Enzyme Engineering for High‐Yielding Amide Formation: Lipase‐Catalyzed Synthesis of N‐Acyl Glycines in Aqueous Media

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 long‐chain fatty acids (C8–C18), giving high yields of N‐decanoyl‐, 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. N‐acyl glycines are essential biosurfactants in cosmetics and pharmaceuticals. Their commercial production involves the use of neurotoxic phosgene. Aiming towards green synthesis of these amides, we developed a novel amide synthesis concept involving glycerol activation and evolved a hydrolytic enzyme by reshaping its catalytic pocket into a highly active amide bond‐forming enzyme, displaying 400‐fold higher catalytic efficiency for N‐lauroylglycine synthesis with over 80 % yield.