Therefore, the research on plastic degradation technology is urgent. In this study, we demonstrated that Phanerochaete chrysosporium (P. chrysposporium), a typical species of white rot fungi, could react as a highly efficient biodegrader of polylactic acid (PLA), and 34.35 % of PLA degradation was obtained during 35-day incubation. A similar mass loss of 19.71 % could be achieved for polystyrene (PS) degradation. Here, we presented the visualization of the plastic deterioration process and their negative reciprocal on cell development, which may be caused by the challenge of using PS as a substrate. The RNA-seq analysis indicated that adaptations in energy metabolism and cellular defense were downregulated in the PS group, while lipid synthesis was upregulated in the PLA-treated group. Possible differentially expressed genes (DEG) of plastic degradation, such as hydrophobic proteins, lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac), Cytochrome P450 (CYP450), and genes involved in styrene or benzoic acid degradation pathways have been recorded, and we proposed a PS degradation pathway.
Lytic polysaccharide monooxygenase (LPMO) is known as an oxidatively cleaving enzyme in recalcitrant polysaccharide deconstruction. Herein, we report a novel AA10 LPMO derived from Bacillus subtilis (BsLPMO10A). A substrate specificity study revealed that the enzyme exhibited an extensive active-substrate spectrum, particularly for polysaccharides linked via β-1,4 glycosidic bonds, such as β-(Man1 → 4Man), β-(Glc1 → 4Glc) and β-(Xyl1 → 4Xyl). HPAEC-PAD and MALDI-TOF-MS analyses indicated that BsLPMO10A dominantly liberated native oligosaccharides with a degree of polymerization (DP) of 3-6 and C1-oxidized oligosaccharides ranging from DP3ox to DP6ox from mixed linkage glucans and beechwood xylan. Due to its synergistic action with a variety of glycoside hydrolases, including glucanase IDSGLUC5-38, xylanase TfXYN11-1, cellulase IDSGLUC5-11 and chitinase BtCHI18-1, BsLPMO10A dramatically accelerated glucan, xylan, cellulose and chitin saccharification. After co-reaction for 72 h, the reducing sugars in Icelandic moss lichenan, beechwood xylan, phosphoric acid swollen cellulose and chitin yielded 3176 ± 97, 7436 ± 165, 649 ± 44, and 2604 ± 130 μmol/L, which were 1.47-, 1.56-, 1.44- and 1.25-fold higher than those in the GHs alone groups, respectively (P < 0.001). In addition, the synergy of BsLPMO10A and GHs was further validated by the degradation of natural feedstuffs, the co-operation of BsLPMO10A and GHs released 3266 ± 182 and 1725 ± 107 μmol/L of reducing sugars from Oryza sativa L. and Arachis hypogaea L. straws, respectively, which were significantly higher than those produced by GHs alone (P < 0.001). Furthermore, BsLPMO10A also accelerated the liberation of reducing sugars from Celluclast® 1.5 L, a commercial cellulase cocktail, on filter paper, A. hypogaea L. and O. sativa L. straws by 49.58 % (P < 0.05), 72.19 % (P < 0.001) and 54.36 % (P < 0.05), respectively. This work has characterized BsLPMO10A with a broad active-substrate scope, providing a promising candidate for lignocellulosic biomass biorefinery.
The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.
The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.
An update to the FY 2005 assessment of the state of technical research progress toward biochemical process goals. This assessment contains research results from 2006 and 2007.
The U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA) are both strongly committed to expanding the role of biomass as an energy source. In particular, they support biomass fuels and products as a way to reduce the need for oil and gas imports; to support the growth of agriculture, forestry, and rural economies; and to foster major new domestic industries-- biorefineries--making a variety of fuels, chemicals, and other products. As part of this effort, the Biomass R&D Technical Advisory Committee, a panel established by the Congress to guide the future direction of federally funded biomass R&D, envisioned a 30 percent replacement of the current U.S. petroleum consumption with biofuels by 2030. Biomass--all plant and plant-derived materials including animal manure, not just starch, sugar, oil crops already used for food and energy--has great potential to provide renewable energy for America s future. Biomass recently surpassed hydropower as the largest domestic source of renewable energy and currently provides over 3 percent of the total energy consumption in the United States. In addition to the many benefits common to renewable energy, biomass is particularly attractive because it is the only current renewable source of liquid transportation fuel. This, of course, makes it invaluable in reducing oil imports--one of our most pressing energy needs. A key question, however, is how large a role could biomass play in responding to the nation's energy demands. Assuming that economic and financial policies and advances in conversion technologies make biomass fuels and products more economically viable, could the biorefinery industry be large enough to have a significant impact on energy supply and oil imports? Any and all contributions are certainly needed, but would the biomass potential be sufficiently large to justify the necessary capital replacements in the fuels and automobile sectors?
Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ 2 ) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing β-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and β-glucan. The endoglucanase activity on CMC and β-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.
The structural complexity and rigidity of cellulosic substrates have given rise to a phenomenal diversity of degradative enzymes — the cellulases. Cellulolytic microorganisms produce a wide variety of different catalytic and noncatalytic enzyme modules, which form the cellulases and act synergistically on their substrate. In some microbes, several types of cellulases are organized into an elaborate multifunctional supramolecular complex, known as the cellulosome. A combination of molecular genetic, biochemical, chemical, crystallographic and microscopic techniques are paving the way for new insights into both the structure of cellulose and the mechanisms of its hydrolysis.
The UniProt consortium was formed in 2002 by groups from the Swiss Institute of Bioinformatics (SIB), the European Bioinformatics Institute (EBI) and the Protein Information Resource (PIR) at Georgetown University, and soon afterwards the website http://www.uniprot.org was set up as a central entry point to UniProt resources. Requests to this address were redirected to one of the three organisations' websites. While these sites shared a set of static pages with general information about UniProt, their pages for searching and viewing data were different. To provide users with a consistent view and to cut the cost of maintaining three separate sites, the consortium decided to develop a common website for UniProt. Following several years of intense development and a year of public beta testing, the http://www.uniprot.org domain was switched to the newly developed site described in this paper in July 2008.
The UniProt consortium is the main provider of protein sequence and annotation data for much of the life sciences community. The http://www.uniprot.org website is the primary access point to this data and to documentation and basic tools for the data. These tools include full text and field-based text search, similarity search, multiple sequence alignment, batch retrieval and database identifier mapping. This paper discusses the design and implementation of the new website, which was released in July 2008, and shows how it improves data access for users with different levels of experience, as well as to machines for programmatic access.http://www.uniprot.org/ is open for both academic and commercial use. The site was built with open source tools and libraries. Feedback is very welcome and should be sent to help@uniprot.org.
The new UniProt website makes accessing and understanding UniProt easier than ever. The two main lessons learned are that getting the basics right for such a data provider website has huge benefits, but is not trivial and easy to underestimate, and that there is no substitute for using empirical data throughout the development process to decide on what is and what is not working for your users.
The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure. Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures.
Our understanding of the origins, the functions and/or the structures of biological sequences strongly depends on our ability to decipher the mechanisms of molecular evolution. These complex processes can be described through the comparison of homologous sequences in a phylogenetic framework. Moreover, phylogenetic inference provides sound statistical tools to exhibit the main features of molecular evolution from the analysis of actual sequences. This chapter focuses on phylogenetic tree estimation under the maximum likelihood (ML) principle. Phylogenies inferred under this probabilistic criterion are usually reliable and important biological hypotheses can be tested through the comparison of different models. Estimating ML phylogenies is computationally demanding, and careful examination of the results is warranted. This chapter focuses on PhyML, a software that implements recent ML phylogenetic methods and algorithms. We illustrate the strengths and pitfalls of this program through the analysis of a real data set. PhyML v3.0 is available from (http://atgc_montpellier.fr/phyml/).
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
We propose an improved version of the neighbor-joining (NJ) algorithm of Saitou and Nei. This new algorithm, BIONJ, follows the same agglomerative scheme as NJ, which consists of iteratively picking a pair of taxa, creating a new mode which represents the cluster of these taxa, and reducing the distance matrix by replacing both taxa by this node. Moreover, BIONJ uses a simple first-order model of the variances and covariances of evolutionary distance estimates. This model is well adapted when these estimates are obtained from aligned sequences. At each step it permits the selection, from the class of admissible reductions, of the reduction which minimizes the variance of the new distance matrix. In this way, we obtain better estimates to choose the pair of taxa to be agglomerated during the next steps. Moreover, in comparison with NJ's estimates, these estimates become better and better as the algorithm proceeds. BIONJ retains the good properties of NJ--especially its low run time. Computer simulations have been performed with 12-taxon model trees to determine BIONJ's efficiency. When the substitution rates are low (maximum pairwise divergence approximately 0.1 substitutions per site) or when they are constant among lineages, BIONJ is only slightly better than NJ. When the substitution rates are higher and vary among lineages,BIONJ clearly has better topological accuracy. In the latter case, for the model trees and the conditions of evolution tested, the topological error reduction is on the average around 20%. With highly-varying-rate trees and with high substitution rates (maximum pairwise divergence approximately 1.0 substitutions per site), the error reduction may even rise above 50%, while the probability of finding the correct tree may be augmented by as much as 15%.
A Trichoderma reesei cDNA encoding a previously unknown protein with a C-terminal cellulose-binding domain was obtained by complementation screening of a T. reesei cDNA library in a sec1 yeast mutant impaired in protein secretion. The T. reesei protein shows amino acid similarity over its entire length to the Agaricus bisporus cellulose-induced protein CEL1 whose function is not known. These two proteins form a new glycosyl hydrolase family, number 61. Expression of the T. reesei cDNA in yeast showed that it encoded a protein with endoglucanase activity and thus the protein was named EGIV and the corresponding gene egl4. Polyclonal antibodies were prepared against EGIV produced in Escherichia coli and detected a 56-kDa protein in the T. reesei culture supernatant. Northern hybridisation revealed that T. reesei egl4 is regulated in the same manner as other cellulase genes of this fungus.
A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.
Obtaining an electron-density map from X-ray diffraction data can be difficult and time-consuming even after the data have been collected, largely because MIR and MAD structure determinations currently require many subjective evaluations of the qualities of trial heavy-atom partial structures before a correct heavy-atom solution is obtained. A set of criteria for evaluating the quality of heavy-atom partial solutions in macromolecular crystallography have been developed. These have allowed the conversion of the crystal structure-solution process into an optimization problem and have allowed its automation. The SOLVE software has been used to solve MAD data sets with as many as 52 selenium sites in the asymmetric unit. The automated structure-solution process developed is a major step towards the fully automated structure-determination, model-building and refinement procedure which is needed for genomic scale structure determinations.
Cellulolytic proteins form a complex of enzymes that work together to depolymerize cellulose to the soluble products cellobiose
and glucose. Fundamental studies on their molecular mechanisms have been facilitated by advances in molecular biology. These
studies have shown homology between cellulases from different microorganisms, and common mechanisms between enzymes whose
modes of action have sometimes been viewed as being different, as suggested by the distribution of soluble products. A more
complete picture of the cellulolytic action of these proteins has emerged and combines the physical and chemical characteristics
of solid cellulose substrates with the specialized structure and function of the cellulases that break it down. This chapter
combines the fundamentals of cellulose structure with enzyme function in a manner that relates the cellulose binding and biochemical
kinetics at the catalytic site of the proteins to the macroscopic behavior of cellulase enzyme systems.
Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20 degrees C and a maximum temperature of growth extending up to 60 to 62 degrees C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45 degrees C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62 degrees C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.
DaliLite is a program for pairwise structure
comparison and for structure database searching. It is a standalone
version of the search engine of the popular Dali server. A web
interface is provided to view the results, multiple alignments and 3D
superimpositions of structures.
Availability: DaliLite has been ported to the Linux and Irix
operating systems and can be compiled in many other UNIX operating
systems. It is found at http://www.embl-ebi.ac.uk/dali/DaliLite.
Contact: holm{at}embl-ebi.ac.uk
Polysaccharide degrading enzymes from commercial T. reesei broth have been subjected to "fingerprint" analysis by high-resolution 2-D gel electrophoresis. Forty-five spots from 11 x 25 cm Pharmacia gels have been analyzed by LC-MS/MS and the resulting peptide sequences were compared to existing databases. Understanding the roles and relationships of component enzymes from the T. reesei cellulase system acting on complex substrates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. These studies suggest follow-on work comparing induced and noninduced T. reesei cells at the proteome level, which may elucidate substrate-specific gene regulation and response.
Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.
A multiple sequence alignment program, MAFFT, has been developed. The CPU time is drastically reduced as compared with existing
methods. MAFFT includes two novel techniques. (i) Homo logous regions are rapidly identified by the fast Fourier transform
(FFT), in which an amino acid sequence is converted to a sequence composed of volume and polarity values of each amino acid
residue. (ii) We propose a simplified scoring system that performs well for reducing CPU time and increasing the accuracy
of alignments even for sequences having large insertions or extensions as well as distantly related sequences of similar length.
Two different heuristics, the progressive method (FFT‐NS‐2) and the iterative refinement method (FFT‐NS‐i), are implemented
in MAFFT. The performances of FFT‐NS‐2 and FFT‐NS‐i were compared with other methods by computer simulations and benchmark
tests; the CPU time of FFT‐NS‐2 is drastically reduced as compared with CLUSTALW with comparable accuracy. FFT‐NS‐i is over
100 times faster than T‐COFFEE, when the number of input sequences exceeds 60, without sacrificing the accuracy.
The structures of superconducting Tl2Ba2CaCu2O8 (T-c similar to 110 K) and YBa2Cu4O8 (T-c similar to 70 K) were investigated by single-crystal X-ray diffraction techniques in a wide temperature range. Some of the structure parameters show anomalous behaviour in the vicinity of the critical temperature. The most pronounced structural change is the shift of the apical O atoms in CuO5 pyramids towards the CuO2 planes, indicating charge redistribution near T-c. This behaviour of the apical O atoms seems to be the common structural feature of hole-doped high-T-c superconductors. Another effect of charge redistribution at T-c concerns a very small decrease of in-plane Cu-O distances and/or O-O distances in CuO5 pyramids. Both effects represent an averaged structure response to charge (hole) redistribution upon the phase transition to the superconducting state.
The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.
The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.
A multiple sequence alignment program, MAFFT, has been developed. The CPU time is drastically reduced as compared with existing methods. MAFFT includes two novel techniques. (i) Homo logous regions are rapidly identified by the fast Fourier transform (FFT), in which an amino acid sequence is converted to a sequence composed of volume and polarity values of each amino acid residue. (ii) We propose a simplified scoring system that performs well for reducing CPU time and increasing the accuracy of alignments even for sequences having large insertions or extensions as well as distantly related sequences of similar length. Two different heuristics, the progressive method (FFT-NS-2) and the iterative refinement method (FFT-NS-i), are implemented in MAFFT. The performances of FFT-NS-2 and FFT-NS-i were compared with other methods by computer simulations and benchmark tests; the CPU time of FFT-NS-2 is drastically reduced as compared with CLUSTALW with comparable accuracy. FFT-NS-i is over 100 times faster than T-COFFEE, when the number of input sequences exceeds 60, without sacrificing the accuracy.
Six xylan-hydrolyzing enzymes have been isolated from the preparations Celloviridin G20x and Xybeten-Xyl, obtained earlier based on the strain Trichoderma longibrachiatum (Trichoderma reesei) TW-1. The enzymes isolated were represented by three xylanases (XYLs), XYL I (20 kDa, pI 5.5), XYL II (21 kDa, pI 9.5), XYL III (30 kDa, pI 9.1); endoglucanase I (EG I), an enzyme exhibiting xylanase activity (57 kDa, pI 4.6); and two exodepolymerases, β-xylosidase (β-XYL; 80 kDa, pI 4.5) and α-L-arabinofuranosidase I (α-L-AF I; 55 kDa, pI 7.4). The substrate specificity of the enzymes isolated was determined. XYL II exhibited maximum specific xylanase activity (190 U/mg). The content of the enzymes in the preparation was assessed. Maximum contributions to the total xylanase activities of preparations Celloviridin G20x and Xybeten-Xyl were made by EG I and XYL II, respectively. Effects of temperature and pH on the enzyme activities, their stabilities under various conditions, and the kinetics of exhaustive hydrolysis of glucuronoxylan and arabinoxylan were studied. Combinations of endodepolymerases (XYL I, XYL II, XYL III, or EG I) and exodepolymerases (α-L-AF I or β-XYL) produced synergistic effects on arabinoxylan cleavage. The reverse was the case when endodepolymerases, such as XYL I or EG I, were combined with α-L-AF I.
The SituationThe StrategyComparison of Petroleum and Biomass ChemistryThe Chemistry of the Lignocellulosic BiorefineryExamples of Integrated Biorefinery ApplicationsSummary
An algorithm is presented for the accurate and rapid generation of multiple protein sequence alignments from tertiary structure comparisons. A preliminary multiple sequence alignment is performed using sequence information, which then determines an initial superposition of the structures. A structure comparison algorithm is applied to all pairs of proteins in the superimposed set and a similarity tree calculated. Multiple sequence alignments are then generated by following the tree from the branches to the root. At each branchpoint of the tree, a structure-based sequence alignment and coordinate transformations are output, with the multiple alignment of all structures output at the root The algorithm encoded in STAMP (Structural Alignment of Multiple Proteins) is shown to give alignments in good agreement with published structural accounts within the dehydrogenase fold domains, globins, and serine proteinases.
In order to reduce the need for visual verification, two similarity indices are introduced to determine the quality of each generated structural alignment. Sc quantifies the global structural similarity between pairs or groups of proteins, whereas Pij′ provides a normalized measure of the confidence in the alignment of each residue. STAMP alignments have the quality of each alignment characterized by Sc and Pij′ values and thus provide a reproducible resource for studies of residue conservation within structural motifs.
Technoeconomic analysis has been used to guide the research and development of lignocellulosic biofuels production processes
for over two decades. Such analysis has served to identify the key technical barriers for these conversion processes so that
research can be targeted most effectively on the pertinent challenges. The tools and methodology used to develop conceptual
conversion processes and analyze their economics are presented here. In addition, the current process design and economic
results are described for dilute acid pretreatment followed by enzymatic hydrolysis and fermentation. Modeled ethanol costs
of 1.33/gallon (in consistent year 2007 dollars) are being targeted for this commercial scale corn stover conversion process in 2012. State of technology models, which take actual research results and project them to commercial scale, estimate an ethanol cost of1.33/gallon (in consistent year 2007 dollars) are being targeted for this commercial scale corn stover conversion process
in 2012. State of technology models, which take actual research results and project them to commercial scale, estimate an
ethanol cost of 2.43/gallon at present. In order to further reduce costs, process improvements must be made in several areas,
including pretreatment, enzymatic hydrolysis, and fermentation. As the biomass industry develops, new fuels and new feedstocks
are being researched. Technoeconomic analysis will play a key role in process development and targeting of technical and economic
barriers for these new fuels and feedstocks.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
Information pertaining to enzymatic hydrolysis of cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of cellulose, adsorption, cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.
The glycoside hydrolase (GH) family 61 is a long-recognized, but still recondite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H. jecorina. The best-quality crystals were formed in the presence of nickel, and the crystal structure was solved to 1.6 A resolution using a single-wavelength anomalous dispersion method with nickel as the source of anomalous scatter. Cel61B lacks a carbohydrate-binding module and is a single-domain protein that folds into a twisted beta-sandwich. A structure-aided sequence alignment of all GH family 61 proteins identified a highly conserved group of residues on the surface of Cel61B. Within this patch of mostly polar amino acids was a site occupied by the intramolecular nickel hexacoordinately bound in the solved structure. In the Cel61B structure, there is no easily identifiable carbohydrate-binding cleft or pocket or catalytic center of the types normally seen in GHs. A structural comparison search showed that the known structure most similar to Cel61B is that of CBP21 from the Gram-negative soil bacterium Serratia marcescens, a member of the carbohydrate-binding module family 33 proteins. A polar surface patch highly conserved in that structural family has been identified in CBP21 and shown to be involved in chitin binding and in the protein's enhancement of chitinase activities. The analysis of the Cel61B structure is discussed in light of our continuing research to better understand the activities and function of GH family 61.
Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
Acid hydrazides react in alkaline solutions with reducing carbohydrates to give yellow anions. The reaction with p-hydroxybenzoic acid hydrazide (PAHBAH), can be used in a simple colorimetric method to detect less than 1 μg glucose or similar sugar. Calcium and very high protein concentrations are the only interfering factors that have been found.
The wealth of information provided by the recent structure determinations of many different glycosyl hydrolases shows that the substrate specificity and the mode of action of these enzymes are governed by exquisite details of their three-dimensional structures rather than by their global fold.
The chapter focuses on the recent advances in understanding the structural and functional organization of individual cellulases, their regulation, and the ways in which the multiple enzyme components of cellulolytic systems cooperate. It overviews the cellulose structures because cellulose is more than a homopolymer of β-1 ,4 linked glucose units. An appreciation of its complex physical organization and its interactions with other plant cell wall components is central for understanding of the mechanisms of cellulase action. Cellulose nearly always occurs in close association with plant cell wall matrix polysaccharides so that enzymes such as xylanases are intimately involved in the attack of cellulose in vivo. Cellulases effect important changes to their substrate before releasing soluble products and the key to understanding cellulase action rest in the examination of these events. Progress in this area is limited by the availability of appropriate analytical tools although new techniques, such as atomic force microscopy are promising. The properties of cellulases are profoundly altered by the presence of trace enzyme contaminants. Future studies in vitro are proposed to be restricted to enzymes from recombinant sources. The reasons for the individual cellulolytic bacteria and fungi requiring many related cellulases with specificities that overlap is still not clear, but perhaps this is because the complexity of the substrates and of the task these microorganisms face is underestimated.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
We have developed an automatic algorithm STRIDE for protein secondary structure assignment from atomic coordinates based on the combined use of hydrogen bond energy and statistically derived backbone torsional angle information. Parameters of the pattern recognition procedure were optimized using designations provided by the crystallographers as a standard-of-truth. Comparison to the currently most widely used technique DSSP by Kabsch and Sander (Biopolymers 22:2577-2637, 1983) shows that STRIDE and DSSP assign secondary structural states in 58 and 31% of 226 protein chains in our data sample, respectively, in greater agreement with the specific residue-by-residue definitions provided by the discoverers of the structures while in 11% of the chains, the assignments are the same. STRIDE delineates every 11th helix and every 32nd strand more in accord with published assignments.
Enzymatic hydrolysis of glycosides can occur by one of two elementary mechanisms identified by the stereochemical outcome of the reaction, inversion or retention. The key active-site residues involved are a pair of carboxylic acids in each case, and strategies for their identification and for probing the details of their roles in catalysis have been developed through detailed kinetic analysis of mutants. Similarly the roles of other active-site residues have also been probed this way, and mutants have been developed that trap intermediates in catalysis, allowing the determination of the three-dimensional structures of several such key species. By manipulating the locations or even the presence of these carboxyl side chains in the active site, the mechanisms of several glycosidases have been completely changed, and this has allowed the development of "glycosynthases," mutant glycosidases that are capable of synthesizing oligosaccharides but unable to degrade them. Surprisingly little progress has been made on altering specificities through mutagenesis, although recent results suggest that gene shuffling coupled with effective screens will provide the most effective approach.
The crystal structure of Tm(5)Re(2)O(12), pentathulium dirhenium dodecaoxide, was determined by synchrotron diffraction on a reticular merohedral twin, revealing space group C2/m with a = 12.3717 (7), b = 5.6744 (3), c = 7.4805 (4) Å, beta = 107.816 (2) degrees and Z = 2. Distorted ReO(6) octahedra form chains with alternating rhenium-rhenium distances of 2.455 (1) and 3.219 (1) Å. Early reports on Ln(2)ReO(5) compounds are critically reviewed in the light of our results for Tm(5)Re(2)O(12).
There is widespread concern that increasing concentrations of carbon dioxide and other greenhouse gases in the earth's atmosphere will ultimately lead to climate changes. Recognizing the important role that fossil fuels have in the economies and lifestyles of people throughout the world, it is reasonable to ask if the global economy can be powered in ways that might have less impact on the environment because they discharge less carbon dioxide. A way of addressing this sensitive issue could be through the biodevelopment of biowaste as an alternative and renewable energy resource. Landfilling and incineration are popular ways to deal with biowaste but both cause negative environmental effects such as the use of valuable land and production of dangerous gases. The structural components of cells, cellulose and hemicellulose, make biowaste very susceptible for bioproduct development and biowaste offers biotechnology an opportunity to assist in maintaining environmental quality.
Glycosylation is a common post-translational modification that can add complexity to the proteome of many cell types. We used enzymatic and chemical methods of deglycosylation to treat a heavily glycosylated exoproteome sample from the filamentous fungus Trichoderma reesei. Deglycosylated samples were resolved on one-dimensional (1-D) and two-dimensional (2-D) gels in order to determine the effect of deglycosylation on the electrophoresis patterns and on the ability to identify proteins by peptide mass matching using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of in-gel tryptic digests. We found that deglycosylation of the protein sample resulted in different protein patterns on 1-D and 2-D gels, reduced the complexity of gel patterns, and enhanced the protein identification of some proteins via MALDI-TOF-MS. Deglycosylation with trifluoromethanesulfonic acid (TFMS) was found to be more effective than enzymatic treatments. These deglycosylation techniques may be employed in whole proteome analysis to locate glycosylated proteins and assist in their identification by MS.