Enzyme miniaturization: Revolutionizing future biocatalysts.

Enzyme miniaturization offers a transformative approach to overcome limitations posed by the large size of conventional enzymes in industrial, therapeutic, and diagnostic applications. However, the evolutionary optimization of enzymes for activity has not inherently favored compact structures, creating challenges for modern applications requiring smaller catalysts. In this review, we surveyed the advantages of miniature enzymes, including enhanced expressivity, folding efficiency, thermostability, and resistance to proteolysis. We described the applications of miniature enzymes as industrial catalysts, therapeutic agents, and diagnostic elements. We highlighted strategies such as genome mining, rational design, random deletion, and de novo design for achieving enzyme miniaturization, integrating both computational and experimental techniques. By investigating these approaches, we aim to provide a framework for advancing enzyme engineering, emphasizing the unique potential of miniature enzymes to revolutionize biocatalysis, gene therapy, and biosensing technologies.