Thermoactivation of a cellobiohydrolase

Abstract

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...

Boosting of enzymatic softwood saccharification by fungal GH5 and GH26 endomannanases

“…4 ), and the ranking of the performance of the enzymes was similar at the two reaction temperatures. The lack of increase in hydrolytic rate by cellulases in Cellic ® CTec3 with temperature (Q 10 close to 1 between 30 and 50 °C) is in agreement with data published by Westh et al [ 39 , 40 ] who showed that at low Avicel concentrations reduction in substrate affinity caused by heating (increase in K M ) cancels thermoactivation (increase in k cat ). In the present study, the substrate concentration was low (2% DM ~ 1% cellulose).…”

The dissociation mechanism of processive cellulases

“…However, these considerations do not account for the full difference with the SEE results. The dissociation k off value computed here (0.79 s −1 ) is on par with single-molecule tracking experiments of TrCel7A (k off = 0.1 to 0.2 s −1 ) (18, 31), with the higher temperature in our simulation potentially accelerating dissociation, as has been speculated for related cellulases (47). Of note, the computational k cat value of 6.9 s −1 (details are in SI Appendix) is well in line with the k cat values measured not only by HS-AFM (29,48) but also, by different biochemical methods (15-17, 28, 32).…”

pH profiles of cellulases depend on the substrate and architecture of the binding region

“…As already stated, the high/low values of substrate loads are well above/below K M for both Avicel and pNPL, and the data therefore reasonably represent the limiting cases. This interpretation is further supported by published binding parameters for the same enzyme–substrate systems (Badino, Kari, Christensen, Borch, & Westh, ; Schiano‐di‐Cola et al, ; Westh et al, ), which suggests almost complete adsorption of enzyme (85–98% bound) at high Avicel load (90 g/L). We used nonlinear regression analyses to fit the pH curves as detailed in the Supporting information.…”