Formation of amide bond catalyzed by lipase in aqueous phase for peptide synthesis

• Lipase catalyzed amidation for peptide synthesis.
• A high yield of dipeptide was obtained in aqueous solution.
• RSM design for optimization of reaction parameters.
• Using the kinetic model compared the catalytic efficiency.
• Kinetic constants show the advantage of lipase on amidation.

A dipeptide N-acetyl-l-phenylalanyl-l-tyrosinamide (N-Ac-Phe-Tyr-NH2), with angiotensin I converting enzyme (ACE) inhibitor activity, was synthesized via porcine pancreatic lipase catalyzed amidation of N-acetyl-phenylalanine ethyl ester with l-tyrosinamide in an aqueous phase. Response surface methodology was employed to evaluate the effects of synthesis parameters. The optimum synthesis conditions obtained an 84.45% yield of N-Ac-Phe-Tyr-NH2 with a reaction time of 3.8 min, a temperature of 20.9 °C, an enzyme amount of 6.5 U, and a substrate molar ratio of 2.5:1 (Tyr:Phe). The kinetics of lipase and α-chymotrypsin catalyzed amidation was compared using the Ping-Pong mechanism. The lipase showed a lower apparent kinetic constant than α-chymotrypsin indicating that the acyl lipase intermediate had a higher affinity toward tyrosinamide in the amidation. In addition, because the lipase can avoid the secondary hydrolysis of synthesized peptide, it is expected to be an effective method for obtaining a good yield of dipeptide.

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