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.
Subject(s)
High-Throughput Screening Assays , Protein Engineering , High-Throughput Screening Assays/methods , Protein Engineering/methods , Enzymes , Enzyme StabilityABSTRACT
The efficient production of high-value-added bioproducts from starchy substances requires α-amylases with hyperthermophilic properties for industrial starch liquefaction. In this study, two hyperthermophilic α-amylases with significant differences in thermostability, PfAmy and TeAmy, were comparatively studied through structural analysis, domain swapping, and site-directed mutagenesis, finding that three residues, His152, Cys166, and His168, located in domain B were the main contributors to hyperthermostability. The effects of these three residues were strongly synergistic, causing the optimum temperature for the mutant K152H/A166C/E168H of TeAmy to shift to 95-100 °C and stabilize at 90 °C without Ca2+. Compared to PfAmy and TeAmy, the mutant K152H/A166C/E168H, respectively, exhibited 1.7- and 2.5-times higher starch hydrolysis activity at 105 °C and pH 5.5 (10411 ± 70 U/mg) and released 1.1- and 1.7-times more maltooligosaccharides from 1% starch. This work has interpreted the hyperthermophilic mechanism of α-amylase and thereby providing a potential candidate for the efficient industrial conversion of starch to bioproducts.