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.

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 10 -10 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.

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 (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/91.html) from Trichoderma reesei; TrCel7A, reducing end‐acting cellobiohydrolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/176.html) from T. reesei.

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.

Cellobiohydrolases (CBHs) from glycoside hydrolase family 6 (GH6) make up an important part of the secretome in many cellulolytic fungi. They are also of technical interest, particularly because they are part of the enzyme cocktails that are used for the industrial breakdown of lignocellulosic biomass. Nevertheless, functional studies of GH6 CBHs are scarce and focused on a few model enzymes. To elucidate functional breadth among GH6 CBHs, we conducted a comparative biochemical study of seven GH6 CBHs originating from fungi living in different habitats, in addition to one enzyme variant. The enzyme sequences were investigated by phylogenetic analyses to ensure that they were not closely related phylogenetically. The selected enzymes were all heterologously expressed in Aspergillus oryzae, purified and thoroughly characterized biochemically. This approach allowed direct comparisons of functional data, and the results revealed substantial variability. For example, the adsorption capacity on cellulose spanned two orders of magnitude and kinetic parameters, derived from two independent steady-state methods also varied significantly. While the different functional parameters covered wide ranges, they were not independent since they changed in parallel between two poles. One pole was characterized by strong substrate interactions, high adsorption capacity and low turnover number while the other showed weak substrate interactions, poor adsorption and high turnover. The investigated enzymes essentially defined a continuum between these two opposites, and this scaling of functional parameters raises interesting questions regarding functional plasticity and evolution of GH6 CBHs.

Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data are available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here, we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from the mesophilic model ascomycete Trichoderma reesei (TrCel7B), which unlike ReCel7B possesses an additional carbohydrate‐binding module (CBM). With a temperature optimum of 80 °C, ReCel7B displayed a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 Å resolution, with Rwork and Rfree factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate‐binding cleft. An increased number of glycosylations were identified in ReCel7B, and we propose a stabilizing mechanism for one of the glycans. Global structure–function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding, and cellulose accessibility in GH7 endoglucanases. Database Structural data are available in RCSB Protein Data Bank database under the accession number https://www.rcsb.org/structure/6SU8. Enzymes ReCel7B, endoglucanase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/4.html) from Rasamsonia emersonii; ReCel7A, cellobiohydrolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/176.html) from Rasamsonia emersonii; TrCel7B, endoglucanase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/4.html) from Trichoderma reesei; TrCel7A, cellobiohydrolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/176.html) from Trichoderma reesei.

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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. K E Y W O R D SArrhenius equation, Cel7A, cellulase, enzyme inactivation, interfacial enzyme activity, optimal temperature 1 | INTRODUCTION One of the most important concepts in technical applications of enzymes is the bell-shaped relationship between temperature and activity, and in the simplest interpretation, this reflects a balance between two independent processes (Laidler & Peterman, 1979). At moderate temperatures, where the enzyme is stable, the rate increases with temperature in same way as other chemical reactions. We will call this thermoactivation of the enzyme process, and in many cases, it follows an exponential course in concurrence with the canonical Arrhenius equation. In practical terms, thermoactivation may be quantified by the so-called Q 10 -value, which is the fractional growth in reaction rate upon a 10°C increment in temperature. As long as the enzyme is stable, thermoactivation corresponding to Q 10 about two has been commonly reported for enzyme reactions (Elias, Wieczorek, Rosenne, & Tawfik, 2014) although widely differing values are also known (Wolfenden, Snider, Ridgway, & Miller, 1999). At higher temperatures, enzyme inactivation (reversible or irreversible) becomes significant. This diminishes thermoactivation, and ultimately leads to a rapid decline in activity as the enzyme denatures. The resulting maximum in activity defines the optimal temperature, T opt . Although poorly defined because it depends on experimental conditions (e.g., duration of the assay), this parameter has proven practical in comparative discussions of enzymes for technical applications.The bell-shaped course of temperature-activity curves has influenced engineering strategies for industrial enzymes. Thus, a feasible way to speed up a certain enzyme application is to engineer variants with increased stability. This will limit activity lo...