Heterologous Expression of Thermotolerant α-Glucosidase in Bacillus subtilis 168 and Improving Its Thermal Stability by Constructing Cyclized Proteins

Abstract

α-glucosidase is an essential enzyme for the production of isomaltooligosaccharides (IMOs). Allowing α-glucosidase to operate at higher temperatures (above 60 °C) has many advantages, including reducing the viscosity of the reaction solution, enhancing the catalytic reaction rate, and achieving continuous production of IMOs. In the present study, the thermal stability of α-glucosidase was significantly improved by constructing cyclized proteins. We screened a thermotolerant α-glucosidase (AGL) with high transglycosylation activity from Thermoanaerobacter ethanolicus JW200 and heterologously expressed it in Bacillus subtilis 168. After forming the cyclized α-glucosidase by different isopeptide bonds (SpyTag/SpyCatcher, SnoopTag/SnoopCatcher, SdyTag/SdyCatcher, RIAD/RIDD), we determined the enzymatic properties of cyclized AGL. The optimal temperature of all cyclized AGL was increased by 5 °C, and their thermal stability was generally improved, with SpyTag-AGL-SpyCatcher having a 1.74-fold increase compared to the wild-type. The results of molecular dynamics simulations showed that the RMSF values of cyclized AGL decreased, indicating that the rigidity of the cyclized protein increased. This study provides an efficient method for improving the thermal stability of α-glucosidase.