Amide syntheses remain a key challenging green chemistry reaction. For instance, green synthesis of N-acyl glycines as biosurfactants and therapeutics is highly desirable to replace chemical pathways using toxic phosgene. Herein, we report a novel concept for enzymatic amidation in an aqueous system via glycerol activation of fatty acids and theirsubsequent aminolysis with glycine to synthesize N-acyl glycines. We then engineer an enzyme (proRML) by reshaping its catalytic pocket to enhance its aminolysis activity and catalytic efficiency by 103-fold and 465-fold, respectively. The evolved proRML (D156S/L258K/L267N/S83D/L58K/ R86K/W88V) catalyzed the amidation of a fatty acid with glycine to give N-lauroylglycine with high yield (80 %). It accepts a broad range of medium-to longchain fatty acids (C 8 -C 18 ), giving high yields of Ndecanoyl-, N-myristoyl-, and N-oleoylglycine. The developed amidation concept may be general, and the engineered enzyme is useful for the green synthesis of N-acyl glycines.

Amide syntheses remain a key challenging green chemistry reaction. For instance, green synthesis of N-acyl glycines as biosurfactants and therapeutics is highly desirable to replace chemical pathways using toxic phosgene. Herein, we report a novel concept for enzymatic amidation in an aqueous system via glycerol activation of fatty acids and theirsubsequent aminolysis with glycine to synthesize N-acyl glycines. We then engineer an enzyme (proRML) by reshaping its catalytic pocket to enhance its aminolysis activity and catalytic efficiency by 103-fold and 465-fold, respectively. The evolved proRML (D156S/L258K/L267N/S83D/L58K/ R86K/W88V) catalyzed the amidation of a fatty acid with glycine to give N-lauroylglycine with high yield (80 %). It accepts a broad range of medium-to longchain fatty acids (C 8 -C 18 ), giving high yields of Ndecanoyl-, N-myristoyl-, and N-oleoylglycine. The developed amidation concept may be general, and the engineered enzyme is useful for the green synthesis of N-acyl glycines.