Chitin binding by Thermobifida fusca cellulase catalytic domains

Cellulose is a linear homopolymer of beta 1-4 linked glucose residues. Chitin is similar to cellulose in structure, and can be described as cellulose with the hydroxyl group on the C2 carbon replaced by an acetylamine group. Both cellulose and chitin form tightly packed, extensively hydrogen-bonded micro-fibrils. Up to now, binding of cellulase catalytic domains (CDs) to chitin has not been reported. In this article, binding of the CDs of Thermobifida fusca Cel6A, Cel6B, Cel48A, Cel5A, and Cel9A to alpha-chitin was investigated. The CDs of endocellulases, Cel6A and Cel5A did not bind to alpha-chitin; one exocellulase, Cel48A CD bound alpha-chitin moderately well; and the exocellulase Cel6B CD and the processive endocellulase Cel9A CD bound extremely tightly to alpha-chitin. Only mutations of Cel6B W329C, W332A and G234S and Cel9A Y206F, Y206S and D261A/R378K caused weaker binding to alpha-chitin than wild-type, and all these mutations were of residues near the catalytic center. One mutant enzyme, Cel9A D261A/R378K had weak chitinase activity, but no soluble products were detected. Chitotriose and chitotetraose were docked successfully to the catalytic cleft of Cel9A. In general, the positioning of the sugar residues in the model structures matched the cellooligosaccharides in the X-ray structure. Our results show that the binding of chitin by a cellulase can provide additional information about its binding to cellulose.     

Reversibility and binding kinetics of Thermobifida fusca cellulases studied through fluorescence recovery after photobleaching microscopy

Cellulases are enzymes capable of depolymerizing cellulose. Understanding their interactions with cellulose can improve biomass saccharification and enzyme recycling in biofuel production. This paper presents a study on binding and binding reversibility of Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A bound onto Bacterial Microcrystalline Cellulose. Cellulase binding was assessed through fluorescence recovery after photobleaching (FRAP) at 23, 34, and 45 °C. It was found that cellulase binding is only partially reversible. For processive cellulases Cel6B and Cel9A, an increase in temperature resulted in a decrease of the fraction of cellulases reversibly bound, while for endocellulase Cel5A this fraction remained constant. Kinetic parameters were obtained by fitting the FRAP curves to a binding-dominated model. The unbinding rate constants obtained for all temperatures were highest for Cel5A and lowest for Cel9A. The results presented demonstrate the usefulness of FRAP to access the fast binding kinetics characteristic of cellulases operating at their optimal temperature.     

Chemical transformation is not rate-limiting in the reaction catalyzed by Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP). Because HPPK is essential for microorganisms but is absent from human and animals, the enzyme is an excellent target for developing antimicrobial agent. Thermodynamic analysis shows that Mg(2+) is important not only for the binding of nucleotides but also for the binding of HMDP. Transient kinetic analysis shows that a step or steps after the chemical transformation are rate-limiting in the reaction catalyzed by HPPK. The pre-steady-state kinetics is composed of a burst phase and a steady-state phase. The rate constant for the burst phase is approximately 50 times larger than that for the steady-state phase. The latter is very similar to the k(cat) value measured by steady-state kinetics. A set of rate constants for the individual steps of the HPPK-catalyzed reaction has been determined by a combination of stopped-flow and quench-flow analyses. These results form a thermodynamic and kinetic framework for dissecting the roles of active site residues in the substrate binding and catalysis by HPPK.     

Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting cellulolytic complexes

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.     

Characterization of a metagenome-derived halotolerant cellulase

Metagenomes of uncultured microorganisms represent a sheer unlimited resource for discovery of novel biocatalysts. Here, we report on the biochemical characterisation of a novel, soil metagenome-derived cellulase (endoglucanase), Cel5A. The deduced amino acid sequence of Cel5A was similar to a family 5, single domain cellulase with no distinct cellulose binding domain from Cellvibrio mixtus. The 1092bp ORF encoding Cel5A was overexpressed in Escherichia coli and the corresponding 42.1 kDa protein purified using three-step chromatography. The recombinant Cel5A protein was highly active against soluble cellulose substrates containing beta-1,4 linkages, such as lichenan and barley beta-glucan, and not active against insoluble cellulose. Glucose was not among the initial hydrolysis products, indicating an endo mode of action. Cel5A displayed a wide range of pH activity with a maximum at pH 6.5 and at least 60% activity at pH 5.5 and 9.0. The enzyme was highly stable at 40 degrees C for up to 11 days, and retained 86-87% activity after incubation with 3M NaCl, 3M RbCl or 4M KCl for 20 h. Cel5A was also active in the presence of diverse divalent cations, detergents and EDTA. This highly stable, salt and pH tolerant cellulase is an ideal candidate for industrial applications.     

Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.     

A comparative analysis of two cDNA clones of the cellulase gene family from anaerobic fungus Piromyces rhizinflata

Cellulase family and some other glycosyl hydrolases of anaerobic fungi inhabiting the digestive tract of ruminants are believed to form an enzyme complex called cellulosome. Study of the individual component of cellulosome may shed light on understanding the organization of this complex and its functional mechanism. We have analysed the primary sequences of two cellulase clones, cel5B and cel6A, isolated from the cDNA library of ruminal fungus, Piromyces rhizinflata strain 2301. The deduced amino acid sequences of the catalytic domain of Cel5B, encoded by cel5B, showed homology with the subfamily 4 of the family 5 (subfamily 5(4)) of glycosyl hydrolases, while cel6A encoded Cel6A belonged to family 6 of glycosyl hydrolases. Phylogenetic tree analysis suggested that the genes of subfamily 5(4) glycosyl hydrolases of P. rhizinflata might have been acquired from rumen bacteria. Cel5B and Cel6A were modular enzymes consisting of a catalytic domain and dockerin domain(s), but not a cellulose binding domain. The occurrence of dockerin domains indicated that both enzymes were cellulosome components. The catalytic domain of the Cel5B (Cel5B') and Cel6A (Cel6A') recombinant proteins were purified. The optimal activity conditions with carboxymethyl cellulose (CMC) as the substrate were pH 6.0 and 50 degrees C for Cel5B', and pH 6.0 and 37-45 degrees C for Cel6A'. Both Cel5B' and Cel6A' exhibited activity against CMC, barley beta-glucan, Lichenan, and oat spelt xylan. Cel5B' could also hydrolyse p-nitrophenyl-beta-d-cellobioside, Avicel and filter paper while Cel6A' did not show any activity on these substrates. It is apparent that Cel6A' acted as an endoglucanase and Cel5B' possessed both endoglucanase and exoglucanase activities. No synergic effect was observed for these recombinant enzymes in vitro on Avicel and CMC.     

Method for characterization of the enzyme profile and the determination of CBH I (Cel 7a) core protein in Trichoderma reesei cellulase preparations

Summary Fast protein liquid chromatography (FPLC) was used to characterize a commercial cellulase preparation (Celluclast 1.5L, Novozymes) in relation to its protein profile and activity against hydroxyethylcellulose (HEC) and other substrates. Co-elution of CBHII (Cel 6A) with other enzyme components of the cellulase system was characterized by immunochemical assays using monoclonal antibodies, whereas the occurrence of EGII (Cel 5A) was assessed based on its ability to cleave the heterosidic bond of 4-methylumbellyferyl-β-d-cellotrioside (MUmbG₃). The main cellulase constituents of Celluclast 1.5L were pooled into six fractions containing EGII (Cel 5A) and EGIII (Cel 12A) (F1), EGII and CBHII (Cel 6A) (F2), CBHII and EGI (Cel 7B) (F3), EGI (F4), and CBHI (Cel 7A) (F5). The occurrence of CBHI core protein within the CBHI fraction of the FPLC profile was determined by hydrophobic interaction chromatography. Using this method, we were able to demonstrate that the batch of Celluclast 1.5L used in this study contained 10.9–18.8% of CBHI as its corresponding free core protein.     

Thermobifida fusca family-6 cellulases as potential designer cellulosome components

During the course of our studies on the structure–function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in ‘designer cellulosomes’. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.     

Surface density of cellobiohydrolase on crystalline celluloses. A critical parameter to evaluate enzymatic kinetics at a solid-liquid interface

The enzymatic kinetics of glycoside hydrolase family 7 cellobiohydrolase (Cel7A) towards highly crystalline celluloses at the solid-liquid interface was evaluated by applying the novel concept of surface density (rho) of the enzyme, which is defined as the amount of adsorbed enzyme divided by the maximum amount of adsorbed enzyme. When the adsorption levels of Trichoderma viride Cel7A on cellulose I(alpha) from Cladophora and cellulose I(beta) from Halocynthia were compared, the maximum adsorption of the enzyme on cellulose I(beta) was approximately 1.5 times higher than that on cellulose I(alpha), although the rate of cellobiose production from cellulose I(beta) was lower than that from cellulose I(alpha). This indicates that the specific activity (k) of Cel7A adsorbed on cellulose I(alpha) is higher than that of Cel7A adsorbed on cellulose I(beta). When k was plotted versus rho, a dramatic decrease of the specific activity was observed with the increase of surface density (rho-value), suggesting that overcrowding of enzyme molecules on a cellulose surface lowers their activity. An apparent difference of the specific activity was observed between crystalline polymorphs, i.e. the specific activity for cellulose I(alpha) was almost twice that for cellulose I(beta). When cellulose I(alpha) was converted to cellulose I(beta) by hydrothermal treatment, the specific activity of Cel7A decreased and became similar to that of native cellulose I(beta) at the same rho-value. These results indicate that the hydrolytic activity (rate) of bound Cel7A depends on the nature of the crystalline cellulose polymorph, and an analysis that takes surface density into account is an effective means to evaluate cellulase kinetics at a solid-liquid interface.     

A slow-tight binding inhibitor of dopamine beta-monooxygenase: a transition state analogue for the product release step

The steady-state kinetic data show that 3-hydroxy-4-phenylthiazole-2(3H)-thione (3H4PTT) is a potent tight-binding inhibitor for dopamine beta-monooxygenase (DbetaM) with a dissociation constant of 0.9 nM. Ackermann-Potter plots of the enzyme dependence of the inhibition revealed that the stoichiometry of the enzyme inhibition by 3H4PTT is 1:1. Pre-steady-state progress curves at varying inhibitor with fixed reductant and enzyme concentrations clearly show the slow binding behavior of the inhibitor. The observed kinetic behavior is consistent with the apparent direct formation of the tightly bound E x I* complex. The k(on) and k(off) for 3H4PTT which were determined under pre-steady-state conditions at variable inhibitor concentrations were found to be (1.85 +/- 0.07) x 10(6) M(-1) s(-1) and (1.9 +/- 0.6) x 10(-3) s(-1), respectively. The dissociation constant calculated from these rates was similar to that determined under steady-state conditions, confirming that 3H4PTT is a kinetically well-behaved inhibitor. The steady-state as well as pre-steady-state kinetic studies at variable DMPD concentrations show that the inhibition is competitive with respect to the reductant, demonstrating the exclusive interaction of 3H4PTT with the oxidized form of the enzyme. The kinetic behavior and the structural properties of 3H4PTT are consistent with the proposal that the E x 3H4PTT complex may mimic the transition state for the product (protonated) release step of the enzyme. Therefore, 3H4PTT could be used as a convenient probe to examine the properties of the E x P complex of the DbetaM reaction and also as an active site titrant for the oxidized enzyme.     

The sorbinil trap: a predicted dead-end complex confirms the mechanism of aldose reductase inhibition

Kinetic and crystallographic studies have demonstrated that negatively charged aldose reductase inhibitors act primarily by binding to the enzyme complexed with oxidized nicotinamide dinucleotide phosphate (E.NADP(+)) to form a ternary dead-end complex that prevents turnover in the steady state. A recent fluorescence study [Nakano and Petrash (1996) Biochemistry 35, 11196-11202], however, has concluded that inhibition by sorbinil, a classic negatively charged aldose reductase inhibitor, results from binding to the enzyme complexed with reduced cofactor (E.NADPH) and not binding to E.NADP(+). To resolve this controversy, we present transient kinetic data which show unequivocally that sorbinil binds to E.NADP(+) to produce a dead-end complex, the so-called sorbinil trap, which prevents steady-state turnover in the presence of a saturating concentration of aldehyde substrate. The reported fluorescence binding results, which we have confirmed independently, are further shown to be fully consistent with the proposed sorbinil trap mechanism. Our conclusions are supported by KINSIM simulations of both pre-steady-state and steady-state reaction time courses in the presence and absence of sorbinil. Thus, while sorbinil binding indeed occurs to both E.NADPH and E.NADP(+), only the latter dead-end complex shows significant inhibition of the steady-state turnover rate. The effect of tight-binding kinetics on the inhibition patterns observed for zopolrestat, another negatively charged inhibitor, is further examined both experimentally and with KINSIM, with the conclusion that all reported aldose reductase inhibition can be rationalized in terms of binding of an alrestatin-like inhibitor at the active site, with no need to postulate a second inhibitor binding site.     

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs.     

Toward the elucidation of the catalytic mechanism of the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A

The catalytic mechanism for the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A was investigated by steady-state and stopped-flow kinetic analyses. The rate constants for binding of the NAD(+) substrate to the enzyme were found to be 4.7 +/- 0.4 microM(-1) s(-1) and 194 +/- 15 s(-1) for k(on) and k(off), respectively. The k(on) and k(off) rate constants for the eEF-2 substrate binding to the enzyme were 320 +/- 39 microM(-1) s(-1) and 131 +/- 22 s(-1), respectively. A potent, competitive inhibitor against the enzyme, 1,8-naphthalimide, bound the enzyme with k(on) and k(off) rates of 82 +/- 9 microM(-1) s(-1) and 51 +/- 6 s(-1), respectively. Furthermore, the binding on and off rates for the reaction products, ADP-ribose and nicotinamide, were too rapid for detection with the stopped-flow technique. Investigation of the pre-steady-state kinetics for the ADP-ribose transferase activity of the toxin-enzyme showed that there is no pre-steady-state complex formed during the catalytic cycle. Binding of NAD+ and smaller compounds representing the various parts of this substrate were investigated by the fluorescence quenching of the intrinsic toxin fluorescence. The binding data revealed a significant structural change in the enzyme upon NAD+ binding that could not be accounted for on the basis of the sum of the structural changes induced by the various NAD+ constituents. Product inhibition studies were conducted with nicotinamide and eEF-2-ADP-ribose, and the results indicate that the reaction involves a random-order ternary complex mechanism. Detailed kinetic analysis revealed that the eEF-2 substrate shows sigmoidal kinetic behavior with the enzyme, and fluorescence resonance energy transfer measurements indicated that wheat germ eEF-2 is oligomeric in solution.     

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.     

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.     

Pre-steady-state kinetics for hydrolysis of insoluble cellulose by cellobiohydrolase Cel7A

The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10-25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ~30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.