Enhancing Enzyme pH Adaptation for Industrial Biocatalysis: From Sequence-Dependent Engineering to Sequence-Independent Stabilization

Abstract Enzymes are indispensable biocatalysts in industrial processes, but their application is often restricted by narrow pH optima. Exposure to acidic or alkaline conditions alters the protonation states of ionizable residues, disrupts catalytic microenvironments and stabilizing interactions, and ultimately reduces activity, catalytic efficiency, or structural integrity. This limitation is particularly important in industrial sectors such as detergents, textiles, biorefineries, and therapeutic formulations, where operation under non-native pH conditions is frequently required. Conventional sequence-dependent strategies, including rational design, directed evolution, mutagenesis, truncation, fusion, and circularization, have improved enzyme pH tolerance in many cases, but they often require extensive screening, depend on detailed structural knowledge, or involve trade-offs between stability and catalytic performance. By contrast, sequence-independent stabilization strategies aim to preserve the enzyme performance without altering the primary catalytic sequence. Among these, co-expression with stabilizing partners and protein additive approaches represent promising molecular shielding concepts that may protect enzyme function under acidic or alkaline stress while offering practical advantages in simplicity, scalability, and process compatibility. This review critically examines these emerging sequence-independent approaches alongside established sequence-dependent methods and highlights their potential to enable broad pH tolerance and more robust industrial biocatalysis.