Combined active-site and global stability engineering improves catalytic activity and thermostability of Krasilnikovia cinnamomea transglutaminase.

Abstract

Streptomyces mobaraensis transglutaminase (SmTG) has limited thermostability, restricting high-temperature applications. We combined molecular-dynamics-guided active-site engineering and saturation mutagenesis with PROSS-assisted global stability design to improve Krasilnikovia cinnamomea transglutaminase (KcTG). The variants showed different activity-thermostability profiles. Untreated PDM4a and PDM4b showed specific activities of 126.89 and 113.80 U/mg and half-lives of 214 and 256 min at 60 °C, respectively. Proteinase K treatment raised the apparent specific activity of PDM4a to 206.67 U/mg but shortened its half-life to less than 2 min. Activity toward CBZ-Gln-Gly did not consistently reflect cross-linking of β-casein or soy protein isolate. Molecular dynamics simulations conducted exclusively on untreated enzymes suggested that catalytic-pocket loop flexibility may contribute to this substrate-dependent behavior. These findings show that complementary active-site and global stability engineering can improve KcTG and highlight the need to evaluate engineered transglutaminases with both model and protein substrates.

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