Enhancing the activity and thermostability of Streptomyces mobaraensis transglutaminase by directed evolution and molecular dynamics simulation

Streptomyces mobaraensis transglutaminase (MTG) has been extensively used in food industry and other biotechnological fields due to its cross-linking modification of proteins. Thus a MTG variant with a higher thermostability as well as an enhanced activity is desired because some processing for MTG applications is often performed at higher temperatures. In this study, the activity and thermostability of wild-type MTG (WT) were enhanced via directed evolution using error-prone PCR, and the mutant MTG (E164L) with the improved specific activity and thermostability was obtained using a high-throughput activity assay. E164L exhibited a 1.95 times specific activity of WT at 50 degrees C. Meanwhile, the half-life of E164L at 50 degrees C was 1.66-fold of WT. The molecular dynamics (MD) simulation results indicated that the mutation Glu164Leu resulted in the weaker interactions of Asp159-Glu164 and Gly228-Leu231, leading to the enhanced instability of Ile240-Asn253 linked to Gly228-Leu231 by eight residues. It further caused the reduced interactions between loop region 1 (Ile240-Asn253) and loop region 2 (His277-Met288), facilitating the access of the substrate molecule to the active site. This study improved the understanding of structure-activity relationship for MTG adapted to high temperature conditions. It also provides theoretical foundation and preliminary information for engineering MTG with enhanced characteristics to meet the industrial requirements.