Phototoxicity of protoporphyrin as related to its subcellular localization in mice livers after short-term feeding with griseofulvin.

The substrate specificities of three cellulases and a beta-glucosidase purified from Thermoascus aurantiacus were examined. All three cellulases partially degraded native cellulose. Cellulase I, but not cellulase II and cellulase III, readily hydrolyzed the mixed beta-1,3; beta-1,6-polysaccharides such as carboxymethyl-pachyman, yeast glucan and laminarin. Both cellulase I and the beta-glucosidase degraded xylan, and it is proposed that the xylanase activity is an inherent feature of these two enzymes. Lichenin (beta-1,4; beta-1,3) was degraded by all three cellulases. Cellulase II cannot degrade carboxymethyl-cellulose, and with filter paper as substrate the end product was cellobiose, which indicates that cellulase II is an exo-beta-1,4-glucan cellobiosylhydrolase. Degradation of cellulose (filter paper) can be catalysed independently by each of the three cellulases; there was no synergistic effect between any of the cellulases, and cellobiose was the principal product of degradation. The mode of action of one cellulase (cellulase III) was examined by using reduced cellulodextrins. The central linkages of the cellulodextrins were the preferred points of cleavage, which, with the rapid decrease in viscosity of carboxymethyl-cellulose, confirmed that cellulase III was an endocellulase. The rate of hydrolysis increased with chain length of the reduced cellulodextrins, and these kinetic data indicated that the specificity region of cellulase III was five or six glucose units in length.     

Specific and nonspecific glucanases from Trichoderma viride

Six endoglucanases (Endo I, II, III, IV, V, and VI), three exoglucanases (Exo I, II, and III), and a beta-glucosidase (beta-gluc I) isolated from a commercial cellulase preparation of Trichoderma viride origin were examined as to their activities on xylan ex oat spelts. Endo I, II, and III as well as Exo II and III showed no activity toward xylan and were classified as specific glucanases. Less specificity was found for the endoglucanases Endo IV, V, and VI, Exo I, and beta-gluc I, whose enzymes were able to hydrolyze xylan. With respect to product formation these xylanolytic cellulases fit the classification of xylanases generally accepted in the literature. Kinetic experiment with xylan, CM-cellulose, and p-nitrophenyl-beta-D-glucoside revealed that Endo IV, V, an VI and Exo I prefer to hydrolyze beta-1, 4-D-glucosidic linkages. beta-Gluc I showed no clear substrate preference.     

Purification, characterization, and synergistic action of endoglucanases from Fusarium lini

Five endoglucanases (1,4-beta-D-glucan-glucanohydrolase, EC 3.2.1.4) were isolated from Fusarium lini. Endo I and II were purified by preparative gel electrophoresis and Endo III, IV, and V were purified in a single-step procedure involving preparative flat-bed isoelectric focusing. All the endoglucanases were homogenous on disk gel electrophoresis and analytical isoelectric focusing in polyacrylamide gel. The pi values were between 6 and 6.6 for Endo III, IV, and V; for Endo I, the pi value was 8. The molecular weights of the enzymes were between 4 x 10(4) and 6.5 x 10(4). The K(m) values for endoglucanases using carboxymethyl cellulose (CM-cellulose) as the substrate were 2-12 mg/mL. The specificity of the enzymes was restricted to beta-1, 4-linkages. All the enzymes showed activity towards D-xylan. The endoglucanases had high viscosity reducing activity with CM-cellulose. Striking synergism was observed for the hydrolysis of CM-cellulose by endoglucanases. Endo II, IV, and V attacked cellopentaose and cellotetraose more readily than cellotriose. Endo II and V hydrolyzed cellotriose, cellotetraose, and cellopentaose, yielding a mixture of cellobiose with a trace amount of glucose; endo IV produced only cellobiose.     

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).     

Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in relation to their activity against various cellulosic substrates

Penicillium echinulatum has been identified as a potential cellulase producer for bioconversion processes but its cellulase system has never been investigated in detail. In this work, the volumetric activities of P. echinulatum cellulases were determined against filter paper (0.27 U/mL), carboxymethylcellulose (1.53 U/mL), hydroxyethylcellulose (4.68 U/mL), birchwood xylan (3.16 U/mL), oat spelt xylan (3.29 U/mL), Sigmacell type 50 (0.10 U/mL), cellobiose (0.19 U/mL), and p-nitrophenyl-glucopiranoside (0.31 U/mL). These values were then expressed in relation to the amount of protein and compared those of Trichoderma reesei cellulases (Celluclast 1.5L FG, Novozymes). Both enzyme complexes were shown to have similar total cellulase and xylanase activities. Analysis of substrate hydrolysates demonstrated that P. echinulatum enzymes have higher beta-glucosidase activity than Celluclast 1.5L FG, while the latter appears to have greater cellobiohydrolase activity. Unlike Celluclast 1.5L FG, P. echinulatum cellulases had enough beta-glucosidase activity to remove most of the cellobiose produced in hydrolysis experiments. However, Celluclast 1.5L FG became more powerful than P. echinulatum cellulases when supplemented with exogenous beta-glucosidase activity (Novozym 188). Both cellulase complexes displayed the same influence over the degree of polymerization of cellulose, revealing that hydrolyzes were carried out under the typical endo-exo synergism of fungal enzymes.     

Cellulases of a marine mollusc, Dolabella sp

1. A crude cellulase extract was prepared from the hepatopancreas of a marine mollusc, Dolabella sp., and partially purified by ammonium sulphate fractionation. 2. The optimum pH values of the partially purified preparation were 6.5 and 8.0 for Walseth cellulose and CM-cellulose respectively. It was most stable at pH6.0 and showed moderate thermostability. 3. The partially purified preparation was subjected to starch-zone electrophoresis, and incompletely resolved into several fractions that contained one or more cellulase components of different substrate specificity. 4. Some of these cellulase fractions showed practically no aryl beta-glucosidase activity and hydrolysed aryl beta-cellobioside with difficulty. From substrates such as higher cello-oligosaccharides, cellodextrin, CM-cellulose, Walseth cellulose and cotton fibre, they produced cellobiose as the major and cellotriose as the minor end products, both of which were resistant to further attack by cellulase. 5. From the slope of the curves of viscosity-reducing power for CM-cellulose, the cellulase components from Dolabella were presumed to be of a ;more-random' or a ;less-random' type in the mode of action. 6. In the hepatopancreas of this mollusc, beta-glucosidases were also present, which hydrolysed cellobiose as well as aryl beta-glucosides. The optimum pH values of these enzymes were about 5.5.     

The action on cellulose and its derivatives of a purified 1,4-beta-glucanase from Trichoderma koningii.

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.     

Substrate specificity and mode of action of a cellulase from Aspergillus niger.

The mode of action and substrate specificity of a cellulase purified from Aspergillus niger were examined. The enzyme showed little capacity to hydrolyse highly ordered cellulose, but readily attacked soluble cellulose derivatives and amorphous alkali-swollen cellulose. Activity towards barley glucan and lichenin was greater than with CM-cellulose. Low activity was detected with CM-pachyman (a substituted beta-1,3-glucose polymer) and xylan. Activity towards yeast glucan, mannan, ethlene glycol chitin, glycol chitosan, laminarin, polygalacturonic acid and pectin could not be demonstrated. Cellobiose and p-nitrophenyl beta-D-glucoside were not hydrolysed, whereas the rate of hydrolysis of the higher members of the reduced cellulodextrins increased with chain length. The central bonds of cellotetraosylsorbitol and cellopentaosylsorbitol were the preferred points of clevage. Kinetic data indicated that the specificity region of the cellulase is five glucose units in length. The evidence indicates that the cellulase is an endoglucanase.     

Purification and partial characterisation of three endo-glucanases from dichomitus squalens

Three endo-glucanases (En I-III) were obtained by chromatofocusing fractionation of a culture supernatant from the white-rot fungus Dichomitus squalens. They were purified further on Phenyl-Sepharose CL-4B, DEAE-Trisacryl, and Ultrogel AcA 54; ∼21-, ∼16-, and ∼9-fold purifications were obtained for En I-III, respectively. The enzymes appeared as homogeneous proteins on disc gel electrophoresis with and without SDS (sodium dodecyl sulphate), and on isoelectric focusing; the respective mol. wts. were 42,000, 56,000, and 47,000, and the isoelectric points 4.8, 4.3, and 4.1. Optimum conditions for the hydrolysis of CM-cellulose were pH 4.8 and 55° for each enzyme, and each was stable over the pH range 4.0–8.5 but inactivated completely within 30 min at 70°. None of the purified enzymes exhibited β-d-glucosidase or cellobiohydrolase activity, but En II was weakly active towards laminaran and xylan. En I and En II acted more randomly on CM-cellulose than did En III. Cellotetraose was degraded by each endo-glucanase, whereas only En III could hydrolyse cellotriose.     

Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover

The production of extracellular cellulases by a newly isolated thermoacidophilic fungus, Aspergillus terreus M11, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The results showed that the high-level cellulase activity was produced at 45 degrees C pH 3 and moisture 80% with corn stover and 0.8% yeast extract as carbon and nitrogen sources. 581 U endoglucanase activity, 243 U filter paper activity and 128 U beta-glucosidase activity per gram of carbon source were obtained in the optimal condition. Endoglucanase and beta-glucosidase exhibited their maximum activity at pH 2 and pH 3, respectively, and both of them showed remarkable stability in the range of pH 2-5. The activities of endoglucanase and beta-glucosidase were up to the maximum at 70 degrees C and maintained about 65% and 53% of their original activities after incubation at 70 degrees C for 6h. The enzyme preparations from this strain were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were up to 63% on 5% Avicel (w/v) for 72 h with 20 U FPase/g substrate.     

Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100-14

A new mutant strain of fungus Trichoderma viride T 100-14 was cultivated on 1% microcrystalline cellulose (Avicel) for 120h and the resulting culture filtrate was prepared for protein identification and purification. To identify the predominant catalytic components, cellulases were separated by an adapted two-dimensional electrophoresis technique. The apparent major spots were identified by high performance liquid chromatography electrospray ionization mass (HPLC-ESI-MS). Seven of the components were previously known, i.e., the endoglucanases Cel7B (EG I), Cel12A (EG III), Cel61A (EG IV), the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), Cel6B (CBH IIb) and the beta-glucosidase. The seven major components in the fermentation broth of T. viride T 100-14 probably constitute the essential enzymes for crystalline cellulose hydrolysis and they were further purified to electrophoretic homogeneity by a series of chromatography column. Hydrolysis studies of the purified elements revealed that three of the cellulases were classified as cellobiohydrolases due to their main activities on p-nitrophenyl-beta-d-cellobioside (pNPC). Three of the cellulases, with the abilities of hydrolyzing both carboxymethyl-cellulose (CMC) and Avicel indicate their endoglucanase activities. It deserved noting that the beta-glucosidase from the T 100-14 displayed an extremely high activity on p-nitrophenyl-beta-D-glycopyranoside (pNPG), which suggested it was a good candidate for the conversion of cellobiose to glucose.     

The cellulase of Trichoderma viride. Purification, characterization and comparison of all detectable endoglucanases, exoglucanases and beta-glucosidases

Six endoglucanases (Endo I; II; III; IV; V; VI), three exoglucanases (Exo I; II; III) and a beta-glucosidase (beta-gluc I) were isolated from a commercial cellulase preparation derived from Trichoderma viride, using gel filtration on Bio-Gel, anion exchange on DEAE-Bio-Gel A, cation exchange on SE-Sephadex and affinity chromatography on crystalline cellulose. Molecular masses were determined by polyacrylamide gel electrophoresis. One group of endoglucanases (Endo I, Endo II and Endo IV) with Mr of 50 000, 45 000 and 23 500 were more random in their attack on carboxymethylcellulose than another group (Endo III, Endo V and Endo VI) showing Mr of 58 000, 57 000 and 53 000 respectively. Endo III was identified as a new type of endoglucanase with relatively high activity on crystalline cellulose and moderate activity on carboxymethylcellulose. Exo II and Exo III with Mr of 60 500 and 62 000 respectively showed distinct adsorption affinities on a column of crystalline cellulose and could be eluted by a pH gradient to alkaline regions. These enzymes were cellobiohydrolases as judged by high-pressure liquid chromatography of the products obtained from incubation with H3PO4-swollen cellulose. It was concluded that these exoglucanases are primarily active on newly generated chain ends. Exo I was essentially another type of exoglucanase which in the first instance was able to split off a cellobiose molecule from a chain end and then hydrolyse this molecule in a second step to two glucose units beta-Gluc I was a new type of aryl-beta-D-glucosidase which had no activity on cellobiose. The enzyme had a Mr of 76 000 and was moderately active on CM-cellulose, crystalline cellulose and xylan and highly active on p-nitrophenyl-beta-D-glucose and p-nitrophenyl-beta-D-xylose.     

Isolation, characterization and manipulation of cellulase genes

The complete hydrolysis of cellulose requires a number of different enzymes including endoglucanase, exoglucanase and beta-glucosidase. These enzymes function in concert as part of a 'cellulase'complex called a cellulosome. In order (i) to develop a better understanding of the biochemical nature of the cellulase complex as well as the genetic regulation of its integral components and (ii) to utilize cellulases either as purified enzymes or as part of an engineered organism for a variety of purposes, researchers have, as a first step, used recombinant DNA technology to isolate the genes for these enzymes from a variety of organisms. This review provides some perspective on the current status of the isolation, characterization and manipulation of cellulase genes and specifically discusses (i) strategies for the isolation of endoglucanase, exoglucanase and beta-glucosidase genes; (ii) DNA sequence characterization of the cellulase genes and their accompanying regulatory elements; (iii) the expression of cellulase genes in heterologous host organisms and (iv) some of the proposed uses for isolated cellulase genes.     

Production and Characterization of Cellulase and beta-Glucosidase from a Mutant of Alternaria alternata

A mutant of Alternaria alternata excreted enhanced levels of carboxymethylcellulase and particularly beta-glucosidase when grown in cellulose liquid media. Both enzymes were purified two- to four-fold by ammonium sulfate precipitation and gel filtration, and the kinetic data showed K(m) values of 16.64 mg/ml of culture fluid for carboxymethylcellulase and 0.14 mM p-nitrophenyl-beta-d-glucoside and 0.81 mM cellobiose for beta-glucosidase at pH 5. Carboxymethylcellulase and extracellular beta-glucosidase functioned optimally at pH 5 to 6 and 4.5 to 5 and at temperatures of 55 to 60 and 70 to 75 degrees C, respectively. Both temperature optima and thermostability of beta-glucosidase were among the highest ever reported for the same enzyme excreted from cellulase and beta-glucosidase hyperproducing microorganisms.     

Purification and properties of a cellulase from Aspergillus niger.

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.     

Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass

Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput fast protein liquid chromatography (HT-FPLC)-based method has been developed to quantify cellobiohydrolase I (CBH I or Cel7A), cellobiohydrolase II (CBH II or Cel6A), and endoglucanase I (EG I or Cel7B) present in hydrolyzates of untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated corn stover (CS). This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall-derived components. The binding isotherms for CBH I, CBH II, and EG I were obtained after incubation for 2 h at 4 °C. Both AFEX and dilute acid pretreatment resulted in increased cellulase binding compared with untreated CS. Cooperative binding of CBH I and/or CBH II in the presence of EG I was observed only for AFEX treated CS. Competitive binding between enzymes was found for certain other enzyme-substrate combinations over the protein loading range tested (i.e., 25-450 mg/g glucan). Langmuir single-site adsorption model was fitted to the binding isotherm data to estimate total available binding sites E(bm) (mg/g glucan) and association constant K(a) (L/mg). Our results clearly demonstrate that the characteristics of cellulase binding depend not only on the enzyme GH family but also on the type of pretreatment method employed.     

Cellulases from Sporocytophaga myxococcoides. Purification and Properties.

Two extracellular cellulases active on carboxymethylcellulose have been isolated from the culture supernatant of Sporocytophaga myxococcoides by a series of gel-filtration and ion-exchange chromatography steps. Cellulase II, being present in highest amount, had a molecular weight determined by gel electrophoresis of 52000, pI 4.75 and a relatively broad pH optimum (5.5--7.5). Cellulase I had a molecular weight of 46000. pI was 7.5 and the pH optimum 6.5--7.5. Both cellulases had a very low carbohydrate content, possibly present as impurities. They had fairly similar amino acid compositions. The specific acitivity of cellulase I was about 6 times higher than that of cellulase II. Both cellulases acted as endoglucanases. A cell-associated cellulase, present in amounts corresponding to about 10% of total activity, was partly purified. It showed similarities with cellulase II.     

Cellulase and beta-glucosidase production by a basidiomycete species.

The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose.     

Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose

An extremely highly active cellobiohydrolase (CBH IIb or Cel6B) was isolated from Chrysosporium lucknowense UV18-25 culture filtrate. The CBH IIb demonstrated the highest ability for a deep degradation of crystalline cellulose amongst a few cellobiohydrolases tested, including C. lucknowense CBH Ia, Ib, IIa, and Trichoderma reesei CBH I and II. Using purified C. lucknowense enzymes (CBH Ia, Ib, and IIb; endoglucanases II and V; beta-glucosidase, xylanase II), artificial multienzyme mixtures were reconstituted, displaying an extremely high performance in a conversion of different cellulosic substrates (Avicel, cotton, pretreated Douglas fir wood) to glucose. These mixtures were much or notably more effective in hydrolysis of the cellulosic substrates than the crude multienzyme C. lucknowense preparation and other crude cellulase samples produced by T. reesei and Penicillium verruculosum. Highly active cellulases are a key factor in bioconversion of plant lignocellulosic biomass to ethanol as an alternative to fossil fuels.     

Cellulase kinetics as a function of cellulose pretreatment

Microcrystalline cellulose (Avicel) was subjected to three different pretreatments (acid, alkaline, and organosolv) before exposure to a mixture of cellulases (Celluclast). Addition of beta-glucosidase, to avoid the well-known inhibition of cellulase by cellobiose, markedly accelerated cellulose hydrolysis up to a ratio of activity units (beta-glucosidase/cellulase) of 20. All pretreatment protocols of Avicel were found to slightly increase its degree of crystallinity in comparison with the untreated control. Adsorption of both cellulase and beta-glucosidase on cellulose is significant and also strongly depends on the wall material of the reactor. The conversion-time behavior of all four states of Avicel was found to be very similar. Jamming of adjacent cellulase enzymes when adsorbed on microcrystalline cellulose surface is evident at higher concentrations of enzyme, beyond 400 U/L cellulase/8 kU/L beta-glucosidase. Jamming explains the observed and well-known dramatically slowing rate of cellulose hydrolysis at high degrees of conversion. In contrast to the enzyme concentration, neither the method of pretreatment nor the presence or absence of presumed fractal kinetics has an effect on the calculated jamming parameter for cellulose hydrolysis.