[Mining of ancestral lipases and enzymatic synthesis of amides]

The amide bond plays a crucial role in the synthesis of proteins, pharmaceuticals, and agrochemicals. It is particularly significant in the pharmaceutical field as a key structural unit in many drugs, such as afatinib (an anticancer drug), teriflunomide (used for multiple sclerosis treatment), acebutolol (used for hypertension and arrhythmia treatment), and paracetamol (an antipyretic and analgesic agent). However, chemical synthesis methods usually require harsh reaction conditions (e.g., high temperature, high pressure, or strongly acidic or alkaline environments) and are often accompanied by high energy consumption and environmental pollution issues. In contrast, enzymatic synthesis of amides has attracted widespread attention due to its mild conditions and environmental friendliness. To mine ancestral lipases with high activity and stability and investigate their enzymatic properties, thus achieving efficient enzymatic synthesis of amide bond-containing compounds. Ancestral lipases were screened based on a gene mining strategy employing ancestral sequence reconstruction (ASR), followed by characterization of their enzymatic properties and construction of an enzymatic reaction system for synthesis of the amide bond. Ancestral enzymes ASR-1 and ASR-7 with higher thermal stability were obtained. Substrate spectrum characterization revealed that ASR-1 preferentially catalyzed the synthesis of aromatic amides, while ASR-6 favored the synthesis of aliphatic amides. ASR-1 was purified via nickel-affinity chromatography and characterized for its enzymatic properties, showing an optimal reaction pH in Tris-HCl 7.5 and an optimal reaction temperature of 40 °C. The reaction conditions for amide synthesis were systematically optimized as 10 mg/mL wet cells, water content ≤10% (V/V), n-dodecane as solvent, 20 mmol/L amine substrate, and 10 mmol/L acyl donor. In addition, the enzyme exhibited good tolerance to nonpolar solvents, achieving an amide yield of 73.25%. This study provides a novel strategy for developing efficient and stable enzymatic processes for amide synthesis, demonstrating the promising application potential of ancestral lipases in green biomanufacturing.