Beyond Sequence Optimization in Enzyme Engineering: Protein Maturation and Operational Stability

Enzyme engineering commonly focuses on catalytic performance and recombinant protein production. Neither focus alone reveals how much expressed protein becomes an active catalyst or how long it remains functional. This review discusses engineering strategies for catalyst formation and maintenance using four representative enzyme groups. Cytochrome P450 enzymes illustrate the roles of cofactor incorporation and redox-system organization. Zymogenic proteases show how precursor maturation and autolysis control the active-enzyme population. Fungal cellulases demonstrate how post-translational modifications and host processing shape the final proteoform. Lytic polysaccharide monooxygenases (LPMOs) link active-site maturation to protection against oxidative damage. Together, these examples point to common strategies for improving catalyst formation and catalytic lifetime. Emerging approaches extend these principles to accessory maturation machinery and controlled activation. Conformational engineering and state-resolved measurements provide further opportunities for enzyme design.