Enhancement of transglutaminase activity by NMR identification of its flexible residues affecting the active site

FEBS Letters

Kashiwagi, Tatsuki¹  Ejima, Daisuke¹  Yokoyama, Kei-ichi¹  Ishikawa, Kohki¹  Shinohara, Mina¹  Suzuki, Ei-ichiro¹  Shimba, Nobuhisa¹ 

[1] Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan

关键词: Transglutaminase;    Nuclear magnetic resonance;    Flexible region;    Site-directed mutagenesis;    Protein engineering;    TGase;    transglutaminase;    MTG;    microbial transglutaminase;    ELT;    enzymatic labeling technique;    GTG;    guinea pig liver transglutaminase;    FTG;    red sea bream liver transglutaminase;   

DOI  :  10.1016/S0014-5793(02)02616-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Incorporation of inter- or intramolecular covalent cross-links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins, such as tofu, boiled fish paste, and sausage. By using nuclear magnetic resonance, we have shown that the residues exhibiting relatively high flexibility in MTG are localized in the N-terminal region; however, the N-terminal region influences the microenvironment of the active site. These results suggest that the N-terminal region is not of primary importance for the global fold, but influences the substrate binding. Therefore, in order to increase the transglutaminase activity, the N-terminal residues were chosen as candidates for site-directed replacement and deletion. We obtained several mutants with higher activity, del1–2, del1–3, and S2R. We propose a strategy for enzyme engineering targeted toward flexible regions involved in the enzymatic activity. In addition, we also briefly describe how the number of glutamine residues in a substrate protein can be increased by mixing more than two kinds of TGases with different substrate specificities.

【 授权许可】

Unknown