Kinetic Analysis of L‐Carnosine Formation by β‐Aminopeptidases

Abstract

AbstractThe β,α‐dipeptide L‐carnosine occurs in high concentrations in long‐lived innervated mammalian tissues and is widely sold as a food additive. On a large scale L‐carnosine is produced by chemical synthesis procedures. We have established two aqueous enzymatic reaction systems for the preparation of L‐carnosine using the dissolved bacterial β‐aminopeptidases DmpA from Ochrobactrum anthropi and BapA from Sphingosinicella xenopeptidilytica as catalysts and investigated the kinetics of the enzyme‐catalyzed peptide couplings. DmpA catalyzed the formation of L‐carnosine from C‐terminally activated β‐alanine derivatives (acyl donor) and L‐histidine (acyl acceptor) in an aqueous reaction mixture at pH 10 with high catalytic rates (Vmax=19.2 μmol min−1 per mg of protein, kcat=12.9 s−1), whereas Vmax in the BapA‐catalyzed coupling reaction remained below 1.4 μmol min−1 per mg of protein (kcat=0.87 s−1). Although the equilibrium of this reaction lies on the side of the hydrolysis products, the reaction is under kinetic control and L‐carnosine temporarily accumulated to concentrations that correspond to yields of more than 50% with respect to the employed acyl donor. However, competing nucleophiles caused unwanted hydrolysis and coupling reactions that led to decreased product yield and to formation of various peptidic by‐products. The substitution of L‐histidine for L‐histidine methyl ester as acyl acceptor shifted the pKa of the amino functionality from 9.25 to 6.97, which caused a drastic reduction in the amount of coupling by‐products in an aqueous reaction system at pH 8.