Date Published: February 22, 2019
Publisher: Public Library of Science
Author(s): Vitor M. Almeida, Sandro R. Marana, Andy T. Y. Lau.
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.
Enzymes have been employed as biotechnological tools for decades. The first patent reporting the utilization of an enzyme was registered in 1894 . Recently, enzyme applications have increased, including use in the production of biofuels, as detergent additives, in sewage treatment, in textile industry processes, in the pulp and paper industry and to improve food and beverage quality . The enzyme world market was estimated at USD 5 billion in 2016 . In addition, enzymes are the main targets of medicines, a market which was worth approximately USD 160 billion in 2017 [1, 2].
To demonstrate that the optimum enzyme temperature depends on the assay conditions, the β-glucosidase Sfβgly was submitted to the “classic” procedure for optimum temperature determination, i.e., its activity was determined at different temperatures (29 to 46 °C) using the same enzyme concentration. Then, the Sfβgly activity during the course of a fixed-temperature assay was determined using the first derivative at specific points of the product versus time curve (S2 Fig). Based on these results, the relative activity of Sfβgly was calculated at distinct times (10 to 120 min), which made possible the examination of how the optimum temperature plots evolved during the assays of the same enzyme (Fig 1).