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The β,αdipeptide Lcarnosine occurs in high concentrations in longlived innervated mammalian tissues and is widely sold as a food additive. On a large scale Lcarnosine is produced by chemical synthesis procedures. We have established two aqueous enzymatic reaction systems for the preparation of Lcarnosine using the dissolved bacterial βaminopeptidases DmpA from Ochrobactrum anthropiand BapA from Sphingosinicella xenopeptidilyticaas catalysts and investigated the kinetics of the enzymecatalyzed peptide couplings. DmpA catalyzed the formation of Lcarnosine from Cterminally activated βalanine derivatives acyl donor and Lhistidine acyl acceptor in an aqueous reaction mixture at pH10 with high catalytic rates Vmax19.2μmolmin−1per mg of protein, kcat12.9s−1, whereas Vmaxin the BapAcatalyzed coupling reaction remained below 1.4μmolmin−1per mg of protein kcat0.87s−1. Although the equilibrium of this reaction lies on the side of the hydrolysis products, the reaction is under kinetic control and Lcarnosine 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 byproducts. The substitution of Lhistidine for Lhistidine methyl ester as acyl acceptor shifted the pKaof the amino functionality from 9.25 to 6.97, which caused a drastic reduction in the amount of coupling byproducts in an aqueous reaction system at pH8.