Optimum temperature may be a misleading parameter in enzyme characterization and application

The optimum temperature is commonly determined in enzyme characterization. A search in the PubMed database for “optimum temperature” and “enzymes” yielded more than 1,700 manuscripts reporting this parameter over the last five years. Here, we show that the optimum temperature is not a constant. The catalytic activity of the mesophylic β-glucosidase Sfβgly was determined at different temperatures using different assay times and enzyme concentrations. We observed that the optimum temperature for Sfβgly changed by 5°C simply by modifying the assay length, and it was inversely correlated with enzyme concentration. These observations rely on the fact that close to the melting temperature, thermal denaturation continuously decreases the active enzyme concentration as the assay progresses. Thus, as the denaturation rate increases with increasing temperature, the bell-shaped curves observed in “activity versus temperature plots” occur only if the enzyme is denatured at and above the optimum temperature, which was confirmed using the thermostable β-glucosidase bglTm. Thus, the optimum temperature hardly reflects an intrinsic enzyme property and is actually a mere consequence of the assay condition. Thus, adoption of the “optimum temperature” determined under bench conditions for large-scale uses, which differ in assay length and enzyme concentration, may result in lower yields and financial losses.