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The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications."}]}],"creator":[{"@id":"https://cir.nii.ac.jp/crid/1070304781186495752","@type":"Researcher","foaf:name":[{"@language":"en","@value":"Ariyoshi, Ryutaro"},{"@language":"ja","@value":"有吉, 龍太郎"},{"@language":"ja-Kana","@value":"アリヨシ, リュウタロウ"}],"jpcoar:affiliationName":[{"@language":"en","@value":"Department of Applied Chemistry, Graduate School of Engineering, Kyushu University"},{"@language":"ja","@value":"九州大学大学院工学府応用化学専攻"}]},{"@id":"https://cir.nii.ac.jp/crid/1070304781186495749","@type":"Researcher","foaf:name":[{"@language":"en","@value":"Sato, Ryo"},{"@language":"ja","@value":"サトウ, リョウ"}],"jpcoar:affiliationName":[{"@language":"en","@value":"Department of Applied Chemistry, Graduate School of Engineering, Kyushu 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