Dissociative enzymes such as cellulases are greatly desired for a variety of applications in the food, fuel, and fiber industries. Cellulases and other cell wall–degrading enzymes are currently being engineered with improved traits for application in the breakdown of lignocellulosic biomass. Biochemical assays using these “designer” enzymes have traditionally been carried out using synthetic substrates such as crystalline bacterial microcellulose (BMCC). However, the use of synthetic substrates may not reflect the actual action of these cellulases on real plant biomass. We examined the potential of suspension cell walls from several plant species as possible alternatives for synthetic cellulose substrates. Suspension cells grow synchronously; hence, their cell walls are more uniform than those derived from mature plants. This work will help to establish a new assay system that is more genuine than using synthetic substrates. In addition to this, we have demonstrated that it is feasible to produce cellulases inexpensively and at high concentrations and activities in plants using a recombinant plant virus expression system. Our long-term goals are to use this system to develop tailored cocktails of cellulases that have been engineered to function optimally for specific tasks (i.e., the conversion of biomass into biofuel or the enhancement of nutrients available in livestock feed). The broad impact would be to provide a facile and economic system for generating industrial enzymes that offer green solutions to valorize biomass in industrialized communities and specifically in developing countries.

Cel48A of Thermobifida fusca (TfCel48A) is a processive exocellulase that contains an active site tunnel and digests lignocellulosic biomass via synergistic interactions between different cellulases. Cel48A possesses a number of aromatic amino acids lining the tunnel entrance, which are highly conserved across a diverse number of microbial species and appear to play a role in the selection and threading of individual strands of cellulose from highly recalcitrant substrates. In this study, we sought to further elucidate the roles of these tunnel entrance aromatic amino acids by creating a series of double mutants and examining their effect on TfCel48A activity, processivity, and synergistic interactions with the well-studied processive endocellulase TfCel9A. Our results provide further insight concerning the mechanism of Cel48A kinetics with soluble and insoluble substrates and could play an influential role in the application of Cel48A and other exocellulases for industrial purposes.

CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate-active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification and degradation of complex carbohydrates. CAZypedia is closely connected with the actively curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.

BACKGROUND:Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The point mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity.RESULTS:In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination.CONCLUSIONS:The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.

Thermostable α-amylases have many industrial applications and are therefore continuously explored from novel sources. We present the characterization of a novel putative α-amylase gene product (Tp-AmyS) cloned from Thermotoga petrophila. The purified recombinant enzyme is highly thermostable and able to hydrolyze starch into dextrin between 90 and 100°C, with optimum activity at 98°C and pH8.5. The activity increased in the presence of Rb1+, K1+ and Ca2+ ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7Å resolution showed common features of the GH-13 family, however was apparently found to be a dimer. Several residues from one monomer interacted with a docked acarbose, an inhibitor of Tp-AmyS, in the other monomer, suggesting catalytic cooperativity within the dimer. The most striking feature of the dimer was that it resembled the dimerization of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases.

Glycoside hydrolase (GH) family 48 is an understudied and increasingly important exoglucanase family found in the majority of bacterial cellulase systems. Moreover, many thermophilic enzyme systems contain GH48 enzymes. Deletion of GH48 enzymes in these microorganisms results in drastic reduction in biomass deconstruction. Surprisingly, given their importance for these microorganisms, GH48s have intrinsically low cellulolytic activity but even in low ratios synergize greatly with GH9 endoglucanases. In this study, we explore the structural and enzymatic diversity of these enzymes across a wide range of temperature optima. We have crystallized one new GH48 module from Bacillus pumilus in a complex with cellobiose and cellohexaose (BpumGH48). We compare this structure to other known GH48 enzymes in an attempt to understand GH48 structure/function relationships and draw general rules correlating amino acid sequences and secondary structures to thermostability in this GH family.

Cellulase activity on insoluble cellulose substrates declines as the substrate is modified. The role of structural changes that result in substrate recalcitrance, such as changes to cellulose crystallinity, requires further investigation. Crystallinity of cellulose samples with varying extents of digestion can only be compared meaningfully using a high throughput - Fourier transform infrared spectroscopy (HTS-FTIR) technique when the many variables involved are carefully controlled. Hence, changes to the HTS-FTIR sample preparation methods previously described in literature were necessary in order to obtain clean raw spectra and reliable measures of cellulose crystallinity. The sample preparation methods of residual cellulose after digestion by individual cellulases and a complex cellulase mixture from T. fusca were improved to remove extraneous overlapping signals, provide accurate extent of digestion, and correct errors caused by varying concentrations. These improved preparation methods enabled measurement of crystallinity index values of residual cellulose which did not show a correlation between cellulose crystallinity and the decline in cellulase activity.

ABSTRACTThe soil bacteriumCytophaga hutchinsoniiactively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes:cel5A,cel5B,cel5C,cel9A,cel9B,cel9C,cel9E, andcel9F. Each mutant retained the ability to digest crystalline cellulose, although the deletion ofcel9Ccaused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking bothcel5Bandcel9Cwere completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth ofC. hutchinsoniion cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane.IMPORTANCEThe bacteriumCytophaga hutchinsoniidigests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms.C. hutchinsoniiuses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels.

Even though only a small fraction of bacterial species can grow on cellulose as a sole carbon source, there is extensive diversity among cellulolytic bacteria for aerobic and anaerobic species. Out of 29 cultured bacterial phyla, 6 contain cellulolytic species. About half of the known cellulolytic bacteria are saprophytes that degrade dead plant material either in the soil, in the rumen, or in termites. Some soil saprophytes are aerobic and some are anaerobic. Many of the other cellulolytic bacteria are plant pathogens, which can degrade living plants. Many fungi degrade cellulose with most being aerobes, but a few anaerobic fungi degrade cellulose and they are found in the rumen. At this time there are only two reported cellulolytic archaea: Desulfurococcus fermentans and Thermogladius cellulolyticus, although there are other archaea that contain cellulase genes.

Molecular dynamics (MD) simulations were used to study substrate recognition by the family 48 exocellulase CelF from Clostridium cellulolyticum. It was hypothesized that residues around the entrance of the active site tunnel of this enzyme might serve to recognize and bind the substrate through an affinity for the cellulose monomer repeat unit, β-d-glucopyranose. Simulations were conducted of the catalytic domain of this enzyme surrounded by a concentrated solution of β-d-glucopyranose, and the full three-dimensional probability distribution for finding sugar molecules adjacent to the enzyme was calculated from the trajectory. A significant probability of finding the sugar stacked against the planar faces of Trp 310 and Trp 312 at the entrance of the active site tunnel was observed. Biotechnol. Bioeng. 2016;113: 1433–1440. © 2015 Wiley Periodicals, Inc.

Lignocellulose, the most abundant renewable carbon source on earth, is the logical candidate to replace fossil carbon as the major biofuel raw material. Nevertheless, the technologies needed to convert lignocellulose into soluble products that can then be utilized by the chemical or fuel industries face several challenges. Enzymatic hydrolysis is of major importance, and we review the progress made in fungal enzyme technology over the past few years with major emphasis on (i) the enzymes needed for the conversion of polysaccharides (cellulose and hemicellulose) into soluble products, (ii) the potential uses of lignin degradation products, and (iii) current progress and bottlenecks for the use of the soluble lignocellulose derivatives in emerging biorefineries.

Cellulases are currently the third largest industrial enzyme class in sales volume and are likely to become the largest when and if the cellulosic ethanol industry matures. Cellulases also are key enzymes in recycling plant cell walls in nature. They are very diverse enzymes, because there are at least 10 different protein folds for cellulases, and in addition there is extensive sequence variation in each of the 12 cellulase families. This diversity probably result from two factors; one is the difficulty in hydrolyzing crystalline cellulose, which is reported to have a half-life greater than 107 years at pH 7, and the other is the extreme heterogeneity of plant cell walls, which are the main substrates of cellulolytic organisms. Thus, cellulolytic organisms may evolve a set of cellulases that are optimal for the types of cell walls that they use. There is evidence for positive selection for some cellulase genes, which is consistent with cellulases being evolved for specific substrates.

Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in degrading biomass substrates. Although abundant, these enzymes have not been fully explored. The recently characterized AA10 family of bacterial lytic polysaccharide monooxygenases (LPMOs) performs oxidative cleavage of crystalline polysaccharide substrates. This oxidative activity synergistically enhances hydrolytic attack on crystalline material without change to soluble substrates, suggesting changes to insoluble substrate recalcitrance. The crystal structures of multiple bacterial AA10 LPMOs have been solved showing a surface-exposed metal ion binding site. This conserved structural feature agrees with data showing a bound copper ion is responsible for mediating oxidative activity.

Lignocellulosic biomass is digested in nature by the synergistic activities of enzymes with complementary properties, and understanding synergistic interactions will improve the efficiency of industrial biomass use for sustainable fuels and chemicals. Cel9A and Cel48A from a model bacterium, Thermobifida fusca (TfCel9A and TfCel48A, respectively), are two cellulases with different properties and have previously been shown to synergize well with each other. TfCel9A is a processive endocellulase with relatively high activity on crystalline cellulose. TfCel48A is a reducing end-directed exocellulase with very low activity on crystalline cellulose. Neither enzyme fits its respective role in the classical synergism model of enzymatic cellulose digestion. Using the results of time course, endpoint, and sequential addition activity assays, we propose a model of synergistic cooperation between the two cellulases. TfCel9A is most effective on fresh bacterial cellulose with a presumably uniform surface at the molecular level. Its processive activity likely erodes the surface and thus reduces its own activity. TfCel48A is able to hydrolyze the TfCel9A-modified substrate efficiently and replenish the uniform surface required by TfCel9A, creating a feedback mechanism. The model of synergistic interactions is comparable to an earlier proposed model for Trichoderma reesei Cel7A and Cel7B, but the roles of endo- and exocellulases are reversed, a finding which suggests that bacteria and fungi may have evolved different approaches to efficient biomass degradation.

Thermobifida fusca Cel6A is an endoglucanase with relatively high activity on crystalline cellulose. Trp 231 is a substrate-binding residue in the −2 sub-site that is within the active-site cleft. Mutating Trp 231 to Ala, Asp, or His causes significant decreases in activity on both oligosaccharides and cellulose. The largest decreases in activity are seen on short oligosaccharides, which is clearly due to altered oligosaccharide binding, as shown by docking studies. The mutation causes a significant drop in the production of soluble products from crystalline cellulose, with little change in the production of insoluble reducing ends. This result is due to weaker rebinding of the mutant enzyme to cellulose after hydrolysis before it completely dissociates, while the initial binding of the mutant catalytic domain is only slightly reduced. The Asp mutant enzyme had a much smaller increase in activity with increased temperature than the wild type, but it is not clear why this occurs. However, this mut...

Molecular dynamics simulations were used to study the possible catalytic role of an unusual conserved water-filled pore structure in the family 48 cellulase enzyme Cel48A from Thermobifida fusca. It was hypothesized that this pore serves as the pathway for the water molecules consumed in the hydrolysis catalyzed by the enzyme to reach the active site in a continuous stream to participate in the processive reactions. Theoretical mutants of this enzyme were created in which all of the residues lining the pore were made hydrophobic, which had the effect in molecular dynamics simulations of emptying the pore of water molecules and preventing any from passing through the pore on the simulation time scale. Mutants with smaller numbers of substitutions of this nature, which could be created experimentally by site-directed mutagenesis, were also identified from simulations, and these proteins were subsequently produced in Escherichia coli, expressed and purified, but were found to not fold in a manner similar to the wild type protein, preventing the determination of the importance of the water pore for activity. It is possible that the presence of a small vacuum in the pore was responsible for the instability of the mutants. In addition, alternate pathways were observed in the simulations that would allow water molecules to reach the active site of the enzyme, suggesting that the hypothesis that the pore has functional significance might be incorrect.

Cellobiohydrolases (CBHs) are typically major components of natural enzyme cocktails for biomass degradation. Their active sites are enclosed in a tunnel, enabling processive hydrolysis of cellulose chains. Glycoside hydrolase Family 6 (GH6) CBHs act from nonreducing ends by an inverting mechanism and are present in many cellulolytic fungi and bacteria. The bacterial Thermobifida fusca Cel6B (TfuCel6B) exhibits a longer and more enclosed active site tunnel than its fungal counterparts. Here, we determine the structures of two TfuCel6B mutants co-crystallized with cellobiose, D274A (catalytic acid), and the double mutant D226A/S232A, which targets the putative catalytic base and a conserved serine that binds the nucleophilic water. The ligand binding and the structure of the active site are retained when compared with the wild type structure, supporting the hypothesis that these residues are directly involved in catalysis. One structure exhibits crystallographic waters that enable construction of a model of the α-anomer product after hydrolysis. Interestingly, the product sites of TfuCel6B are completely enclosed by an "exit loop" not present in fungal GH6 CBHs and by an extended "bottom loop". From the structures, we hypothesize that either of the loops enclosing the product subsites in the TfuCel6B active site tunnel must open substantially for product release. With simulation, we demonstrate that both loops can readily open to allow product release with equal probability in solution or when the enzyme is engaged on cellulose. Overall, this study reveals new structural details of GH6 CBHs likely important for functional differences among enzymes from this important family.

Lignocellulosic biomass is a potential source of renewable, low-carbon-footprint liquid fuels. Biomass recalcitrance and enzyme cost are key challenges associated with the large-scale production of cellulosic fuel. Kinetic modeling of enzymatic cellulose digestion has been complicated by the heterogeneous nature of the substrate and by the fact that a true steady state cannot be attained. We present a two-parameter kinetic model based on the Michaelis-Menten scheme ( Michaelis, L., and Menten, M. L. ( 1913 ) Biochem. Z. , 49 , 333 - 369 ) with a time-dependent activity coefficient analogous to fractal-like kinetics formulated by Kopelman ( Kopelman, R. ( 1988 ) Science 241 , 1620 - 1626 ). We provide a mathematical derivation and experimental support to show that one of the parameters is a total activity coefficient and the other is an intrinsic constant that reflects the ability of the cellulases to overcome substrate recalcitrance. The model is applicable to individual cellulases and their mixtures at low-to-medium enzyme loads. Using biomass degrading enzymes from cellulolytic bacterium Thermobifida fusca , we show that the model can be used for mechanistic studies of enzymatic cellulose digestion. We also demonstrate that it applies to the crude supernatant of the widely studied cellulolytic fungus Trichoderma reesei ; thus it can be used to compare cellulases from different organisms. The two parameters may serve a similar role to Vmax, KM, and kcat in classical kinetics. A similar approach may be applicable to other enzymes with heterogeneous substrates and where a steady state is not achievable.