Possibilities for recycling cellulases after use in cotton processing: part II: Separation of cellulases from reaction products and released dyestuffs by ultrafiltration

The adsorption and activity of a total cellulase (Trichoderma reesei) was measured and compared on undyed and dyed cotton fabrics. Recovery of enzymes from the reaction mixture and by desorption from the cotton substrate was evaluated. About 80% of the initial protein could be recovered. The removal of released products (soluble reducing sugars and dyes) from the treatment liquor and subsequent concentration of cellulase proteins was performed using an ultrafiltration membrane. Strong protein-dye interactions made it impossible to separate efficiently the dyes from the enzyme-containing treatment liquors. The use of surfactants did not enhance cellulase desorption from cotton fabric. Although anionic surfactants have a deactivating effect on cellulases, this effect seems to be reversible, since after ultrafiltration the cellulase activity was similar to that of enzymes desorbed with buffer only. Humicola insolens cellulases were shown to be much more sensitive to anionic surfactant than T. reesei cellulases. The use of cellulases that bind reversibly to cellulose is suggested for achieving more efficient cellulase recycling and for reducing backstaining by dye-cellulase complexes.     

An ultrastructural study of the interaction of a fungal endoglucanase from Humicola insolens with cotton fibres

Because cellulases are finding more applications in the textile and detergent industries, their effect on cotton fibres must be evaluated. For this purpose, the action of a recombinant cellulase, endoglucanase V from the fungus Humicola insolens, has been followed by scanning electron microscopy (SEM) in classical longitudinal views as well as in cross-sections of cotton fibres. The experiments were conducted at large enzyme dilution typical of conditions where cellulases are used for biopolishing, i.e. for the removal of defects created by mechanical abrasion. Endoglucanase V appears to restrict its action to the hydrolysis of the loose fibrils created at the surface of the fibres and no indication of extensive enzyme penetration and damage to the interior of the fibres could be detected by SEM. The adsorption sites for endoglucanase V on cotton fibres were examined by transmission electron microscopy (TEM) on ultrathin cross-sections after immuno-gold labeling of the enzyme. This approach showed that the enzymes did not penetrate the fibres but remained at their surface. The use of an immuno-gold labeled cellulase provides a new way to probe the surface features of cotton fibres This revised version was published online in August 2006 with corrections to the Cover Date.     

Reactivities of cellulases from fungi towards ribbon-type bacterial cellulose and band-shaped bacterial cellulose

We have investigated the reactivities of various cellulases onribbon-type bacterial cellulose (BC I) and band-shaped bacterial cellulose (BCII) so as to clarify the properties of different cellulases. BC I waseffectively hydrolyzed by exo-type cellulases from different fungi from twicetofour times as much as BC II, but endo-type cellulases showed little differencein reactivity on those substrates. One of the endo-type cellulases, EG II fromTrichoderma reesei, degraded BC II more rapidly thanexo-type cellulases even in the production of reducing sugars. The degree ofpolymerization (DP) of BC II was rapidly decreased by endo-type cellulases atanearly stage, while exo-type cellulases did not cause the decrease of DP atthe initial stage, though the decrease of DP was observed after an incubation of24 h. All exo-type cellulases adsorbed on BC I and BC II,whileendo-type cellulases except for EG II adsorbed slightly on both substrates. Itwas interesting to observe EG II adsorbed on BC I but not on BC II. It issuggested that the adsorption of enzyme on cellulose is important for thedegradation of BC I, but not for BC II. It is proposed that the ratio of aspecific activity of each enzyme between BC I and BC II represents thedifference in the mode of action of cellulase. Furthermore, the K _RW value, which we can calculate from thedecrease of DP/reducing sugar produced, is effective for discriminating themode of action of cellulase, especially the evaluation of randomness in thehydrolysis of cellulose by endo- and exo-type cellulases.     

Determination of cellulase colocalization on cellulose fiber with quantitative FRET measured by acceptor photobleaching and spectrally unmixing fluorescence microscopy

The determination of cellulase distribution on the surface of cellulose fiber is an important parameter to understand when determining the interaction between cellulase and cellulose and/or the cooperation of different types of cellulases during the enzymatic hydrolysis of cellulose. In this communication, a strategy is presented to quantitatively determine the cellulase colocalization using the fluorescence resonance energy transfer (FRET) methodology, which is based on acceptor photobleaching and spectrally unmixing fluorescence microscopy. FRET monitoring of cellulase colocalization was achieved by labeling cellulases with an appropriate pair of FRET dyes and by adopting an appropriate FRET model. We describe here that the adapted acceptor photobleaching FRET method can be successfully used to quantify cellulase colocalization regarding their binding to a cellulose fiber at a resolution <10 nm. This developed quantitative FRET method is promising for further studying the interactions between cellulase and cellulose and between different types of cellulases.     

The study of cell wall structure and cellulose–cellulase interactions through fluorescence microscopy

The efficient decomposition of biomass into carbohydrates for the sustainable generation of biofuels has become the focus of much research. Yet, limited understanding exists on how the enzymes that catalyze the biochemical conversion of biomass, such as cellulases, interact with cellulose microfibrils and how cellulose structure is changed by cellulolytic enzymes. This has spurred the application of high-resolution imaging techniques, such as atomic force microscopy or fluorescence microscopy, to visualize the biomolecular interactions and structural changes that occur at the micro/nanoscale. In particular, fluorescence microscopy offers advantages such as high sensitivity and the ability to monitor species under biologically relevant conditions. Furthermore, the introduction of techniques, such as single molecule or super-resolution fluorescence microscopy, has allowed imaging biomolecules and macromolecular structures with near molecular resolution. These advantages make fluorescence microscopy ideally suited for the study of cell wall structure and cellulose–cellulase interactions. The application of fluorescence microscopy has already yielded key insights into the arrangement of structural polysaccharides in the plant cell wall, the reversibility and binding kinetics of cellulases, their molecular motion on crystalline cellulose, and the structural changes that occur as cellulose is depolymerized by cellulases. Yet, the application of fluorescence to study cellulose–cellulase interactions remains limited. This review aims at (1) providing an overview of fluorescence microscopy techniques suitable for the study of cellulose–cellulase interactions; (2) the applications of these techniques to date and the key insights obtained; and (3) the opportunities for future studies of the interaction of cell wall degrading enzymes with cellulosic materials.     

Anaerobic digestion of lignocellulosic biomass and wastes

Anaerobic digestion represents one of several commercially viable processes to convert woody biomass, agricultural wastes, and municipal solid wastes to methane gas, a useful energy source. This process occurs in the absence of oxygen, and is substantially less energy intensive than aerobic biological processes designed for disposal purposes. The anaerobic conversion process is a result of the synergistic effects of various microorganisms, which serve as a consortium. The rate-limiting step of this conversion process has been identified as the hydrolysis of cellulose, the major polymeric component of most biomass and waste feedstocks. Improvements in process economics therefore rely on improving the kinetic and physicochemical characteristics of cellulose degrading enzymes. The most thoroughly studied cellulase enzymes are produced by aerobic fungi, namely Trichoderma reesei. However, the pH and temperature optima of fungal cellulases make them incompatible for use in anaerobic digestion systems, and the major populations of microorganisms involved in cellulase enzyme production under anaerobic digestion conditions are various bacterial producers. The current state of understanding of the major groups of bacterial cellulase producers is reviewed in this paper. Also addressed in this review are recently developed methods for the assessment of actual cellulase activity levels, reflective of the digester "hydrolytic potential," using a series of detergent extractive procedures.     

Cloning of Thermostable Cellulase Genes of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> and Their Secretive Expression in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.     

Synergism betweenClostridiwn Thermocellum cellulases cloned inEscherichia coli

We have obtained a synergistic effect during degradation of Avicel and filter paper by Clostridium thermocellum cellulases (two endoglucanases and one cellobiohydrolase) cloned in Escherichia coli. The highest degree of synergism was found at early stages of reaction, during the first 20 h: 2.5 and 2.9 on Avicel and filter paper, respectively. During combined action of all three cellulases the main product is cellobiose.     

Hydrolysis of Corn Stover by Talaromyces cellulolyticus Enzymes: Evaluation of the Residual Enzymes Activities Through the Process

The obtainment of sugars from lignocellulosic residues represents a sustainable and versatile platform for the production of a number of bio-based products. Cellulases are a family of enzymes which can effectively hydrolyze the biomass polysaccharides at mild conditions. Cellulolytic fungi belonging to the genera Trichoderma and Aspergillus are the most commonly source of commercial cellulases used so far. More recently, Talaromyces cellulolyticus was also scored as a promising cellulases producer. In comparison to the Trichoderma and Aspergillus systems, Talaromyces enzymes have been less investigated. The present research dealt with the conversion of steam-pretreated corn stover by commercial blend of T. cellulolyticus enzymes with respect to the common blends. The paper also investigated the stability of the enzyme preparation and tested the use of additives (namely Tween 80, Tween 20, and BSA) to improve the enzymes performances and the hydrolysis efficiency. The results indicated that, at the same process conditions, T. cellulolyticus cellulases were more effective and yielded 20% more sugars compared to control blends. Furthermore, the cellulase components displayed a synergistic interaction with hemicellulases. The results indicate that cellulases from T. cellulolyticus are less affected by the high dry matter consistency and the use of additives could increase the total activity by around 50% and β-glucosidase capacity by 10–15%.

     

β-Glucosidase production by Trichoderma reesei

The hydrolysis of cellulose to the water-soluble products cellobiose and glucose is achieved via synergistic action of cellulolytic proteins. The three types of enzymes involved in this process are endoglucanases, cellobiohydrolases, and β-glucosidases. One of the best fungal cellulase producers is Trichoderma reesei RUT C30. However, the amount of β-glucosidases secreted by this fungus is insufficient for effective cellulose conversion. We investigated the production of cellulases and β-glucosidases in shake-flask cultures by applying three pH-controlling strategies: (1) the pH of the production medium was adjusted to 5.8 after the addition of seed culture with no additional pH adjustment performed, (2) the pH was adjusted to 6.0 daily, and (3) the pH was maintained at 6.0 by the addition of Tris-maleate buffer to the growth medium. Different carbon sources—Solka Floc 200, glucose, lactose, and sorbitol—were added to standard Mandels nutrients. The lowest β-glucosidase activities were obtained when no pH adjustment was done regardless of the carbon source employed. Somewhat higher levels of β-glucosidase were measured in the culture filtrates when daily pH adjustment was carried out. The effect of buffering the culture medium on β-glucosidase liberation was most prominent when a carbon source inducing the production of other cellulases was applied.     

Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate

Conversion of lignocellulose to biofuels is partly inefficient due to the deleterious impact of cellulose crystallinity on enzymatic saccharification. We demonstrate how the synergistic activity of cellulases was enhanced by altering the hydrogen bond network within crystalline cellulose fibrils. We provide a molecular-scale explanation of these phenomena through molecular dynamics (MD) simulations and enzymatic assays. Ammonia transformed the naturally occurring crystalline allomorph I(β) to III(I), which led to a decrease in the number of cellulose intrasheet hydrogen bonds and an increase in the number of intersheet hydrogen bonds. This rearrangement of the hydrogen bond network within cellulose III(I), which increased the number of solvent-exposed glucan chain hydrogen bonds with water by ~50%, was accompanied by enhanced saccharification rates by up to 5-fold (closest to amorphous cellulose) and 60-70% lower maximum surface-bound cellulase capacity. The enhancement in apparent cellulase activity was attributed to the "amorphous-like" nature of the cellulose III(I) fibril surface that facilitated easier glucan chain extraction. Unrestricted substrate accessibility to active-site clefts of certain endocellulase families further accelerated deconstruction of cellulose III(I). Structural and dynamical features of cellulose III(I), revealed by MD simulations, gave additional insights into the role of cellulose crystal structure on fibril surface hydration that influences interfacial enzyme binding. Subtle alterations within the cellulose hydrogen bond network provide an attractive way to enhance its deconstruction and offer unique insight into the nature of cellulose recalcitrance. This approach can lead to unconventional pathways for development of novel pretreatments and engineered cellulases for cost-effective biofuels production.     

Cellulase Production Under Solid-State Fermentation by Trichoderma reesei RUT C30: Statistical Optimization of Process Parameters

Sugar cane bagasse was used as substrate for cellulase production using Trichoderma reesei RUT C30, and the culture parameters were optimized for enhancing cellulase yield. The culture parameters, such as incubation temperature, duration of incubation, and inducer concentration, were optimized for enhancing cellulase yield using a Box-Behnken experimental design. The optimal level of each parameter for maximum cellulase production by the fungus was determined. Predicted results showed that cellulase production was highest (25.6 FPAase units per gram dry substrate) when the inducer concentration was 0.331 ml/gds, and the incubation temperature and time were 33 degrees C and 67 h, respectively. Crude inducer generated by cellulase action was found to be very effective in inducing cellulases. Validation of predicted results was done, and the experimental values correlated well with that of the predicted.     

Automated filter paper assay for determination of cellulase activity

Recent developments in molecular breeding and directed evolution have promised great developments in industrial enzymes as demonstrated by exponential improvements in β-lactamase and green fluorescent protein (GFP). Detection of and screening for improved enzymes are relatively easy if the target enzyme is expressible in a suitable high-throughput screening host and a clearly defined and usable screen or selection is available, as with GFP and β-lactamase. Fungal cellulases, however, are difficult to measure and have limited expressibility in heterologous hosts. Furthermore, traditional cellulase assays are tedious and time-consuming. Multiple enzyme components, an insoluble substrate, and generally slow reaction rates have plagued cellulase researchers interested in creating cellulase mixtures with increased activities and/or enhanced biochemical properties. Although the International Union of Pure and Applied Chemists standard measure of cellulase activity, the filter paper assay (FPA), can be reproduced in most laboratories with some effort, this method has long been recognized for its complexity and susceptibility to operator error. Our current automated FPA method is based on a Cyberlabs C400 robotics deck equipped with customized incubation, reagent storage, and plate-reading capabilities that allow rapid evaluation of cellulases acting on cellulose and has a maximum throughput of 84 enzyme samples per day when performing the automated FPA.     

The Effect of Lignin Removal by Alkaline Peroxide Pretreatment on the Susceptibility of Corn Stover to Purified Cellulolytic and Xylanolytic Enzymes

Pretreatment of corn stover with alkaline peroxide (AP) at pH 11.5 resulted in reduction of lignin content in the residual solids as a function of increasing batch temperature. Scanning electron microscopy of these materials revealed notably more textured surfaces on the plant cell walls as a result of the delignifying pretreatment. As expected, digestion of the delignified samples with commercial cellulase preparations showed an inverse relationship between the content of lignin present in the residual solids after pretreatment and the extent of both glucan and xylan conversion achievable. Digestions with purified enzymes revealed that decreased lignin content in the pretreated solids did not significantly impact the extent of glucan conversion achievable by cellulases alone. Not until purified xylanolytic activities were included with the cellulases were significant improvements in glucan conversion realized. In addition, an inverse relationship was observed between lignin content after pretreatment and the extent of xylan conversion achievable in a 24-h period with the xylanolytic enzymes in the absence of the cellulases. This observation, coupled with the direct relationship between enzymatic xylan and glucan conversion observed in a number of cases, suggests that the presence of lignins may not directly occlude cellulose present in lignocelluloses but rather impact cellulase action indirectly by its association with xylan.     

A Simplified Filter Paper Assay Method of Cellulase Enzymes Based on HPLC Analysis

A simplified filter paper assay (FPA) method of cellulase enzymes was proposed based on high-performance liquid chromatography (HPLC) measurement. The method was according to the sum of glucose and cellobiose concentrations measured by HPLC that was able to be correlated with filter paper units (FPU) of the cellulase enzymes assayed by the traditional FPA method, regardless of the differences in the sources, activities, and components of the cellulases. This simple and quick assay method for the cellulase enzymes provided another parameter of the ratio of glucose to cellobiose (G/C ratio) representing the capacity of cellulase enzymes degrading cellulose into fermentable monomeric sugars.     

Thermophilic Bacillus coagulans Requires Less Cellulases for Simultaneous Saccharification and Fermentation of Cellulose to Products than Mesophilic Microbial Biocatalysts

Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g^?1 cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g^?1 cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.     

Use of Spectroscopic and Imaging Techniques to Evaluate Pretreated Sugarcane Bagasse as a Substrate for Cellulase Production Under Solid-State Fermentation

The enzymatic cocktail of cellulases is one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels and other chemicals. Here, the influence of liquid hot water, dilute acid, alkali, and combined acid/alkali pretreatments on sugarcane bagasse (SCB) used for cellulase production was investigated by means of spectroscopic and imaging techniques. Chemical composition and structural characteristics, such as crystallinity (determined by X-ray diffraction), functional groups (Fourier transform infrared spectroscopy), and microstructure (scanning electron microscopy), were used to correlate SCB pretreatments with enzymatic biosynthesis by a strain of the filamentous fungus Aspergillus niger under solid-state fermentation. The combined acid/alkali pretreatment resulted in a SCB with higher cellulose content (86.7 %). However, the high crystallinity (74 %) of the resulting biomass was detrimental to microbial uptake and enzyme production. SCB pretreated with liquid hot water yielded the highest filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), and xylanase activities (0.4, 14.9, and 26.1 U g^?1, respectively). The results showed that a suitable pretreatment for SCB to be used as a substrate for cellulase production should avoid severe conditions in order to preserve amorphous cellulose and to enhance the physical properties that assist microbial access.     

Utilization of waste cellulose—III. Comparative study of the activity of the cellulases of Trichoderma viride and Aspergillus niger towards different cellulosic substrates

Filter paper, carboxymethylcellulase and β-glucosidase activities have been determined and compared for cellulases originating from Trichoderma viride (TV) and Aspergillus niger (AN). The formation of glucose and of total reducing sugar has been measured as a function of time for the hydrolysis of cellulose I by the same quantity of FP units from TV, AN or a mixture of both strains. Long term efficiency is lower for AN but an important synergistic effect has been observed for the mixture of the enzymes. This synergistic action has been assigned to a better balance of endo- and exoglucanases and essentially to the addition to TV of thermally stable endoglucanases from AN. The β-glucosidases formed in large quantity by AN have been found to be thermally unstable and susceptible to product inhibition. They do not play any role in the observed synergistic action.     

Real-time observation of the swelling and hydrolysis of a single crystalline cellulose fiber catalyzed by cellulase 7B from Trichoderma reesei

The biodegradation of cellulose involves the enzymatic action of cellulases (endoglucanases), cellobiohydrolases (exoglucanases), and β-glucosidases that act synergistically. The rate and efficiency of enzymatic hydrolysis of crystalline cellulose in vitro decline markedly with time, limiting the large-scale, cost-effective production of cellulosic biofuels. Several factors have been suggested to contribute to this phenomenon, but there is considerable disagreement regarding the relative importance of each. These earlier investigations were hampered by the inability to observe the disruption of crystalline cellulose and its subsequent hydrolysis directly. Here, we show the application of high-resolution atomic force microscopy to observe the swelling of a single crystalline cellulose fiber and its-hydrolysis in real time directly as catalyzed by a single cellulase, the industrially important cellulase 7B from Trichoderma reesei. Volume changes, the root-mean-square roughness, and rates of hydrolysis of the surfaces of single fibers were determined directly from the images acquired over time. Hydrolysis dominated the early stage of the experiment, and swelling dominated the later stage. The high-resolution images revealed that the combined action of initial hydrolysis followed by swelling exposed individual microfibrils and bundles of microfibrils, resulting in the loosening of the fiber structure and the exposure of microfibrils at the fiber surface. Both the hydrolysis and swelling were catalyzed by the native cellulase; under the same conditions, its isolated carbohydrate-binding module did not cause changes to crystalline cellulose. We anticipate that the application of our AFM-based analysis on other cellulolytic enzymes, alone and in combination, will provide significant insight into the process of cellulose biodegradation and greatly facilitate its application for the efficient and economical production of cellulosic ethanol.     

A comparative study of different cellulase preparations in the enzymatic treatment of cotton fabrics

Twenty-nine cellulase preparations from different sources were compared interms of their abrasive activities (the ability to remove Indigo from denim) and their ability tosaccharify cellulose. Nodirectrelationship could be found between these two abilities. The preparations were divided into three groups: (1) with a high yield of reducing sugars after 24 h hydrolysis of Avicel cellulose but low abrasive activity; (2) universal cellulases that could both effectively hydrolyze cellulose and remove Indigo from denim; and (3) cellulase samples with high abrasive activity but low saccharification ability. Cellobiohydrolase (CBH) and xylanase were purified from different fungi by chromatofocusing on a Mono P column and subjected to limited proteolysis with papain yielding cellulose-binding and core (catalytic) domains. The adsorption ability and backstaining index of both CBH and xylanase core proteins were notably lower than the respective parameters for the in itial nondigested enzymes indicating that protein adsorption on the surface of cotton fibers is a crucial factor causing Indigo backstaining during the enzymatic denim washing procedure.