Enhancing catalytic activity of thermostable 4-α-glucanotransferase from Thermus filiformis through semi-rational design

4-alpha-glucanotransferases (4GT) are valuable enzymatic tools with application in the food and pharmaceutical industries, particularly for producing thermoreversible starch gels. The screening of thermostable 4GT enzymes with high catalytic activity presents a significant challenge. In this study, a comprehensive screening of potential 4GT in the UniProt database led to the identification of a 4GT from Thermus filiformis (TfGT) with superior catalytic activity and thermal stability. To further improve its catalytic efficiency, a semi-rational design approach based on protein sequence conservation analysis was employed. The optimized mutant, M3 (Q60S/ Y452I/R455K), exhibited a 3.76-fold increase in specific activity compared to the wild type (WT) enzyme and retained more than 50 % of its activity after incubation at 70 degrees C for 24 h. Additionally, molecular dynamics simulations revealed that the enhanced activity of M3 was largely due to the reshaping of the substrate tunnel, which facilitated substrate entry to the active pocket and promoted the reaction. This study not only provides a robust approach for enhancing 4GT enzyme performance but also paves the way for broader industrial applications of 4GT enzymes.