Assessing enzyme stability for biocatalysis

Despite the many advantages of enzymes, their stability remains a significant limitation in biocatalytic applications. A systematic evaluation of stability is a critical first step towards developing effective stabilization strategies. Enzyme stability can be broadly categorized into three types: thermodynamic stability, kinetic stability, and operational stability. Each category offers distinct insights into enzyme performance. Thermodynamic stability reflects the enzyme’s intrinsic tendency to maintain its folded structure under equilibrium conditions. Kinetic stability refers to the rate of irreversible deactivation over time, while operational stability describes the enzyme’s ability to retain catalytic activity under actual process conditions, accounting for all relevant deactivation mechanisms. This review compiles the key parameters, experimental methodologies, and tools to assess each stability type. To this end, we highlight case studies from the past decade that demonstrate the absence of a universal approach to evaluating enzyme stability and we present examples of AI- and ML-driven tools for different types of stability predictions. The specific application should guide the choice of method and metric, whether the objective is to understand fundamental deactivation mechanisms or to optimize enzyme performance under industrial conditions.