High-Throughput Screening Techniques for the Selection of Thermostable Enzymes

Abstract

The acquisition of a thermostable enzyme is an indispensable prerequisite for its successful implementation in industrial applications and the development of novel functionalities. Various protein engineering approaches, including rational design, semirational design, and directed evolution, have been employed to enhance thermostability. However, all of these approaches require sensitive and reliable high-throughput screening (HTS) technologies to efficiently and rapidly identify variants with improved properties. While numerous reviews focus on modification strategies for enhancing enzyme thermostability, there is a dearth of literature reviewing HTS methods specifically aimed at this objective. Herein, we present a comprehensive overview of various HTS methods utilized for modifying enzyme thermostability across different screening platforms. Additionally, we highlight significant recent examples that demonstrate the successful application of these methods. Furthermore, we address the technical challenges associated with HTS technologies used for screening thermostable enzyme variants and discuss valuable perspectives to promote further advancements in this field. This review serves as an authoritative reference source offering theoretical support for selecting appropriate screening strategies tailored to specific enzymes with the aim of improving their thermostability.

Crafting Genetic Diversity: Unlocking the Potential of Protein Evolution

“…The combination of a largersized and higher-quality library enables more efficient exploration of the protein space, particularly when the exploration is not constrained by screening/selection capacity. This review deliberately omits the discussion of screening or selection as it has been extensively covered in our textbook [5] and several recent, excellent reviews [66][67][68][69][70][71]. Readers are encouraged to consult these references for a comprehensive understanding of this aspect.…”