Adsorption of enzymes with hydrolytic activity on polyethylene terephthalate.

Polyethylene terephthalate (PET) degrading enzymes have recently obtained an increasing interest as a means to decompose plastic waste. Here, we have studied the binding of three PET hydrolases on a suspended PET powder under conditions of both enzyme- and substrate excess. A Langmuir isotherm described the binding process reasonably and revealed a prominent affinity for the PET substrate, with dissociation constants consistently below 150 nM. The saturated substrate coverage approximately corresponded to a monolayer on the PET surface for all three enzymes. No distinct contributions from specific ligand binding in the active site could be identified, which points towards adsorption predominantly driven by non-specific interactions in contrast to enzymes naturally evolved for the breakdown of insoluble polymers. However, we observed a correlation between the progression of enzymatic hydrolysis and increased binding capacity, probably due to surface modifications of the PET polymer over time. Our results provide functional insight, suggesting that rational design should target the specific ligand interaction in the active site rather than the already high, general adsorption capacity of these enzymes.

Physical constraints and functional plasticity of cellulases.

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. In this work, we produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the substrate binding strength and the activation barrier. The scaling occurred despite the investigated enzymes being structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection has condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from its substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes.

A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases.

Lytic polysaccharide monooxygenases (LPMOs) are known to act synergistically with glycoside hydrolases in industrial cellulolytic cocktails. However, a few studies have reported severe impeding effects of C1-oxidizing LPMOs on the activity of reducing-end cellobiohydrolases. The mechanism for this effect remains unknown, but it may have important implications as reducing-end cellobiohydrolases make up a significant part of such cocktails. To elucidate whether the impeding effect is general for different reducing-end cellobiohydrolases and study the underlying mechanism, we conducted a comparative biochemical investigation of the cooperation between a C1-oxidizing LPMO from Thielavia terrestris and three reducing-end cellobiohydrolases; Trichoderma reesei (TrCel7A), T. terrestris (TtCel7A), and Myceliophthora heterothallica (MhCel7A). The enzymes were heterologously expressed in the same organism and thoroughly characterized biochemically. The data showed distinct differences in synergistic effects between the LPMO and the cellobiohydrolases; TrCel7A was severely impeded, TtCel7A was moderately impeded, while MhCel7A was slightly boosted by the LPMO. We investigated effects of C1-oxidations on cellulose chains on the activity of the cellobiohydrolases and found reduced activity against oxidized cellulose in steady-state and pre-steady-state experiments. The oxidations led to reduced maximal velocity of the cellobiohydrolases and reduced rates of substrate complexation. The extent of these effects differed for the cellobiohydrolases and scaled with the extent of the impeding effect observed in the synergy experiments. Based on these results, we suggest that C1-oxidized chain ends are poor attack sites for reducing-end cellobiohydrolases. The severity of the impeding effects varied considerably among the cellobiohydrolases, which may be relevant to consider for optimization of industrial cocktails.

A steady-state approach for inhibition of heterogeneous enzyme reactions.

The kinetic theory of enzymes that modify insoluble substrates is still underdeveloped, despite the prevalence of this type of reaction both in vivo and industrial applications. Here, we present a steady-state kinetic approach to investigate inhibition occurring at the solid-liquid interface. We propose to conduct experiments under enzyme excess (E0 ≫ S0), i.e. the opposite limit compared with the conventional Michaelis-Menten framework. This inverse condition is practical for insoluble substrates and elucidates how the inhibitor reduces enzyme activity through binding to the substrate. We claim that this type of inhibition is common for interfacial enzyme reactions because substrate accessibility is low, and we show that it can be analyzed by experiments and rate equations that are analogous to the conventional approach, except that the roles of enzyme and substrate have been swapped. To illustrate the approach, we investigated the major cellulases from Trichoderma reesei (Cel6A and Cel7A) acting on insoluble cellulose. As model inhibitors, we used catalytically inactive variants of Cel6A and Cel7A. We made so-called inverse Michaelis-Menten curves at different concentrations of inhibitors and found that a new rate equation accounted well for the data. In most cases, we found a mixed type of surface-site inhibition mechanism, and this probably reflected that the inhibitor both competed with the enzyme for the productive binding-sites (competitive inhibition) and hampered the processive movement on the surface (uncompetitive inhibition). These results give new insights into the complex interplay of Cel7A and Cel6A on cellulose and the approach may be applicable to other heterogeneous enzyme reactions.

Substrate binding in the processive cellulase Cel7A: Transition state of complexation and roles of conserved tryptophan residues.

Cellobiohydrolases effectively degrade cellulose and are of biotechnological interest because they can convert lignocellulosic biomass to fermentable sugars. Here, we implemented a fluorescence-based method for real-time measurements of complexation and decomplexation of the processive cellulase Cel7A and its insoluble substrate, cellulose. The method enabled detailed kinetic and thermodynamic analyses of ligand binding in a heterogeneous system. We studied WT Cel7A and several variants in which one or two of four highly conserved Trp residues in the binding tunnel had been replaced with Ala. WT Cel7A had on/off-rate constants of 1 × 105 m-1 s-1 and 5 × 10-3 s-1, respectively, reflecting the slow dynamics of a solid, polymeric ligand. Especially the off-rate constant was many orders of magnitude lower than typical values for small, soluble ligands. Binding rate and strength both were typically lower for the Trp variants, but effects of the substitutions were moderate and sometimes negligible. Hence, we propose that lowering the activation barrier for complexation is not a major driving force for the high conservation of the Trp residues. Using so-called Φ-factor analysis, we analyzed the kinetic and thermodynamic results for the variants. The results of this analysis suggested a transition state for complexation and decomplexation in which the reducing end of the ligand is close to the tunnel entrance (near Trp-40), whereas the rest of the binding tunnel is empty. We propose that this structure defines the highest free-energy barrier of the overall catalytic cycle and hence governs the turnover rate of this industrially important enzyme.

Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules.

The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity. Here, we have investigated the functional diversity of CBMs found within the GH6 family. This was done by constructing five chimeric enzymes based on the model enzyme, TrCel6A, from the soft-rot fungus Trichoderma reesei. The natural CBM of this enzyme was exchanged with CBMs from other GH6 enzymes originating from different cellulose degrading fungi. The chimeric enzymes were expressed in the same host and investigated in adsorption and quasi-steady-state kinetic experiments. Our results quantified functional differences of these phylogenetically distant binding modules. Thus, the partitioning coefficient for substrate binding varied 4-fold, while the maximal turnover (kcat) showed a 2-fold difference. The wild-type enzyme showed the highest cellulose affinity on all tested substrates and the highest catalytic turnover. The CBM from Serendipita indica strongly promoted the enzyme's ability to form productive complexes with sites on the substrate surface but showed lower turnover of the complex. We conclude that the CBM plays an important role for the functional differences between GH6 wild-type enzymes.

Rate-limiting step and substrate accessibility of cellobiohydrolase Cel6A from Trichoderma reesei.

The cellobiohydrolase (CBH) Cel6A is an important component of enzyme cocktails for industrial degradation of lignocellulosic biomass. However, the kinetics of this enzyme acting on its natural, insoluble substrate remains sparsely investigated. Here, we studied Cel6A from Trichoderma reesei with respect to adsorption, processivity, and kinetics both in the steady-state and pre-steady-state regimes, on microcrystalline and amorphous cellulose. We found that slow dissociation (koff ) was limiting the overall reaction rate, and we suggest that this leads to an accumulation of catalytically inactive complexes in front of obstacles and irregularities on the cellulose surface. The processivity number of Cel6A was low on both investigated substrates (5-10), and this suggested a rugged surface with short obstacle-free path lengths. The turnover of the inner catalytic cycle (the reactions of catalysis in one processive step) was too fast to be fully resolved, but a minimum value of about 20 s-1 could be established. This is among the highest values reported hitherto for a cellulase, and it underscores the catalytic efficiency of Cel6A. Conversely, we found that Cel6A had a poor ability to recognize attack sites on the cellulose surface. On amorphous cellulose, for example, Cel6A was only able to initiate hydrolysis on about 4% of the sites to which it could adsorb. This probably reflects high requirements of Cel6A to the architecture of the site. We conclude that compared to the other CBH, Cel7A, secreted by T. reesei, Cel6A is catalytically more efficient but less capable of attacking a broad range of structurally distinct sites on the cellulose surface. ENZYMES: TrCel6A, nonreducing end-acting cellobiohydrolase (EC3.2.1.91) from Trichoderma reesei; TrCel7A, reducing end-acting cellobiohydrolase (EC3.2.1.176) from T. reesei.

Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum.

BACKGROUND: The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. RESULTS: The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. CONCLUSIONS: We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.

Thermoactivation of a cellobiohydrolase.

We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a "high affinity" variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two-fold faster around 70 °C. This better thermoactivation of the high-affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability.

The influence of different linker modifications on the catalytic activity and cellulose affinity of cellobiohydrolase Cel7A from Hypocrea jecorina.

Various cellulases consist of a catalytic domain connected to a carbohydrate-binding module (CBM) by a flexible linker peptide. The linker if often strongly O-glycosylated and typically has a length of 20-50 amino acid residues. Functional roles, other than connecting the two folded domains, of the linker and its glycans, have been widely discussed, but experimental evidence remains sparse. One of the most studied cellulose degrading enzymes is the multi-domain cellobiohydrolase Cel7A from Hypocrea jecorina. Here, we designed variants of Cel7A with mutations in the linker region to elucidate the role of the linker. We found that moderate modification of the linker could result in significant changes in substrate affinity and catalytic efficacy. These changes were quite different for different linker variants. Thus, deletion of six residues near the catalytic domain had essentially no effects on enzyme function. Conversely, a substitution of four glycosylation sites near the middle of the linker reduced substrate affinity and increased maximal turnover. The observation of weaker binding provides some support of recent suggestions that linker glycans may be directly involved in substrate interactions. However, a variant with several inserted glycosylation sites near the CBM also showed lower affinity for the substrate compared to the wild-type, and we suggest that substrate interactions of the glycans depend on their exact location as well as other factors such as changes in structure and dynamics of the linker peptide.

Direct kinetic comparison of the two cellobiohydrolases Cel6A and Cel7A from Hypocrea jecorina.

Cellulose degrading fungi such as Hypocrea jecorina secrete several cellulases including the two cellobiohydrolases (CBHs) Cel6A and Cel7A. The two CBHs differ in catalytic mechanism, attack different ends, belong to different families, but are both processive multi-domain enzymes that are essential in the hydrolysis of cellulose. Here we present a direct kinetic comparison of these two enzymes acting on insoluble cellulose. We used both continuous- and end-point assays under either enzyme- or substrate excess, and found distinct kinetic differences between the two CBHs. Cel6A was catalytically superior with a maximal rate over four times higher than Cel7A. Conversely, the ability of Cel6A to attack diverse structures on the cellulose surface was inferior to Cel7A. This latter difference was pronounced as the density of attack sites for Cel7A was almost an order of magnitude higher compared to Cel6A. We conclude that Cel6A is a fast but selective enzyme and that Cel7A is slower, but promiscuous. One consequence of this is that Cel6A is more effective when substrate is plentiful, while Cel7A excels when substrate is limiting. These diverse kinetic properties of Cel6A and Cel7A might elucidate why both cellobiohydrolases are prominent in cellulolytic degrading fungi.

Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes.

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 103 -104 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc.

Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases.

We measured hydrolytic rates of four purified cellulases in small increments of temperature (10-50 °C) and substrate loads (0-100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants of these enzymes designed to elucidate the role of the carbohydrate binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased, and as a result, the effect of temperature depended strongly on the substrate load. Thus, temperature had little or no effect on the hydrolytic rate in dilute substrate suspensions, whereas strong temperature activation (Q10 values up to 2.6) was observed at saturating substrate loads. The CBM had a dual effect on the activity. On one hand, it diminished the tendency of heat-induced desorption, but on the other hand, it had a pronounced negative effect on the maximal rate, which was 2-fold larger in variants without CBM throughout the investigated temperature range. We conclude that although the CBM is beneficial for affinity it slows down the catalytic process. Cel7A from the thermophilic organism was moderately more activated by temperature than the mesophilic analog. This is in accord with general theories on enzyme temperature adaptation and possibly relevant information for the selection of technical cellulases.

Reversibility of substrate adsorption for the cellulases Cel7A, Cel6A, and Cel7B from Hypocrea jecorina.

Adsorption of cellulases on the cellulose surface is an integral part of the catalytic mechanism, and a detailed description of the adsorption process is therefore required for a fundamental understanding of this industrially important class of enzymes. However, the mode of adsorption has proven intricate, and several key questions remain open. Perhaps most notably it is not clear whether the adsorbed enzyme is in dynamic equilibrium with the free population or irreversibly associated with no or slow dissociation. To address this, we have systematically investigated adsorption reversibility for two cellobiohydrolases (Cel7A and Cel6A) and one endoglucanase (Cel7B) on four types of pure cellulose substrates. Specifically, we monitored dilution-induced release of adsorbed enzyme in samples that had previously been brought to a steady state (constant concentration of free enzyme). In simple dilution experiments (without centrifugation), the results consistently showed full reversibility. In contrast to this, resuspension of enzyme-substrate pellets separated by centrifugation showed extensive irreversibility. We conclude that these enzymes are in a dynamic equilibrium between free and adsorbed states but suggest that changes in the physical properties of cellulose caused by compaction of the pellet hampers subsequent release of adsorbed enzyme. This latter effect may be pertinent to both previous controversies in the literature on adsorption reversibility and the development of enzyme recycling protocols in the biomass industry.

In situ stability of substrate-associated cellulases studied by DSC.

This work shows that differential scanning calorimetry (DSC) can be used to monitor the stability of substrate-adsorbed cellulases during long-term hydrolysis of insoluble cellulose. Thermal transitions of adsorbed enzyme were measured regularly in subsets of a progressing hydrolysis, and the size of the transition peak was used as a gauge of the population of native enzyme. Analogous measurements were made for enzymes in pure buffer. Investigations of two cellobiohydrolases, Cel6A and Cel7A, from Trichoderma reesei, which is an anamorph of the fungus Hypocrea jerorina, showed that these enzymes were essentially stable at 25 °C. Thus, over a 53 h experiment, Cel6A lost less than 15% of the native population and Cel7A showed no detectable loss for either the free or substrate-adsorbed state. At higher temperatures we found significant losses in the native populations, and at the highest tested temperature (49 °C) about 80% Cel6A and 35% of Cel7A was lost after 53 h of hydrolysis. The data consistently showed that Cel7A was more long-term stable than Cel6A and that substrate-associated enzyme was less long-term stable than enzyme in pure buffer stored under otherwise equal conditions. There was no correlation between the intrinsic stability, specified by the transition temperature in the DSC, and the long-term stability derived from the peak area. The results are discussed with respect to the role of enzyme denaturation for the ubiquitous slowdown observed in the enzymatic hydrolysis of cellulose.

A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases.

A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39-0.40s(-1)) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.