title: Enhancement of transglutaminase activity by NMR identification of its flexible residues affecting the active site.
abstract: 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.
authors.affiliation: Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Japan.
authors.forename: Nobuhisa
authors.initials: N
authors.lastname: Shimba
authors.forename: Mina
authors.initials: M
authors.lastname: Shinohara
authors.forename: Kei ichi
authors.initials: Ki
authors.lastname: Yokoyama
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Kashiwagi
authors.forename: Kohki
authors.initials: K
authors.lastname: Ishikawa
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ejima
authors.forename: Ei ichiro
authors.initials: Ei
authors.lastname: Suzuki
mesh_terms: D001665:Binding Sites; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005511:Food Handling; D005973:Glutamine / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017510:Protein Folding; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005973:Glutamine; D011503:Transglutaminases