Kinetic Analysis of L-Carnosine Formation by β-Aminopeptidases

The (beta,alpha-dipeptide L-carnosine occurs in high concentrations in long-lived innervated mammalian tissues and is widely sold as a food additive. Oil 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 beta-amino-peptidases 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 beta-alanine derivatives (acyl donor) and L-histidine (acyl acceptor) in an aqueous reaction Mixture at pH10 with high catalytic rates (V-max = 19.2 mu mol min(-1) per mg of protein, k(cat) = 12.9 s(-1)), whereas V-max in the BapA-catalyzed coupling reaction remained below 1.4 mu mol min(-1) per mg of protein (k(cat) = 0.87 s(-1)). Although the equilibrium Of this reaction lies oil 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 pK(a) 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.