Enhance Thermostability of β-Cyclodextrin Glycosyltransferase by Hydrophobic Interactions in the β-Sheet Structure.

β-Cyclodextrin (β-CD) is an attractive material with broad market potential across various industries. β-CD is produced from starch by β-cyclodextrin glycosyltransferase (β-CGTase), while the thermal stability of β-CGTase is one of the critical limitations in industrial β-CD production. Enhancing the thermal stability of β-CGTase is essential for increasing the β-CD yield and reducing production costs. In this study, site-directed mutagenesis was applied to the β-sheet region near the hinge of β-CGTase from STB01. By substitution of the native residue with hydrophobic amino acids, single mutant T487 V and double mutant T487 V/T500 V were obtained. The half-lives of T487 V and T487 V/T500 V at 55 °C were 51.7 and 68.4 min, respectively, 36.4 and 80.5% longer than that of the wild-type. Hydrophobic interactions within the β-sheet enhanced the thermal stability without compromising the catalytic efficiency of β-CGTase, although substrate affinity varied among the mutants. The double mutant T487 V/T500 V improved β-CD production, achieving a 62.5% conversion rate and a reduced reaction time. These findings provide valuable insights into β-CGTase thermal stability and substrate interaction with significant implications for industrial β-CD production.