The rates and yields in the synthesis of N-protected amino compounds catalyzed by penicillin G acylase are very different when using different protecting reagents, D- or L-mandelic acid methyl ester, or D- or L-phenylglycine methyl ester. L-mandelic acid methyl ester (L-MAME) gave the best adsorption of the nucleophile, the best transferase/hydrolase activity ratios, and the lowest rate of hydrolysis of the synthetic product when using several amino compounds (antibiotic nucleus, hydroxylamine, amino acids, and amino acid derivatives). For example, L-MAME gave synthetic yields up to 32-fold higher than D-phenylglycine for enzymic protection of tyrosinamide. Furthermore, N-protections of chiral amines by penicillin G acylase are extremely stereospecific; thus, optically pure L-mandelyl-L-amino acids were produced at high yields by protection of racemic mixtures of amino acids (alanine, phenylglycine, etc.) catalyzed by penicillin G acylase under very mild experimental conditions (aqueous media, neutral pH, and moderate temperature).