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The basidiomycete Coprinopsis cinerea produces five cellobiohydrolases belonging to glycoside hydrolase family 6 (GH6). Among these enzymes, C. cinerea cellulase 6C (CcCel6C), but not C. cinerea cellulase 6A (CcCel6A), can efficiently hydrolyze carboxymethyl cellulose and is constitutively expressed in C. cinerea. In contrast, CcCel6A possesses a cellulose-binding domain, and is strongly induced by cellobiose. Here, we determined the crystal structures of the CcCel6A catalytic domain complexed with a Hepes buffer molecule, with cellobiose, and with p-nitrophenyl β-D-cellotrioside (pNPG3). A notable feature of the GH6 cellobiohydrolases is that the active site is enclosed by two loops to form a tunnel, and the loops have been demonstrated to open and close in response to ligand binding. The enclosed tunnel of CcCel6A-Hepes is seen as the open form, whereas the tunnels of CcCel6A-cellobiose and CcCel6A-pNPG3 adopt the closed form. pNPG3 was not hydrolyzed by CcCel6A, and bound in subsites +1 to +4. On the basis of this observation, we constructed two mutants, CcCel6A D164A and CcCel6C D102A. Neither CcCel6A D164A nor CcCel6C D102A hydrolyze phosphoric acid-swollen cellulose. We have previously determined the crystal structures of CcCel6C unbound and in complex with ligand, both of which adopt the open form. In the present study, both CcCel6A and CcCel6C mutants were identified as the closed form. However, the motion angle of CcCel6C was more than 10-fold greater than that of CcCel6A. The width of the active site cleft of CcCel6C was narrowed, owing to a tweezer-like motion., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Aims: To develop a novel PCR-based method able to detect potential cellulolytic filamentous fungi and to classify them exploiting the amplification of the cellobiohydrolase gene (cbh-I) and its polymorphism., Methods and Results: A mixed approach including the combination of (i) fungal cultivation and isolation, (ii) classification of fungal isolates through the amplification of the cbh gene using a fluorescently labelled primer (f-CBH-PCR) and (iii) final fungal identification based on amplification and sequencing of the ITS1-5.8S rDNA-ITS2 region of the selected fungal strains was developed. By this approach, it was possible to screen 77 fungal strains belonging to 14 genera and 26 species., Conclusions: The f-CBH-PCR permitted the discrimination of fungal species, producing typical f-CBH profiles., Significance and Impact of the Study: In this study, the cbh gene was used as a preliminary classification tool able to differentiate among themselves the fungal members isolated from indoor museum items and surrounding environment. Such mixed approach consented the fast identification of all isolated fungal strains. The f-CBH-PCR method demonstrated its discrimination power, and it can be considered as a new molecular system suitable for the classification of fungal strains isolated from different environments., (© 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.)

A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I gene (cbh1). RNAi-mediated gene silencing was successfully used to further improve the recombinant lipase production via down-regulation of CBHI which comprised more than 60% of the total extracellular proteins in T. reesei. The gene and protein expression of CBHI and recombinant lipase were analyzed by real-time PCR, SDS-PAGE and activity assay. The results demonstrated that RNAi-mediated gene silencing could effectively suppress cbh1 gene expression and the reduction of CBHI could result in obvious improvement of heterologous lipase production. The reconstructed strains with decreased CBHI production exhibited 1.8- to 3.2-fold increase in lipase activity than that of parental strain. The study herein provided a feasible and advantageous method of increasing heterologous target gene expression in T. reesei through preventing the high expression of a specific endogenenous gene by RNA interference., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A critical role of the Family 7 cellobiohydrolase (Cel7A) carbohydrate binding domain (CBD) is to bind to a cellulose surface and increase the enzyme concentration on the surface. Several residues of Trichoderma reesei Cel7A CBD, including Y5, N29, Y31, Y32 and Q34, contribute to cellulose binding, as revealed by early experimental studies. To investigate the interactions between these important residues and cellulose, we applied a thermodynamic integration method to calculate the cellulose-Cel7A CBD binding free energy changes caused by Y5A, N29A, Y31A, Y32A and Q34A mutations. The experimental binding trend was successfully predicted, proving the effectiveness of the complex model. For the two polar residue mutants N29A and Q34A, the changes in the electrostatics are comparable to those of van der Waals, while for three Y to A mutants, the free energy differences mainly come from van der Waals interactions. However, in both cases, the electrostatics dominates the interactions between individual residues and cellulose. The side chains of these residues are rigidified after the complex is formed. The binding free energy changes for the two mutants Y5W and Y31W were also determined, and for these the van der Waals interaction was strengthened but the electrostatics was weakened.

A mechanistically based kinetic model for the enzymatic hydrolysis of cellulosic biomass has been developed that incorporates the distinct modes of action of cellulases on insoluble cellulose polymer chains. Cellulose depolymerization by an endoglucanase (endoglucanase I, EG(I) ) and an exoglucanase (cellobiohydrolase I, CBH(I)) is modeled using population-balance equations, which provide a kinetic description of the evolution of a polydisperse distribution of chain lengths. The cellulose substrate is assumed to have enzyme-accessible chains and inaccessible interior chains. EG(I) is assumed to randomly cleave insoluble cellulose chains. For CBH(I), distinct steps for adsorption, complexation, processive hydrolysis, and desorption are included in the mechanistic description. Population-balance models that employ continuous distributions track the evolution of the spectrum of chain lengths, and do not require solving equations for all chemical species present in the reacting mixture, resulting in computationally efficient simulations. The theoretical and mathematical development needed to describe the hydrolysis of insoluble cellulose chains embedded in a solid particle by EG(I) and CBH(I) is given in this article (Part I). Results for the time evolution of the distribution of chain sizes are provided for independent and combined enzyme hydrolysis. A companion article (Part II) incorporates this modeling framework to study cellulose conversion processes, specifically, solution kinetics, enzyme inhibition, and cooperative enzymatic action., (Copyright © 2011 Wiley Periodicals, Inc.)

The efficient deconstruction of lignocellulosic biomass remains a significant barrier to the commercialization of biofuels. Whereas most commercial plant cell-wall-degrading enzyme preparations used today are derived from fungi, the cellulosomal enzyme system from Clostridium thermocellum is an equally effective catalyst, yet of considerably different structure. A key difference between fungal enzyme systems and cellulosomal enzyme systems is that cellulosomal enzyme systems utilize self-assembled scaffolded multimodule enzymes to deconstruct biomass. Here, the possible function of the X1 modules in the complex multimodular enzyme system cellobiohydrolase A (CbhA) from C. thermocellum is explored. The crystal structures of the two X1 modules from C. thermocellum CbhA have been solved individually and together as one construct. The role that calcium may play in the stability of the X1 modules has also been investigated, as well as the possibility that they interact with each other. Furthermore, the results show that whereas the X1 modules do not seem to act as cellulose disruptors, they do aid in the thermostability of the CbhA complex, effectively allowing it to deconstruct cellulose at a higher temperature.

The projected cost for the enzymatic hydrolysis of cellulosic biomass continues to be a barrier for the commercial production of liquid transportation fuels from renewable feedstocks. Predictive models for the kinetics of the enzymatic reactions will enable an improved understanding of current limitations, such as the slow-down of the overall conversion rate, and may point the way for more efficient utilization of the enzymes in order to achieve higher conversion yields. A mechanistically based kinetic model for the enzymatic hydrolysis of cellulose was recently reported in Griggs et al. (2011) (Part I). In this article (Part II), the enzyme system is expanded to include solution-phase kinetics, particularly cellobiose-to-glucose conversion by β-glucosidase (βG), and novel adsorption and product inhibition schemes have been incorporated, based on current structural knowledge of the component enzymes. Model results show cases of cooperative and non-cooperative hydrolysis for an enzyme system consisting of EG(I) and CBH(I). The model is used to explore various potential rate-limiting phenomena, such as substrate accessibility, product inhibition, sterically hindered enzyme adsorption, and the molecular weight of the cellulose substrate., (Copyright © 2011 Wiley Periodicals, Inc.)

Efforts to improve the activity of cellulases, which catalyze the hydrolysis of insoluble cellulose, have been hindered by uncertainty surrounding the mechanistic origins of rate-limiting phenomena and by an incomplete understanding of complementary enzyme function. In particular, direct kinetic measurements of individual steps occurring after enzymes adsorb to the cellulose surface have proven to be experimentally elusive. This work describes an experimental and analytical approach, derived from a detailed mechanistic model of cellobiohydrolase action, for determining rates of initial- and processive-cut product generation by Trichoderma longibrachiatum cellobiohydrolase I (TlCel7A) as it catalyzes the hydrolysis of bacterial microcrystalline cellulose (BMCC) alone and in the presence of Talaromyces emersonii endoglucanase II (TemGH5). This analysis revealed that the rate of TlCel7A-catalyzed hydrolysis of crystalline cellulose is limited by the rate of enzyme complexation with glycan chains, which is shown to be equivalent to the rate of initial-cut product generation. This rate is enhanced in the presence of endoglucanase enzymes. The results confirm recent reports about the role of morphological obstacles in enzyme processivity and also provide the first direct evidence that processive length may be increased by the presence of companion enzymes, including small amounts of TemGH5. The findings of this work indicate that efforts to improve cellobiohydrolase activity should focus on enhancing the enzyme's ability to complex with cellulose chains, and the analysis employed provides a new technique for investigating the mechanism by which companion enzymes influence cellobiohydrolase activity.

The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An enzyme preparation has been produced on the basis of Penicillium canescens strains with the activity of cellibiohydrolase I, II; endo-1,4-beta-gluconase of Penicillium verruculosum; and beta-glucosidase of Aspergillus niger. It was shown that for the most effective hydrolysis of aspen wood pulp the optimal ratio of cellobiohydrolase and endo- 1,4-3-gluconase in enzyme preparations was 8 : 2 (by protein). It was also established that the homologous xylanase secreted by the Penicillium canescens fungus is a required component for the enzyme complex for hydrolysis of the hemicellulose matrix of aspen wood.

Cellulases hydrolyze β-1,4-glucosidic linkages of insoluble cellulose at the solid/liquid interface, generating soluble cellooligosaccharides. We describe here our method for real-time observation of the behavior of cellulase molecules on the substrate, using high-speed atomic force microscopy (HS-AFM). When glycoside hydrolase family 7 cellobiohydrolase from Trichoderma reesei (TrCel7A) was incubated with crystalline cellulose, many enzyme molecules were observed to move unidirectionally on the surface of the substrate by HS-AFM. The velocity of the moving molecules of TrCel7A on cellulose I crystals was estimated by means of image analysis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Due to its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum (T. harzianum) has considerable potential in biomass hydrolysis application. Cellulases from Trichoderma reesei have been widely used in studies of cellulose breakdown. However, cellulases from T. harzianum are less-studied enzymes that have not been characterized biophysically and biochemically as yet. Here, we examined the effects of pH and temperature on the secondary and tertiary structures, compactness, and enzymatic activity of cellobiohydrolase Cel7A from T. harzianum (Th Cel7A) using a number of biophysical and biochemical techniques. Our results show that pH and temperature perturbations affect Th Cel7A stability by two different mechanisms. Variations in pH modify protonation of the enzyme residues, directly affecting its activity, while leading to structural destabilization only at extreme pH limits. Temperature, on the other hand, has direct influence on mobility, fold, and compactness of the enzyme, causing unfolding of Th Cel7A just above the optimum temperature limit. Finally, we demonstrated that incubation with cellobiose, the product of the reaction and a competitive inhibitor, significantly increased the thermal stability of Th Cel7A. Our studies might provide insights into understanding, at a molecular level, the interplay between structure and activity of Th Cel7A at different pH and temperature conditions.

Natural cellulolytic enzyme systems as well as leading commercial cellulase cocktails are dominated by enzymes that degrade cellulose chains in a processive manner. Despite the abundance of processivity among natural cellulases, the molecular basis as well as the biotechnological implications of this mechanism are only partly understood. One of the major limitations lies in the fact that it is not straightforward to measure and quantify processivity in what essentially are biphasic experimental systems. Here, we describe and discuss both well-established methods and newer methods for measuring cellulase processivity. In addition, we discuss recent insights from studies on chitinases that may help direct further studies on processivity in cellulases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Glycosylation of cellobiose hydrolase I (CBHI), is a key step in the processing and degradation of cellulose. Here the pathways and barriers of the reaction are explored using the free energy from adaptive reaction coordinate forces (FEARCF) reaction dynamics method coupled with SCC-DFTB/MM. In many respects CBHI follows the expected general GH7 family mechanism that involves the Glu-X-Asp-X-X-Glu motif. However, critical electronic and conformational details, previously not known, were discovered through our computations. The central feature that ensures the success of the glycosylation reaction are the Glu212 nucleophile's hydrogen bond to the hydroxyl on C2, of the glucose in the -1 position of the cellulosic strand. This Glu212 function restricts the C2 hydroxyl in such a way as to favor the formation of the (4)E ring pucker of the -1 position glucose. A frontier molecular orbital analysis of the structures along the reaction surface proves the existence of an oxocarbenium ion, which has both transition state and intermediate character. The transition state structure is able to descend down the glycosylation pathway through the critical combination of Asp214 (HOMO), ring oxygen (LUMO), and Glu212 (HOMO), anomeric carbon (LUMO) interactions. Using the fully converged FEARCF SCC-DFTB/MM reaction surface, we find a barrier of 17.48 kcal/mol separating bound cellulose chain from the glycosylated CBHI. Taking recrossing into account gives k(cat) = 0.415 s(-1) for cellobiohydrolase glycosylation., (© 2011 American Chemical Society)

Cost-effective release of fermentable sugars from non-food biomass through biomass pretreatment/enzymatic hydrolysis is still the largest obstacle to second-generation biorefineries. Therefore, the hydrolysis performance of 21 bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase (BsCel5), family 9 Clostridium phytofermentans processive endoglucanase (CpCel9), and family 48 C. phytofermentans cellobiohydrolase (CpCel48) was studied on partially ordered low-accessibility microcrystalline cellulose (Avicel) and disordered high-accessibility regenerated amorphous cellulose (RAC). Faster hydrolysis rates and higher digestibilities were obtained on RAC than on Avicel. The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was the most important for high cellulose digestibility regardless of substrate type. This study provides important information for the construction of a minimal set of bacterial cellulases for the consolidated bioprocessing bacteria, such as Bacillus subtilis, for converting lignocellulose to biocommodities in a single step., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol., (© 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates in soil. The production of extracellular hydrolytic enzymes was studied during the growth of two saprotrophic basidiomycetes, Hypholoma fasciculare and Phanerochaete velutina, across the surface of nonsterile soil microcosms, along with the effects of these basidiomycetes on fungi and bacteria within the soil. Higher activities of α-glucosidase, β-glucosidase, cellobiohydrolase, β-xylosidase, phosphomonoesterase and phosphodiesterase, but not of arylsulphatase, were recorded beneath the mycelia. Despite the fact that H. fasciculare, with exploitative hyphal growth, produced much denser hyphal cover on the soil surface than P. velutina, with explorative growth, both fungi produced similar amounts of extracellular enzymes. In the areas where the mycelia of H. fasciculare and P. velutina interacted, the activities of N-acetylglucosaminidase, α-glucosidase and phosphomonoesterase, the enzymes potentially involved in hyphal cell wall damage, and the utilization of compounds released from damaged hyphae of interacting fungi, were particularly increased. No significant differences in fungal biomass were observed between basidiomycete-colonized and noncolonized soil, but bacterial biomass was reduced in soil with H. fasciculare. The increases in the activities of β-xylosidase, β-glucosidase, phosphomonoesterase and cellobiohydrolase with increasing fungal:bacterial biomass ratio indicate the positive effects of fungal enzymes on nutrient release and bacterial abundance, which is reflected in the positive correlation of bacterial and fungal biomass content., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Cellobiohydrolase I (Cel7A) of the fungus Trichoderma reesei (now classified as an anamorph of Hypocrea jecorina) hydrolyzes crystalline cellulose to soluble sugars, making it of key interest for producing fermentable sugars from biomass for biofuel production. The activity of the enzyme is pH-dependent, with its highest activity occurring at pH 4-5. To probe the response of the solution structure of Cel7A to changes in pH, we measured small angle neutron scattering of it in a series of solutions having pH values of 7.0, 6.0, 5.3, and 4.2. As the pH decreases from 7.0 to 5.3, the enzyme structure remains well defined, possessing a spatial differentiation between the cellulose binding domain and the catalytic core that only changes subtly. At pH 4.2, the solution conformation of the enzyme changes to a structure that is intermediate between a properly folded enzyme and a denatured, unfolded state, yet the secondary structure of the enzyme is essentially unaltered. The results indicate that at the pH of optimal activity, the catalytic core of the enzyme adopts a structure in which the compact packing typical of a fully folded polypeptide chain is disrupted and suggest that the increased range of structures afforded by this disordered state plays an important role in the increased activity of Cel7A through conformational selection.

Trichoderma reesei is a well-known cellulase producer and widely applied in enzyme industry. To increase its ability to efficiently decompose cellulose, the beta-glucosidase activity of its enzyme cocktail needs to be enhanced. In this study, a beta-glucosidase I coding sequence from Penicillium decumbens was ligated with the cellobiohydrolase I (cbh1) promoter of T. reesei and introduced into the genome of T. reesei strain Rut-C30 by Agrobacterium-mediated transformation. In comparison to that from the parent strain, the beta-glucosidase activity of the enzyme complexes from two selected transformants increased 6- to 8-fold and their filter paper activity (FPAs) was enhanced by 30% on average. The transformant's saccharifying ability towards pretreated cornstalk was also significantly enhanced. To further confirm the effect of heterologous beta-glucosidase on the cellulase activity of T. reesei, the heterologously expressed pBGL1 was purified and added to the enzyme complex produced by T. reesei Rut-C30. Supplementation of the Rut-C30 enzyme complex with pBGL1 brought about 80% increase of glucose yield during the saccharification of pretreated cornstalk. Our results indicated that the heterologous expression of a beta-glucosidase gene in T. reesei might produce balanced cellulase preparation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of crystalline cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic form of cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.

In order to establish which are the contribution of linear (total), hyperbolic (partial) or parabolic inhibitions by cellobiose, and also a special case of substrate inhibition, the kinetics of cellobiohydrolase Cel7A obtained from Trichoderma reesei was investigated. Values of kinetic parameters were estimated employing integrated forms of Michaelis-Menten equations through the use of non-linear regression, and criteria for selecting inhibition models are discussed. With cellobiose added at the beginning of the reaction, it was found that cellulose hydrolysis follows a kinetic model, which takes into account a mixed hyperbolic inhibition, by cellobiose with the following parameter values: K (m) 5.0 mM, K (ic) 0.029 mM, K (iu) 1.1 mM, k (cat) 3.6 h(-1) and k (cat') 0.2 h(-1). Cellulose hydrolysis without initial cellobiose added also follows the same inhibition model with similar values (4.7, 0.029 and 1.5 mM and 3.2 and 0.2 h(-1), respectively). According to Akaike information criterion, more complex models that take into account substrate and parabolic inhibitions do not increase the modulation performance of cellulose hydrolysis.

Saccharophagus degradans 2-40 is a marine gamma proteobacterium that can produce polyhydroxyalkanoates from lignocellulosic biomass using a complex cellulolytic system. This bacterium has been annotated to express three surface-associated β-glucosidases (Bgl3C, Ced3A, and Ced3B), two cytoplasmic β-glucosidases (Bgl1A and Bgl1B), and unusual for an aerobic bacterium, two cytoplasmic cellobiose/cellodextrin phosphorylases (Cep94A and Cep94B). Expression of the genes for each of the above enzymes was induced when cells were transferred into a medium containing Avicel as the major carbon source except for Bgl1B. Both hydrolytic and phosphorolytic degradation of cellobiose by crude cell lysates obtained from cellulose-grown cells were demonstrated and all of these activities were cell-associated. With the exception of Cep94B, each purified enzyme exhibited their annotated activity upon cloning and expression in E. coli. The five β-glucosidases hydrolyzed a variety of glucose derivatives containing β-1, (2, 4, or 6) linkages but did not act on any α-linked glucose derivatives. All but one β-glucosidases exhibited transglycosylation activity consistent with the formation of an enzyme-substrate intermediate. The biochemistry and expression of these cellobiases indicate that external hydrolysis by surface-associated β-glucosidases coupled with internal hydrolysis and phosphorolysis are all involved in the metabolism of cellobiose by this bacterium.

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha- helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-β-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cellulose in dead wood and as such they attracted considerable attention. The aims of this work were to quantify the activity and spatial distribution of extracellular enzymes in coarse wood colonised by the white-rot basidiomycete Fomes fomentarius and in adjacent fruitbodies of the fungus and to analyse the diversity of the fungal and bacterial community in a fungus-colonised wood and its potential effect on enzyme production by F. fomentarius. Fungus-colonised wood and fruitbodies were collected in low management intensity forests in the Czech Republic. There were significant differences in enzyme production by F. fomentarius between Betula pendula and Fagus sylvatica wood, the activity of cellulose and xylan-degrading enzymes was significantly higher in beech wood than in birch wood. Spatial analysis of a sample B. pendula log segment proved that F. fomentarius was the single fungal representative found in the log. There was a high level of spatial variability in the amount of fungal biomass detected, but no effects on enzyme activities were observed. Samples from the fruiting body showed high β-glucosidase and chitinase activities compared to wood samples. Significantly higher levels of xylanase and cellobiohydrolase were found in samples located near the fruitbody (proximal), and higher laccase and Mn-peroxidase activities were found in the distal ones. The microbial community in wood was dominated by the fungus (fungal to bacterial DNA ratio of 62-111). Bacterial abundance composition was lower in proximal than distal parts of wood by a factor of 24. These results show a significant level of spatial heterogeneity in coarse wood. One of the explanations may be the successive colonization of wood by the fungus: due to differential enzyme production, the rates of biodegradation of coarse wood are also spatially inhomogeneous.

A highly efficient cellobiohydrolase (CBH)-secreting basidiomycetous fungus, Agaricus arvensis KMJ623, was isolated and identified based on its morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular CBH was purified to homogeneity from A. arvencis culture supernatant using sequential chromatography. The relative molecular mass of A. arvencis CBH was determined to be 65 kDa by SDSPAGE and 130 kDa by size-exclusion chromatography, indicating that the enzyme is a dimer. A. arvencis CBH showed a catalytic efficiency (kcat/Km) of 31.8 mM⁻¹ s⁻¹ for p-nitrophenyl-beta-D-cellobioside, the highest level seen for CBH-producing microorganisms. Its internal amino acid sequences showed significant homology with CBHs from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, A. arvencis CBH is distinguished from other CBHs by its high catalytic efficiency.

QM/MD simulations are performed to study mutational effects on the glycosylation step of the oligosaccharide hydrolysis catalyzed by Trichoderma reesei cellobiohydrolase I. The potential of mean force along the reaction pathway is determined by the umbrella sampling method. A detailed mechanism is developed to illustrate the decrease in activity of the mutants. Our calculations demonstrate that (1) the E212Q mutation increases the overall activation barrier by ~4.0 kcal/mol, while the D214N mutation causes ~0.4 kcal/mol increase of the barrier, and (2) there is only one transition state identified in the wild type (WT) and D214N mutant, while two transition states exist in the E212Q mutant for the glycosylation process. The results explain the experimental observation that the E212Q mutant loses most of its hydrolysis capability, while the D214N mutant only reduces it slightly compared to the WT. Further analysis suggests that the proton transfer from Glu(217) to O(4) and the glycosidic bond cleavage between subsites +1 and -1 are concerted, facilitating the subsequent nucleophilic attack of Glu(212) on C(1)' in subsite -1. Our QM/MD study illustrates the importance of the prearrangement of the active site and provides atomic details of the enzymatic catalytic mechanism.

The celB gene of Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli to create a recombinant biocatalyst for hydrolyzing lignocellulosic biomass at high temperature. The GH5 domain of CelB hydrolyzed 4-nitrophenyl-β-D-cellobioside and carboxymethyl cellulose with optimum activity at pH 4.7-5.5 and 80°C. The recombinant GH5 and CBM3-GH5 constructs were both stable at 80°C with half-lives of 23 h and 39 h, respectively, and retained >94% activity after 48 h at 70°C. Enzymatic hydrolysis of corn stover and cellulose pretreated with the ionic liquid 1-ethyl-3-methylimidazolium acetate showed that GH5 and CBM3-GH5 primarily produce cellobiose, with product yields for CBM3-GH5 being 1.2- to 2-fold higher than those for GH5. Confocal microscopy of bound protein on cellulose confirmed tighter binding of CBM3-GH5 to cellulose than GH5, indicating that the enhancement of enzymatic activity on solid substrates may be due to the substrate binding activity of CBM3 domain., (Published by Elsevier Ltd.)

This is the first report of isolation of fungi present in fatty and defatted castor bean meal as well as the first of crop's selection to test the cellulolytic potential, in order to verify the diversity and potential of cellulolytic fungi in castor bean waste (Ricinus communis L.). For the screening on solid medium, it was used carboxymethylcellulose (CMC) as the sole carbon source. The microcrystalline cellulose (Avicel) was used as a substrate for submerged fermentation for production of cellobiohydrolase (FPase) and the CMC to produce endoglucanases (CMCase) and β-glycosidases (BG). 189 cultures of fungi were isolated, including 40 species of filamentous fungi and three yeasts. The Aspergillus was the most frequent found genus. Regarding the distribution of isolated species from defatted castor bean meal, the A. niger was the most frequent one; and within the fatty castor bean meal, the Emericela variecolor prevailed among other species. Among the 67 fungal cultures tested in the initial screening on solid media to assess the cellulolytic potential, 54 disclosed Cellulolytic Index (CI) ranging from 1.04 to 6.00 mm. The isolates were selected for enzyme production in liquid medium with values above 2.0 CI. They were obtained with A. japonicus URM5620 FPase activity (4.99 U/ml) and BG (0.05 U/ml), and Rhodotorula glutinis URM5724 activity of CMCase 3.58 U/ml. These cases occurred after 168 h of submersion for both species of fungi. In our study, we could conclude that the castor bean is a promising source of fungi capable of producing cellulolytic enzymes.

The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.

Processive cellobiohydrolases (CBHs) are the key components of fungal cellulase systems. Despite the wealth of structural data confirming the processive mode of action, little quantitative information on the processivity of CBHs is available. Here, we developed a method for measuring cellulase processivity. Sensitive fluorescence detection of enzyme-generated insoluble reducing groups on cellulose after labeling with diaminopyridine enabled quantification of the number of reducing-end exo-mode and endo-mode initiations. Both CBHs TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium employed reducing-end exo- and endo-mode initiation in parallel. Processivity values measured for TrCel7A and PcCel7D on cellulose hydrolysis were more than an order of magnitude lower than the values of intrinsic processivity that were found from the ratio of catalytic constant (k(cat)) and dissociation rate constant (k(off)). We propose that the length of the obstacle-free path available for a processive run on cellulose chain limits the processivity of CBHs on cellulose. TrCel7A and PcCel7D differed in their k(off) values, whereas the k(cat) values were similar. Furthermore, the k(off) values for endoglucanases (EGs) were much higher than the k(off) values for CBHs, whereas the k(cat) values for EGs and CBHs were within the same order of magnitude. These results suggest that the value of k(off) may be the primary target for the selection of cellulases.

A high cellobiohydrolase (CBH)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 according to the morphology and comparison of internal transcribed spacer rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, a maximum CBH activity of 2.6 U mg-protein(-1), one of the highest among CBH-producing microorganisms, was obtained. The optimum temperature and pH for CBH production were 30 °C and 4.0, respectively. The increased production of CBH in P. purpurogenum culture at 30 °C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. The internal amino acid sequences of P. purpurogenum CBH showed a significant homology with hydrolases from glycoside hydrolase family 7. The extracellular CBH was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast-protein liquid chromatography. The purified CBH was a monomeric protein with a molecular weight of 60 kDa and showed broad substrate specificity with maximum activity towards p-nitrophenyl β-D: -cellobiopyranoside. P. purpurogenum CBH showed t (1/2) value of 4 h at 60 °C and V (max) value of 11.9 μmol min(-1) mg-protein(-1) for p-nitrophenyl-D: -cellobiopyranoside. Although CBHs have been reported, the high specific activity distinguishes P. purpurogenum CBH.

Enzymatic liquefaction was studied by measuring continuously the flowability change of high-solids lignocellulose substrates using a real time viscometric method. Hydrolysis experiments of hydrothermally pretreated wheat straw were carried out with purified enzymes from Trichoderma reesei; Cel7A, Cel6A, Cel7B, Cel5A, Cel12A and Xyn11A. Results obtained at 15% (w/w) solids revealed that endoglucanases, in particular Cel5A, are the key enzymes to rapidly reduce the viscosity of lignocellulose substrate. Cellobiohydrolases had only minor and the xylanase practically no effect on the viscosity. Efficient, fast liquefaction was obtained already at a dosage of 1.5 mg of Cel5A/gdrysolids. Partial replacement or supplementation of Cel5A by the other major hydrolytic enzymes did not improve the liquefaction. The reduction of viscosity did not correlate with the saccharification obtained in the same reaction, suggesting that efficient liquefaction is rather dependent on the site than the frequency of enzymatic cleavages., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

High-pressure homogenization, as a way to further mechanically disrupt plant cells and cell walls compared to conventional blending, has been applied to thermally treated and comminuted carrot and tomato material in the presence of 5% olive oil. Mixes of both vegetables in a 1:1 ratio were also included. Both the effect of homogenization pressure and the effect of multiple process cycles were studied. The different microstructures generated were linked to different rheological properties analyzed by oscillatory and steady state measurements. The results showed that while carrot tissue requires a high shear input to be disrupted into cells and cell fragments, tomato cells were broken across the cell walls already at moderate shear input, and the nature of the tomato particles changed to amorphous aggregates, probably composed of cell contents and cell wall polymers. All the plant stabilized emulsions generated were stable against creaming under centrifugation. While for tomato a low-pressure multiple cycle and a high-pressure single-cycle process led to comparable microstructures and rheological properties, carrot showed different rheological properties after these treatments linked to differences in particle morphology. Mixes of carrot and tomato showed similar rheological properties after homogenizing in a single or in a split-stream process. Practical Application: Following consumers' demand, the food industry has shown a growing interest in manufacturing products free of gums and stabilizers, which are often perceived as artificial. By tailored processing, fresh plant material could be used to structure food products in a more natural way while increasing their nutritional quality.

A screening for cellobiohydrolase (CBH) activity was performed and Fomitopsis pinicola KMJ812 was selected for further characterization as it produced a high level of CBH activity. An extracellular CBH was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants. The molecular mass of the F. pinicola CBH was determined to be 64 kDa by SDS-PAGE and by size-exclusion chromatography, indicating that the enzyme is a monomer. The F. pinicola CBH showed a t1/2 value of 42 h at 70 degrees C and catalytic efficiency of 15.8 mM-1 S-1 (kcat/ Km) for p-nitrophenyl-beta-D-cellobioside, one of the highest levels seen for CBH-producing microorganisms. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, the F. pinicola CBH is distinguished from other CBHs by its high catalytic efficiency and thermostability.

We describe an efficient SCHEMA recombination-based approach for screening homologous enzymes to identify stabilizing amino acid sequence blocks. This approach has been used to generate active, thermostable cellobiohydrolase class I (CBH I) enzymes from the 390 625 possible chimeras that can be made by swapping eight blocks from five fungal homologs. Constructing and characterizing the parent enzymes and just 32 'monomeras' containing a single block from a homologous enzyme allowed stability contributions to be assigned to 36 of the 40 blocks from which the CBH I chimeras can be assembled. Sixteen of 16 predicted thermostable chimeras, with an average of 37 mutations relative to the closest parent, are more thermostable than the most stable parent CBH I, from the thermophilic fungus Talaromyces emersonii. Whereas none of the parent CBH Is were active >65°C, stable CBH I chimeras hydrolyzed solid cellulose at 70°C. In addition to providing a collection of diverse, thermostable CBH Is that can complement previously described stable CBH II chimeras (Heinzelman et al., Proc. Natl Acad. Sci. USA 2009;106:5610-5615) in formulating application-specific cellulase mixtures, the results show the utility of SCHEMA recombination for screening large swaths of natural enzyme sequence space for desirable amino acid blocks.

The hydrolysis of cellulose is the bottleneck in cellulosic ethanol production. The cellobiohydrolase CelS from Clostridium thermocellum catalyzes the hydrolysis of cello-oligosaccharides via inversion of the anomeric carbon. Here, to examine key features of the CelS-catalyzed reaction, QM/MM (SCCDFTB/MM) simulations are performed. The calculated free energy profile for the reaction possesses a 19 kcal/mol barrier. The results confirm the role of active site residue Glu87 as the general acid catalyst in the cleavage reaction and show that Asp255 may act as the general base. A feasible position in the reactant state of the water molecule responsible for nucleophilic attack is identified. Sugar ring distortion as the reaction progresses is quantified. The results provide a computational approach that may complement the experimental design of more efficient enzymes for biofuel production.

Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), β-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.

Understanding carbohydrate ring pucker is critical to rational design in materials and pharmaceuticals. Recently we have generalized our adaptive reaction coordinate force biasing method to perform calculations on multidimensional reaction coordinates. We termed this the Free Energies from Adaptive Reaction Coordinate Forces (FEARCF) method. Using FEARCF in SCC-DFTB QM/MM non-Boltzmann simulations, we are able to calculate multidimensional ring pucker free energies of conformation. Here we apply this to the six-membered glucopyranose ring located in an eight-membered β 1-4 linked octaose oligosaccharide (cellooctaose). The cellooctaose was built following the conformation of the saccharides bound to cellobiohydrolase I (CBHI) of Trichoderma reesei as reported in the 7CEL crystal structure obtained from the PDB. We calculate the free energy of ring puckering of the glucopyranose ring at the -1 position in vacuum, in water, and bound to the protein. We find that the protein induces (4)E and (4)H(3) conformations that are much more stable than the usually preferred (4)C(1) conformer. Furthermore, for the (4)H(3) conformation in the catalytic binding domain, there is significant electronic rearrangement that drives the structure toward the transition state of the glycosylation reaction.

The crystal structure of the carbohydrate-binding module (CBM) 4 Ig fused domain from the cellulosomal cellulase cellobiohydrolase A (CbhA) of Clostridium thermocellum was solved in complex with cellobiose at 2.11 A resolution. This is the first cellulosomal CBM4 crystal structure reported to date. It is similar to the previously solved noncellulosomal soluble oligosaccharide-binding CBM4 structures. However, this new structure possesses a significant feature-a binding site peptide loop with a tryptophan (Trp118) residing midway in the loop. Based on sequence alignment, this structural feature might be common to all cellulosomal clostridial CBM4 modules. Our results indicate that C. thermocellum CbhA CBM4 also has an extended binding pocket that can optimally bind to cellodextrins containing five or more sugar units. Molecular dynamics simulations and experimental binding studies with the Trp118Ala mutant suggest that Trp118 contributes to the binding and, possibly, the orientation of the module to soluble cellodextrins. Furthermore, the binding cleft aromatic residues Trp68 and Tyr110 play a crucial role in binding to bacterial microcrystalline cellulose (BMCC), amorphous cellulose, and soluble oligodextrins. Binding to BMCC is in disagreement with the structural features of the binding pocket, which does not support binding to the flat surface of crystalline cellulose, suggesting that CBM4 binds the amorphous part or the cellulose "whiskers" of BMCC. We propose that clostridial CBM4s have possibly evolved to bind the free-chain ends of crystalline cellulose in addition to their ability to bind soluble cellodextrins., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The study of cellulolytic enzymes has traditionally been carried out using endpoint measurements by quantitation of reaction products using high-performance liquid chromatography (HPLC) or overall determination of produced reducing ends. To measure catalytic activity, model substrates such as solubilized cellulose derivates, soluble chromogenic, and fluorogenic oligomeric substrates are often employed even though they do not reflect the natural insoluble substrate hydrolysis. Thermochemical methods using, for example, isothermal titration calorimetry (ITC) yield data where the primary observable is heat production. This can be converted to the rate of reaction and allows direct and continuous monitoring of the hydrolysis of complex substrates. To overcome the low molar enthalpy of the hydrolysis of the glycosidic bond, which is typically on the order of -2.5 kJ mol(-1), an enzymatic signal amplification method has been developed to measure even slow hydrolytically active enzymes such as cellobiohydrolases. This method is explained in detail for the amplification of the heat signal by more than 130 times by using glucose oxidase and catalase. The kinetics of this complex coupled reaction system is thoroughly investigated, and the potential use to generate kinetic models of enzymatic hydrolysis of unmodified cellulosic substrates is demonstrated., (2010 Elsevier Inc. All rights reserved.)

Despite intensive research, the mechanism of the rapid retardation in the rates of cellobiohydrolase (CBH) catalyzed cellulose hydrolysis is still not clear. Interpretation of the hydrolysis data has been complicated by the inability to measure the catalytic constants for CBH-s acting on cellulose. We developed a method for measuring the observed catalytic constant (k(obs)) for CBH catalyzed cellulose hydrolysis. It relies on in situ measurement of the concentration of CBH with the active site occupied by the cellulose chain. For that we followed the specific inhibition of the hydrolysis of para-nitrophenyl-beta-D-lactoside by cellulose. The method was applied to CBH-s TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium and their isolated catalytic domains. Bacterial microcrystalline cellulose, Avicel, amorphous cellulose, and lignocellulose were used as substrates. A rapid decrease of k(obs) in time was observed on all substrates. The k(obs) values for PcCel7D were about 1.5 times higher than those for TrCel7A. In case of both TrCel7A and PcCel7D, the k(obs) values for catalytic domains were similar to those for intact enzymes. A model where CBH action is limited by the average length of obstacle-free way on cellulose chain is proposed. Once formed, productive CBH-cellulose complex proceeds with a constant rate determined by the true catalytic constant. After encountering an obstacle CBH will "get stuck" and the rate of further cellulose hydrolysis will be governed by the dissociation rate constant (k(off)), which is low for processive CBH-s.

Changes in cellulose microstructure have been proposed to occur throughout hydrolysis that impact enzyme access and hydrolysis rates. However, there are very few direct observations of such changes in ongoing reactions. In this study, changes in the microstructure of cellulose are measured by simultaneous confocal and atomic force microscopy and are correlated to hydrolysis extents and quantities of bound enzyme in the reaction. Minimally processed and never-dried cellulose I was hydrolyzed by a purified cellobiohydrolase, Trichoderma reesei Cel7A. Early in the reaction ( approximately 30% hydrolysis), at high hydrolysis rates and high bound cellulase quantities, untwisting of cellulose microfibrils was observed. As the hydrolysis reaction neared completion (>80% hydrolysis), extensively thinned microfibrils (diameters of 3-5 nm) and channels (0.3-0.6 nm deep) along the lengths of the microfibrils were observed. The prominent microstructural changes in cellulose due to cellobiohydrolase action are discussed in the context of the overall hydrolysis reaction.

To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-beta-D: -lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.

We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and beta-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.

Cellobiohydrolase genes cbhI and cbhII were isolated from Trichoderma viride AS3.3711 and T. viride CICC 13038, respectively, using RT-PCR technique. The cbhI gene from T. viride AS3.3711 contains 1,542 nucleotides and encodes a 514-amino acid protein with a molecular weight of approximately 53.96 kDa. The cbhII gene from T. viride CICC 13038 was 1,413 bp in length encoding 471 amino acid residues with a molecular weight of approximately 49.55 kDa. The CBHI protein showed high homology with enzymes belonging to glycoside hydrolase family 7 and CBHII is a member of Glycoside hydrolase family 6. CBHI and CBHII play a role in the conversion of cellulose to glucose by cutting the disaccharide cellobiose from the non-reducing end of the cellulose polymer chain. The two cellobiohydrolase (CBHI, CBHII) genes were successfully expressed in Saccharomyces cerevisiae H158. Maximal activities of transformants Sc-cbhI and Sc-cbhII were 0.03 and 0.089 units ml(-1) under galactose induction, respectively. The optimal temperatures of the recombinant enzymes (CBHI, CBHII) were 60 and 70 degrees C, respectively. The optimal pHs of recombinant enzymes CBHI and CBHII were at pH 5.8 and 5.0, respectively.

Cellulose synthesis, but not its degradation, is generally thought to be required for plant cell growth. In this work, we cloned a dinoflagellate cellulase gene, dCel1, whose activities increased significantly in G(2)/M phase, in agreement with the significant drop of cellulose content reported previously. Cellulase inhibitors not only caused a delay in cell cycle progression at both the G(1) and G(2)/M phases in the dinoflagellate Crypthecodinium cohnii, but also induced a higher level of dCel1p expression. Immunostaining results revealed that dCel1p was mainly localized at the cell wall. Accordingly, the possible role of cellulase activity in cell cycle progression was tested by treating synchronized cells with exogenous dCelp and purified antibody, in experiments analogous to overexpression and knockdown analyses, respectively. Cell cycle advancement was observed in cells treated with exogenous dCel1p, whereas the addition of purified antibody resulted in a cell cycle delay. Furthermore, delaying the G(2)/M phase independently with antimicrotubule inhibitors caused an abrupt and reversible drop in cellulase protein level. Our results provide a conceptual framework for the coordination of cell wall degradation and reconstruction with cell cycle progression in organisms with cell walls. Since cellulase activity has a direct bearing on the cell size, the coupling between cellulase expression and cell cycle progression can also be considered as a feedback mechanism that regulates cell size.

Family 48 glycoside hydrolases (cellobiohydrolases) are among the most important cellulase components for crystalline cellulose hydrolysis mediated by cellulolytic bacteria. Open reading frame (Cphy_3368) of Clostridium phytofermentans ISDg encodes a putative family 48 glycoside hydrolase (CpCel48) with a family 3 cellulose-binding module. CpCel48 was successfully expressed as two soluble intracellular forms with or without a C-terminal His-tag in Escherichia coli and as a secretory active form in Bacillus subtilis. It was found that calcium ion enhanced activity and thermostability of the enzyme. CpCel48 had high activities of 15.1 U micromol(-1) on Avicel and 35.9 U micromol(-1) on regenerated amorphous cellulose (RAC) with cellobiose as a main product and cellotriose and cellotetraose as by-products. By contrast, it had very weak activities on soluble cellulose derivatives (e.g., carboxymethyl cellulose (CMC)) and did not significantly decrease the viscosity of the CMC solution. Cellotetraose was the smallest oligosaccharide substrate for CpCel48. Since processivity is a key characteristic for cellobiohydrolases, the new initial false/right attack model was developed for estimation of processivity by considering the enzyme's substrate specificity, the crystalline structure of homologous Cel48 enzymes, and the configuration of cellulose chains. The processivities of CpCel48 on Avicel and RAC were estimated to be approximately 3.5 and 6.0, respectively. Heterologous expression of secretory active cellobiohydrolase in B. subtilis is an important step for developing recombinant cellulolytic B. subtilis strains for low-cost production of advanced biofuels from cellulosic materials in a single step.

We report here a successful expression of a single-module GH-7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (Te Cel7A) in Saccharomyces cerevisiae. The heterologous expression system allowed structure-guided protein engineering to improve the thermostability and activity of Te Cel7A. Altogether six different mutants aimed at introducing additional disulphide bridges to the catalytic module of Te Cel7A were designed. These included addition of five individual S-S bridges in or between the loops extending from the beta-sandwich fold, and located either near the active site tunnel or forming the tunnel in Te Cel7A. A triple mutant containing the three best S-S mutations was also engineered. Three out of five single S-S mutants all had clearly improved thermostability which was also reflected as improved Avicel hydrolysis efficiency at 75 degrees C. The best mutant was the triple mutant whose unfolding temperature was improved by 9 degrees C leading to efficient microcrystalline cellulose hydrolysis at 80 degrees C. All the additional S-S bonds contributed mainly to the thermostability of the Te Cel7A, but one of the mutants (N54C/P191C) also showed, somewhat surprisingly, improved activity even at room temperature.

We probe the molecular-level behavior of the Family 1 carbohydrate-binding module (CBM) from a commonly studied fungal cellulase, the Family 7 cellobiohydrolase (Cel7A) from Trichoderma reesei, on the hydrophobic face of crystalline cellulose. With a fully atomistic model, we predict that the CBM alone exhibits regions of thermodynamic stability along a cellulose chain corresponding to a cellobiose unit, which is the catalytic product of the entire Cel7A enzyme. In addition, we determine which residues and the types of interactions that are responsible for the observed processivity length scale of the CBM: Y5, Q7, N29, and Y32. These results imply that the CBM can anchor the Cel7A enzyme at discrete points along a cellulose chain and thus aid in both recognizing cellulose chain ends for initial attachment to cellulose as well as aid in enzymatic catalysis by diffusing between stable wells on a length scale commensurate with the catalytic, processive cycle of Cel7A during cellulose hydrolysis. Comparison of other Family 1 CBMs show high functional homology to the four amino acids responsible for the processivity length scale on the surface of crystalline cellulose, which suggests that Family 1 CBMs may generally employ this type of approach for translation on the cellulose surface. Overall, this work provides further insight into the molecular-level mechanisms by which a CBM recognizes and interacts with cellulose.