Trichoderma Cellulase Research Articles

Effects of a Delayed Harvest and Additives on the Fermentation Quality of Corn Stalk Silage

This study investigated the effects of harvest time, cellulase, lactic acid bacteria, and ensiling with alfalfa hay on the chemical parameters, bacterial community composition, and fermentation of corn stalk silage. Corn stalks were ensiled after the ear harvest at 0 days (D0) and 20 days (D20). Each harvest was treated with alfalfa hay [AL, 5% of fresh matter (FM)], Lactobacillus plantarum (Lp) (1 × 106 CFU/g of FM), Trichoderma cellulase (TC) (100 mg/kg of FM), or both Lp and TC (Lp+TC). The silos were opened after 60 days of fermentation. The dry matter (DM) content of raw materials increased from 18.79% to 28.32% with the harvest time. The acid detergent fiber (ADF) content also significantly increased (p < 0.05), while water-soluble carbohydrates (WSCs) and in vitro dry matter digestibility (IVDMD) significantly decreased (p < 0.05). After 60 days, the neutral detergent fiber (NDF) and ADF contents increased, and all chemical composition and fermentation state parameters of silages had significant differences (p < 0.05) with the harvest time. Compared with the control group, the AL treatment resulted in higher (p < 0.05) DM and crude protein (CP) contents, and the Lp+TC treatment resulted in a lower NDF (p < 0.05) content. For D0, the TC and Lp+TC treatments resulted in a higher WSC content and lower NDF and ADF contents than in D20. Meanwhile, the ammonia-nitrogen (NH3-N) content in each treatment was less than 10% of the DM. The pH values were all approximately 3.70. The AL treatment significantly increased the lactic acid (LA) content and lactic/acetic acid ratio (LA/AA). Additionally, the silages had a similar microbial environment before and after fermentation. We recommend harvesting corn stalks for silage immediately after the corn ear harvest. Adding cellulase improved the corn stalk feed value, and ensiling with alfalfa hay improved its fermentation quality.

Effect of cellulose crystallinity modification by starch gel treatment for improvement in ethanol fermentation rate by non-GM yeast cell factories

This paper studies the reduction of crystallinity degree (CD) of cellulose treated with starch gel (SG), and the correlation of CD with the fermentation efficiency of cellulose to fuel-grade ethanol. Cellulose bioconversion from wood sawdust, consisting of three processes, was conducted in the same batch (one-step). The XRD and TEM analysis revealed 11% reduction in cellulose CD after its treatment with SG. One-step bioconversion process was performed employing two cell factories (CF) of non-engineered S. cerevisiae. CFs contained non- engineered S. cerevisiae cells covered with either SG entrapping Trichoderma reesei or cellulases prepared in the laboratory and immobilized in SG. The consolidated fermentation of treated cellulose resulted in an increase of bioethanol concentration (60-90%) in 2-day fermentation and the maximum ethanol concentration reached was approximately 5mL/L (3.95g/L). The fermentation efficiency for grade-fuel ethanol production was improved by cellulose pretreatment using SG to achieve reduced CD.

Comparative analyses of enzymatic activity, structural study and docking of fungal cellulases

Aspergillus cellulase was observed to show superior hydrolysis of microcrystalline cellulose (Avicel PH101) when compared to Trichoderma cellulase. In silico physicochemical characterization revealed the acidic nature of selected fungal cellulases. GRAVY index for Aspergillus and Trichoderma cellulase was −0.314 and −0.374, respectively. Negative values of GRAVY index for both the fungal cellulases indicate that these enzymes have better interaction with water. Secondary structures such as alpha helices (34.44%) and random coils (54.21%) were found to be dominant in A. niger and T. longibrachiatum cellulase, respectively. Validation results for homology modeled 3D structures of selected cellulases indicated their fine quality. The analyses revealed that 96.7 and 92.4% residues of the constructed cellulase models of A. niger and T. longibrachiatum were placed in the favored region, respectively with >82 ERRAT overall quality factor and have >0.7 QMEAN 4 score. ProSA evaluation revealed that the Z-score for A. niger and T. longibrachiatum cellulase was −9.2 and −6.78, respectively. Energy minimization results revealed that the potential energies −7.140×105kJ/mol and −1.228×106kJ/mol were found remaining constant after 744 and 1431 steps for A. niger and T. longibrachiatum cellulases, respectively. Molecular docking study revealed that TRP 200, THR 201 and GLU 160 residues from A. niger cellulase and ASP 220, GLN 196, TYR 192 and TYR 168 residues from T. longibrachiatum cellulase interact with the cellulose. Studies such as this could prove to be helpful in terms of formulating cellulase enzyme for the purpose of industrial use.

Exploring the Synergy between Cellobiose Dehydrogenase from Phanerochaete chrysosporium and Cellulase from Trichoderma reesei

Recent demands for the production of lignocellulose biofuels boosted research on cellulase. Hydrolysis efficiency and production cost of cellulase are two bottlenecks in “biomass to biofuels” process. The Trichoderma cellulase mixture is one of the most commonly used enzymes for cellulosic hydrolysis. During hydrolytic process cellobiose accumulation causes feedback inhibition against most cellobiohydrolases and endoglucanases. In this study, we demonstrated the synergism effects between cellobiose dehydrogenase (CDH) and cellulase both in vitro and in vivo. The CDH from Phanerochaete chrysosporium was heterologously expressed in Pichia pastoris. Supplementation of the purified CDH in Trichoderma cellulase increased the cellulase activities. Especially β-glucosidase activity was increased by 30–100% varying at different time points. On the other hand, the cdh gene was heterologously expressed in Trichoderma reesei to explore the synergism between CDH and cellulases in vivo. The analyses of gene expression and enzymatic profiles of filter paper activity, carboxymethylcellulase (CMCase) and β-glucosidase show the increased cellulase activity and the enhanced cellulase production in the cdh-expressing strains. The results elucidate a possible mechanism for diminishing the cellobiose inhibition of cellulase by CDH. These findings provide a novel perspective to make more economic enzyme cocktails for commercial application or explore alternative strategies for generating cellulase-producing strains with higher efficiency.

Controlled enzymolysis preparation of nanocrystalline cellulose from pretreated cotton fibers

Natural cotton fibers were pretreated with DMSO, NaOH, or ultrasonic waves and hydrolyzed by cellulase (Trichoderma vride G) to prepare nanocrystalline cellulose (NCC). The as-prepared samples were characterized by TEM, DLS, XRD, and FT-IR. The influences of the pretreatment on the yield and morphology of NCC were investigated. The alterations of the crystalline phase and chemical structure of NCC were also measured during the enzymolysis process. The experimental results proved that the pretreated cotton fibers could be hydrolyzed by the Trichoderma cellulase to prepare a nanosized strip (10 to 40 nm ´ 70 to 280 nm) and grainy (20 nm or 6 nm) crystalline cellulose, in which the different morphologies resulted from the different manners of pretreatment. The unaltered crystalline phase and chemical composition of NCC prepared by enzymolysis of pretreated cotton fibers were measured. The highest yield of NCC reached was 32.4%.

Cellulolytic Microbes in the Yanbaru, a Subtropical Rainforest with an Endemic Biota on Okinawa Island, Japan

Cellulolytic microbes in the soil of the Yanbaru, a subtropical forest with an endemic biota, on Okinawa Island, were isolated and characterized in a search for novel microbial strains with biotechnological potential. Soil samples of the Yanbaru were suspended in sterilized water, inoculated on mineral salt agar overlaid with a filter paper as carbon source, and cultivated aerobically at 30 °C. After 2 weeks of cultivation, emerging colonies were isolated and subjected to phylogenetic and enzyme analyses. The phylogenetic analyses revealed bacterial and fungal isolates belonging to nine and three genera respectively. All isolates possessed cellulase activity, and several strains showed strong activity comparable to Trichoderma cellulase. Many isolates also exhibited xylanase activity.

Enzymatic hydrolysis of cellulose from steam-pretreated Lespedeza stalk (Lespedeza crytobotrya) with four Trichoderma cellulases

The hydrolytic potential of cellulases produced by Trichoderma viride, Trichoderma pseudokoningii, Trichoderma koningii, and Trichoderma reesei with addition of exogenous β-glucosidase was evaluated on cellulose of steam-pretreated Lespedeza. The T. viride enzyme achieved the highest glucose conversion (90.09%), while T. pseudokoningii cellulase achieved the highest ratio of cellobiose to glucose (4.94%) at the end of hydrolysis. Enzymatic adsorption on the substrate was evaluated on filter paper activity and β-glucosidase activity in the corresponding digest with the obtained T. cellulases. T. viride cellulase possessed an efficient adsorption-desorption on the substrate and reached the highest FPA difference (0.72 U/mL) among enzyme activities, indicating to its excellent hydrolysis capability. However, β-glucosidase in T. viride cellulase system showed close bonding on the substrate, suggesting that efficiencies of adsorption-desorption on the cellulose are different between the entire cellulase system and β-glucosidase. T. viride cellulase, with active endogenous β-glucosidase (1.60 U/mL), has compatible synergism with the additional exogenous β-glucosidase.

Evaluation of low molecular weight fractions and crude enzyme preparation from a Trichoderma cellulase complex as a treatment for fibrous feeds

An experiment was conducted to evaluate effects of an enzyme filtrate or a crude enzyme preparation on in vitro disappearance of six common fibrous feeds being: alfalfa hay, brome grass hay, cottonseed hulls, soybean hulls, wheat bran and wheat straw. A commercial liquid feed grade enzyme product originating from a strain of Trichoderma reesei was used. To obtain filtrates, the crude enzyme preparation was filtered through membranes with molecular weight cutoffs of 5 and 10 kDa. Feeds were exposed to enzymes, filtrates, or a buffer, for 20 h. Filtrates and crude enzyme preparation were diluted in 12.5 mM sodium citrate buffer. In vitro fermentation followed treatment of the feeds for 24 and 48 h. Release of glucose and total monosaccharides was quantified following treatment of alfalfa hay and soybean hulls at 2 and 24 h, and release of phenolic monomers was measured at 24 h of incubation. For in vitro dry matter disappearance (IVDMD), treatment x time interactions occurred for alfalfa hay ( P=0.01) and cottonseed hulls ( P=0.04). Higher ( P<0.01) IVDMD occurred at 48 h of incubation vs 24 h. The IVDMD of alfalfa hay and wheat bran was not affected by treatments. The crude enzyme preparation (CE) increased IVDMD compared to Ctrl for brome grass hay, cottonseed hulls, soybean hulls and wheat straw. Likewise, CE resulted in higher IVDMD than the 5 and 10 kDa treatments for the same feeds. Release of total monosaccharides from soybean hulls and alfalfa hay was higher ( P<0.001) after 24 h than after 2 h of treatment. Although the 10 kDa treatment resulted in release of monosaccharides from soybean hulls, the greatest response was from CE after the 24 h treatment. Phenolics were released from alfalfa hay and wheat straw as a result of the treatments during a single 24 h incubation, with the biggest response from the 10 kDa and CE. When the enzyme complex from a commercial T. reesei enzyme product was partitioned by molecular weight and used as a treatment for brome grass hay, cottonseed hulls, soybean hulls and wheat straw, both the filtrates and the crude enzyme preparation increased IVDMD, with the crude enzyme preparation treatment causing the biggest IVDMD response. Release of monosaccharides and phenolics from the substrates also occurred as a result of the treatments. The addition of fungal enzymes or their subunits may prove a viable management opportunity to increase rumen digestibility of forages to enhance animal performance.

A functionally based model for hydrolysis of cellulose by fungal cellulase

A new functionally based kinetic model for enzymatic hydrolysis of pure cellulose by the Trichoderma cellulase system is presented. The model represents the actions of cellobiohydrolases I, cellobiohydrolase II, and endoglucanase I; and incorporates two measurable and physically interpretable substrate parameters: the degree of polymerization (DP) and the fraction of beta-glucosidic bonds accessible to cellulase, F(a) (Zhang and Lynd, 2004). Initial enzyme-limited reaction rates simulated by the model are consistent with several important behaviors reported in the literature, including the effects of substrate characteristics on exoglucanase and endoglucanase activities; the degree of endo/exoglucanase synergy; the endoglucanase partition coefficient on hydrolysis rates; and enzyme loading on relative reaction rates for different substrates. This is the first cellulase kinetic model involving a single set of kinetic parameters that is successfully applied to a variety of cellulosic substrates, and the first that describes more than one behavior associated with enzymatic hydrolysis. The model has potential utility for data accommodation and design of industrial processes, structuring, testing, and extending understanding of cellulase enzyme systems when experimental date are available, and providing guidance for functional design of cellulase systems at a molecular scale. Opportunities to further refine cellulase kinetic models are discussed, including parameters that would benefit from further study.

Treatment of recycled fiber with Trichoderma cellulases

Recycled fibers have become increasingly important as raw material in the pulp and paper industry. Although utilization of secondary fibers has environmental and economical advantages, unfortunately the deteriorated drainability of secondary fibers causes decreased productivity. In this work, two commercial Trichoderma cellulase enzyme preparations were used to improve pulp properties of recycled paper. A blend of various cellulase components (Pergalase A40) was compared with endoglucanase III (IndiAge Super L) based on their effect on drainage, water retention value, fiber length distribution and paper properties (air permeability, tensile index, burst index, tear index). Enzyme characteristics were investigated in order to establish the industrial applicability. IndiAge Super L containing only EG III proved to be more stable than Pergalase A40 against fluctuation of temperature and pH. Experimental design (cross-classification) was used to examine the influence of enzyme dosage and treatment time on drainage. The effects of enzymatic treatments on pulp and paper properties showed that the type of enzyme component is probably very important. Moreover, existence or absence of cellulose-binding domain might play a determinative role.

Investigation of the cell-wall loosening protein expansin as a possible additive in the enzymatic saccharification of lignocellulosic biomass.

Expansins are a class of proteins that enable and regulate the extension of plant cell walls, thereby, among other possible functions, permitting the growth of plant cells (1–6). Although the precise molecular mechanism of expansin action remains to be determined, considerable evidence points to their mediating the exchange of interpolymer interactions (between cellulose and matrix-polymers, or between cellulose microfibrils themselves) that allows microfibrils to “creep” past each other in response to mechanical stress applied by the expanding cell inside (1,2). Because expansins have not been found to have any detectable glycosidase activity (7), it seems likely that the interactions being broken and then reformed under expansin influence are noncovalent, although the involvement of some extremely specific glycosyl transferase activity cannot be ruled out (1). Although expansins do not hydrolyze cellulose, they have been reported to enhance the hydrolysis of microcystalline cellulose by low levels of a mixed Trichoderma cellulase preparation (8).

Ion-exchange chromatographic purification and quantitative analysis of Trichoderma reesei cellulases cellobiohydrolase I, II and endoglucanase II by fast protein liquid chromatography

Trichoderma cellulases appear in several isoforms which makes their purification and analysis difficult. We used fast protein liquid chromatography (FPLC) to purify three major cellulases and to quantitate these enzymes in reconstituted mixtures during cellulose hydrolysis studies (in lack of specific substrates and because of the synergism between the enzymes such analysis is very difficult, if at all possible, with conventional activity measurements). For the analysis methods linear calibration was achieved from 10–15 pmol to 0.5–1 nmol (from 0.5–0.8 to 27–64 μg) for the different enzymes. Due to the high resolution chromatographic media used, our purification methods are simpler and quicker than the usual protocols for cellulase purification. Several isoforms of cellobiohydrolase (CBH) I were purified. The isoforms had different isoelectric points (p I) but their catalytic and adsorption properties were similar. A remarkable feature of CBH I and endoglucanase (EG) II was that their electrophoretically pure preparations gave double peaks during ion-exchange chromatography in certain pH intervals where the two peaks (probably representing two conformations) were transformed into each other by changing pH. This behaviour of cellulases has never been reported before and further explains the difficulties in cellulase purification.

The effect of oxidative pretreatment on cellulose degradation by Poria placenta and Trichoderma reesei cellulases

The possible role of hydrogen peroxide in brown-rot decay was investigated by studying the effects of pretreatment of spruce wood and microcrystalline Avicel cellulose with H2O2 and Fe2+ (Fenton's reagent) on the subsequent enzymatic hydrolysis of the substrates. A crude endoglucanase preparation from the brown-rot fungus Poria placenta, a purified endoglucanase from Trichoderma reesei and a commercial Trichoderma cellulase were used as enzymes. Avicel cellulose and spruce dust were depolymerized in the H2O2/Fe2+ treatment. Mainly hemicelluloses were lost in the treatment of spruce dust. The effect of the pretreatment on subsequent enzymatic hydrolysis was found to depend on the nature of the substrate and the enzyme preparation used. Pretreatment with H2O2/Fe2+ clearly increased the amount of enzymatic hydrolysis of spruce dust with both the endoglucanases and the commercial cellulase. In all cases the amount of hydrolysis was increased about threefold. The hydrolysis of Avicel with the endoglucanases was also enhanced, whereas the hydrolysis with the commercial cellulase was decreased.

Comparative evaluation of hydrolytic efficiency toward microcrystalline cellulose of Penicillium and Trichoderma cellulases

Composition, stability at different pH and temperatures and hydrolytic efficiency toward microcrystalline cellulose (MCC) of Penicillium cellulase were evaluated. The pH optimum (4·5-5·0) of filter paper activity (FPA) and temperature optimum (50–55°C) were determined. Maximum stability of FPA was observed at pH 4·0–5·0 and temperature 40–50°C. A minor contribution of stirring (3–4%) in inactivation of Penicillium preparation was shown. Kinetic parameters of MCC hydrolysis by Penicillium cellulase were determined at optimal conditions (pH 4·5, 50°C): K m = 50 g/l, V max = 330 μM/min per gram of protein. More effective saccharification of MCC by Penicillium preparations compared to Trichoderma reesei cellulases with the same level of total cellulolytic activity (FPA=1·0 U/ml) was demonstrated. Glucose concentration in 24 h hydrolysate was 41 g/l for Penicillium cellulase, 15 g/l for T. reesei (Privolzhski Fermentation Plant) and 26 g/l for T. reesei (Genencor). Hydrolysates of Penicillum cellulase contained only 9% of cellobiose compared to 24–40% of cellobiose for T. reesei cellulases. Factors contributing to superior properties of Penicillium cellulase — higher level of cellobiase activity and higher operational stability — are discussed.

Specific quantification of trichoderma reesei cellulases in reconstituted mixtures and its application to cellulase–cellulose binding studies

Specific quantifications of the major cellulolytic components of the Trichoderma reesei enzyme complex, i.e., endoglucanases I and III and cellobiohydrolases I and II, are described and, employing a defined mixture of these four cellulases reconstituted according to the composition of the native Trichoderma cellulase complex, used to determine the binding of each individual component onto filter paper. During substrate degradation by this enzyme mixture, the specific adsorption of each individual cellulase gradually increases and no preferential binding of one enzyme component in any particular phase of cellulose hydrolysis is found. T. reesei cellobiohydrolases I and II admixed with endoglucanases I and III represent a "full-value" cellulase system that is capable of degrading semicrystalline cellulose efficiently. In comparison with the crude Trichoderma enzyme complex, almost identical adsorption properties and similar hydrolytic efficiency are found for the reconstituted mixture. (c) 1994 John Wiley & Sons, Inc.

Production of Trichoderma cellulase in laboratory and pilot scale

Trichoderma reesei mutants QM 9414, MCG 77, MCG 80, RUT C 30, CL-847, VTT-D, and SVG are the preferred microorganism for the production of a complete cellulase which can hydrolyze crystalline cellulose. All these strains secrete cellulolytic proteins (exocellulases, endocellulases, and cellobiase) when they are grown on a mineral medium supplemented with pure cellulose (Solka Floc, cotton, Avicel) or pretreated lignocellulose (wood, straw). Some of the mutants produce cellulase with lactose as the sole carbon source. Submersed shake culture fermentations were mainly used for strain selection and preliminary optimization of culture conditions. Laboratory fermentations were performed in stirred tank reactors with volumes up to 3000 liters. On average, 250 mg of cellulase protein is formed per gram of metabolized carbon source, the enzyme has a specific activity of about 0·5 to 1·0 filter paper units per mg protein. The volumetric productivity rate is in the range of 50–150 FPU liter h −1. The enzyme concentration strongly depends on the substrate concentration and increases from 2–3 FPU ml −1 at 2% to 7–18 FPU ml −1 at 8%. High enzyme concentrations and volumetric rates are obtained by fed batch fermentations. If compared under identical conditions, all T. reesei mutants developed after 1978 show approximately the same capacity to produce cellulase. The very great differences reported in the literature result from modifications of the fermentation parameters and difficulties in objectively measuring the enzyme concentration FPU ml −1. On an industrial scale, Trichoderma cellulases are produced by solid state as well as by submersed fermentation. A list of commercially available cellulases and their potential application is given. If compared on a protein base (mg cellulase protein g −1 cellulose), the different cellulases show very different capacities for hydrolyzing cellulose.

Quantification and identification of the main components of the Trichoderma cellulase complex with monoclonal antibodies using an enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies has been developed to measure the concentration of three main cellulase components from Trichoderma reesei, cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and endoglucanase I (EG I), in both commercial enzyme preparations as well as in samples from laboratory fermentations. The sensitivity of the assay is 1-10 ng protein, depending on the type of cellulase. The coefficient of variability is between 10% and 20%. By a combination of two different domain-specific monoclonals against CBH I or II it is also possible to quantify the concentration of intact and truncated forms of these two enzymes, respectively. The use of the ELISA to quantify the formation of the three cellulase components under different cultivation conditions is described.

In vivo Degradation and Extracellular Polymer-Binding of Xyloglucan Nonasaccharide, a Naturally-Occurring Anti-Auxin

Summary 1-10 nM Xyloglucan nonasaccharide (XG9) and its naturally-occurring acylated derivatives (XG9·Ac) antagonise the growth-promoting action of 1μM 2,4-dichlorophenoxyacetic acid in pea stem segments. To investigate the mechanisms involved in controlling the in vivo concentrations of these biologically-active oligosaccharides, we 3 H-labelled XG9 and XG9·Ac in their xylose or fucose residues, fed them at 10 nM to rapidly-growing Spinacia cell cultures, and monitored their fate by gel-permeation chromatography. Very little breakdown to low-M r products took place, and little 3 H entered or bound to the cells. However, a proportion of the 3 H appeared to become linked to a soluble, extracellular polymer. The linkage was stable to 31% acetic acid and to 0.5M NaOH at 25°C, but Trichoderma cellulase was able to disrupt it, liberating a [ 3 H]oligosaccharide similar to XG9. We speculate that XG9 and XG9·Ac became attached to soluble extracellular xyloglucans by transglycosylation.

A binding-site-deficient, catalytically active, core protein of endoglucanase III from the culture filtrate of Trichoderma reesei

From the culture filtrate of Trichoderma reesei we have isolated a novel endoglucanase (38 kDa) which was shown to be identical to endoglucanase III (E III, 50 kDa), but lacking the first 61 N-terminal amino acids. This core protein, designated E III core, is fully active against soluble substrates, such as carboxymethylcellulose, whereas both activity against and adsorption to microcrystalline cellulose (Avicel) is markedly decreased. Sedimentation velocity experiments revealed that the intact E III enzyme has much higher asymmetry than the E III core protein, suggesting that the N-terminal region split off constitutes a protruding part of the native enzyme. These results lead to the proposal that native E III consists of two functionally separated domains: a catalytically active core and a protruding N-terminal domain which acts in the binding to insoluble cellulose. The N-terminal peptide missing in E III core corresponds to the heavily glycosylated common structural element found also in the N-terminus of cellobiohydrolase II and in the C-termini of cellobiohydrolase I and endoglucanase I. A similar bifunctional organization could thus be the rule for Trichoderma cellulases, endoglucanases as well as cellobiohydrolases.

Evaluation of the hydrolytic potential of a crude cellulase from mixed cultivation of Trichoderma reesei and Aspergillus phoenicis

Mixed cultivation of Trichoderma reesei Rut C30 and Aspergillus phoenicis produced a cellulase complex which showed enhanced activity against cellulose. Initial rates of glucose production were improved up to twofold over cellulase from Trichoderma alone. For the mixed culture cellulase, the pH optimum was found to be slightly different when measured on the basis of glucose production rate (4.5–4.75), than if measured on the basis of production of total reducing sugars (4.0–4.25). The initial rate of glucose and reducing sugar production increased with temperature over the range tested (40–60°C). Stability of individual enzyme component activities were tested at 50 and 60°C. β-Glucosidase (cellobiase or β- d-glucoside glucohydrolase, EC 3.2.1.21) activity was most labile at elevated temperatures ( t 1 2 = 10 h at 60°C and 48 h at 50°C). The mixed culture enzyme was more resistant to end product inhibition by glucose than the Trichoderma enzyme. Hydrolysis of steam exploded aspen wood (SEAW) by the mixed culture cellulase was markedly improved over that achieved by the Trichoderma cellulase.