Discovering novel transglutaminases from Streptomyces species for efficient protein cross-linking in foods

Transglutaminase (TG) is a key enzyme in food processing by catalyzing protein crosslinking. This study identified novel hyperactive TGs from Streptomyces species to enhance crosslinking efficiency. We integrated phylogenetic analysis for initial screening, followed by virtual docking for further selection. Of the eight variants tested, all exhibited specific activity against CBZ-Gln-Gly, with TG derived from Streptomyces wuyuanensis (SwTG) displayed a specific activity of 78.3 U/mg, which is 2.3-fold higher than that of the commonly used Streptomyces mobaraensis derived TG (SmTG). The top three variants were selected for crosslinking experiments, showing that SwTG, Streptomyces sp. TN58 derived TG (StTG), and Streptomyces roseoverticillatus (SrTG) outperformed SmTG in casein crosslinking, while only StTG showed obvious higher activity than SmTG in crosslinking minced meat. These findings suggest that the specific activity of TGs does not always correlate with their ability to induce protein crosslinks. Further investigation using molecular dynamics simulations revealed that larger enzyme volumes with lower flexibility could hinder substrate binding, resulting in weak crosslinking activity for food proteins. Additionally, surface charge was found to be another key factor that disrupt substrate binding. This study presents alternative TGs for generating food crosslinks and offers valuable insights into the mechanisms behind TG-induced crosslinking.