Maltose gradient-induced biosensor-based high-throughput screening for directed evolution of maltogenic amylase from Bacillus stearothermophilus

Maltogenic amylase is a starch-hydrolyzing enzyme commonly used in bread baking and high-concentration maltose syrup production. However, low catalytic activity limits its industrial application. Improving catalytic activity based on molecular modification and directed evolution requires a High-Throughput Screening (HTS) method. In this study, a maltose gradient-induced (MaGI) biosensor was designed and applied for the directed evolution of maltogenic amylase AmyM, showing a good positive correlation between enzyme activity and fluorescence. The MaGI biosensor detected maltose and maltogenic amylase activity efficiently and specifically. Two mutants, Q440N and S442N/Q661L, were identified through the screening of 3000 mutants using the MaGI biosensor, showing a significant increase in catalytic activity of 35.56 % and 24.51 %, respectively, compared to the wild-type. Meanwhile, the t 1/2 of Q440N and S442N/Q661L at 60 °C increased by 58.53 % and 66.66 %, respectively. In industrial applications, the enhancement of catalytic activity and stability is conducive to improving production efficiency and reducing costs. MD simulation has found that when modifying multidomain enzymes, distal mutations can enhance catalytic activity. In conclusion, the developed MaGI biosensor is a promising tool for high-throughput and specific detection of maltose.