Optimized protocols to measure stability of ligninolytic enzymes.

Abstract

Biocatalysis is considered a critical component for developing a sustainable bioeconomy, and stability is a crucial enzyme property for biotechnological and industrial applications. Enzymes with higher thermostability are more durable and desirable in industrial settings due to their resilience across various operational conditions, which helps reduce overall enzyme costs. Understanding an enzyme's thermal stability ensures its long-term efficacy and performance. Thermodynamic stability reflects the equilibrium between the native, functional protein, and unfolded state, and the kinetic or long-term stability is associated with the irreversible inactivation of the enzyme. Therefore, the thermostability of biocatalysts can be characterized by their melting temperature (T_m) when 50 % of the enzyme is unfolded and the half-life time (t_1/2), reporting the time gap to the loss of 50 % of the activity at a specific temperature. This parameter is crucial for assessing the feasibility of an enzymatic-based (bio)process, as it indicates the enzyme's temperature-dependent deactivation and operational stability over time. The optimum temperature of an enzyme (T_opt) usually reflects its (thermo)stability, particularly the stability of the native state. Here, we describe protocols for accessing the thermodynamic and kinetic stability of different ligninolytic enzymes, including laccases and DyP-type peroxidases. We provide practical examples and emphasize the challenges encountered during experimental procedures and data analysis. While these protocols are tailored to these specific enzymes, they can be broadly applied to other proteins and enzymes.