Integrative Approaches in Protein Structure/Function Analysis, Design, and Engineering

Over the past decade, protein design has drawn considerable attention and emerged as a promising tool for sustainable and efficient biocatalytic processes, driven by breakthroughs in DNA sequencing, gene synthesis, bioinformatics, and artificial intelligence. Enabled by strategies such as directed evolution, structure/function analysis, and (semi-)rational design, engineered enzymes can now accommodate nonnatural substrates, catalyze novel reactions, and operate under harsh conditions including elevated temperatures and organic solvents.This chapter provides a comprehensive overview of major strategies in protein design including directed evolution, rational design, and semi-rational design. Emphasis is placed on recent advances in high-throughput screening technologies, identification of functionally critical residues, and optimization of site-specific mutations. Furthermore, the integration of artificial intelligence and multiscale modeling to enzyme design is discussed, highlighting data-driven decision-making and deep learning-assisted prediction of enzymatic performance. Looking forward, biocatalysis is poised to further expand its applications in valorization of renewable resources, biofuels, and synthetic chemistry. Powered by ongoing advances in AI and computational protein design, the development of enzymes with unprecedented catalytic functions is anticipated to accelerate rapidly.