The Sabatier principle states that optimal catalysis occurs when interactions between catalyst and substrate are of intermediary strength. Although qualitative in nature, this concept has proven extremely useful within (nonbiochemical) heterogeneous catalysis. In the current work, we show that the principle may be applied to an interfacial enzyme reaction. Specifically, we studied the breakdown of cellulose by different cellulases (wild types and variants) and found that the results could be rationalized in so-called volcano plots that are emblematic of the principle. This implies that the rate of the complex enzymatic reaction can be described by a single parameter (binding strength), and we show how this may help elucidating e.g. rate-controlling steps and relationships of substrate load and enzymatic efficacy. On a more general level, we propose that the Sabatier principle may be widely applicable to interfacial enzyme processes and hence open an avenue to the application within biocatalysis of some of the principles and practices originally developed for heterogeneous catalysis.

Background: Temperature concomitantly modulates kinetic and adsorption properties in heterogeneous enzyme catalysis. Results: Affinity-activity relationships for four Cel7A cellobiohydrolases are characterized over a broad temperature interval. Conclusion: Cellobiohydrolases are strongly activated by temperature at high, but not at low, substrate loads. Significance: Fundamental insight into cellulolytic mechanisms at high (industrially relevant) temperatures is gained.

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.

Lytic polysaccharide monooxygenases (LPMOs) have attracted attention due to their ability to boost cellulolytic enzyme cocktails for application in biorefineries. However, the interplay between LPMOs and individual glycoside hydrolases remains poorly understood. We investigated how the activity of two cellobiohydrolases (Cel7A and Cel6A) and an endoglucanase (Cel7B) from Trichoderma reesei were affected by a C1-oxidizing LPMO from Thielavia terrestris (TtAA9). We quantified products from a mixture of LPMO and glycoside hydrolase and estimated separate contributions of products by each of the enzymes. Hereby, we assessed if an observed synergy reflected a promotion of the activity of hydrolase, LPMO, or both. We consistently found that TtAA9 affected the investigated hydrolases differently. It strongly impeded the turnover of the reducing end cellobiohydrolase, TrCel7A, moderately promoted the turnover of the nonreducing end cellobiohydrolase TrCel6A, and promoted the turnover of the endoglucanase, TrCel7B up to 5-fold. The promoting effect on the endoglucanase increased with hydrolysis extent, indicating that the promoting effect became more important as the recalcitrance of the substrate increased. Experiments with mixtures containing multiple glycoside hydrolases suggested that the LPMO primarily promoted the activity of the endoglucanase, whereas promotion of TrCel6A was secondary.