Rational enzyme design by reducing the number of hotspots and library size.

Biocatalysts that are eco-friendly, sustainable, and highly specific have great potential for applications in the production of fine chemicals, food, detergents, biofuels, pharmaceuticals, and more. However, due to factors such as low activity, narrow substrate scope, poor thermostability, or incorrect selectivity, most natural enzymes cannot be directly used for large-scale production of the desired products. To overcome these obstacles, protein engineering methods have been developed over decades and have become powerful and versatile tools for adapting enzymes with improved catalytic properties or new functions. The vastness of the protein sequence space makes screening a bottleneck in obtaining advantageous mutated enzymes in traditional directed evolution. In the realm of mathematics, there are two major constraints in the protein sequence space: (1) the number of residue substitutions (); and (2) the number of codons encoding amino acids as building blocks (). This feature review highlights protein engineering strategies to reduce screening efforts from two dimensions by reducing the numbers and , and also discusses representative seminal studies of rationally engineered natural enzymes to deliver new catalytic functions.