원문링크: https://pubs.acs.org/doi/10.1021/acs.chemrev.0c01201

Research Background

The ground-breaking strategies for enzyme engineering i.e. directed evolution and rational design, have found widespread applications in improvement of inherent activities of proteins. Although many advances have been accomplished by these powerful approaches, the chemical diversity of the functional biopolymers is limited to the 20 canonical amino acids, which limit the functional groups that can be applied to the optimization of enzymes. The sequence space of proteins can be significantly expanded via chemical modification and/or incorporation of non-canonical amino acids (ncAAs). Despite the notable achievements in this area, a comprehensive review that critically evaluates the methodologies of chemical protein modification and ncAA incorporation and their utility in the fields of enzyme engineering and biocatalysis was missing from the literature.

Research Outcomes

This review offers a comprehensive survey of the literature describing how chemical modifications of proteins and/or the incorporation of non-canonical amino acids can advance our understanding of enzyme catalysis or lead to biocatalysts with augmented properties. The article is divided into three main approaches for modifying protein structure and function: (i) chemical modification of cAAs, (ii) ncAA incorporation, and (iii) chemical modification of incorporated ncAAs. This review provides a unique overview of all recent attempts and ideas to overcome the bottlenecks of traditional approaches and emphasizing important contributions across these areas. It covers the examples of enzyme mechanistic studies, functional enhancement, and creation novel artificial enzymes providing a valuable resource to a broad research community.

Future plans

We aim to engineer enzymes for functional enhancement through rational ncAA incorporation, which could not be achieved through traditional enzyme engineering approaches. These studies could be further expanded using a directed evolution approach with ncAAs to optimize enzyme properties in the presence of an expanded amino acid alphabet.

Abstract

The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chemical diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chemical modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and critical evaluation of the applications, recent advances, and technical breakthroughs in biocatalysis for three approaches: (i) chemical modification of cAAs, (ii) incorporation of ncAAs, and (iii) chemical modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.