Peptide synthesis with immobilized carboxypeptidase Y

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Abstract
Enzymatic peptide synthesis was investigated using carboxypeptidase Y immobilized with glutaraldehyde on 10 μm microparticulate amino‐silica. Carboxypeptidase Y was immobilized with 98.5% recovery of active enzyme to yield the immobilized enzyme having 0.55 units esterase activity/mg amino‐silica support. The stability of the immobilized enzyme was examined as a function of pH, temperature, and reactant concentrations. Immobilized Carboxypeptidase Y was used in stirred batch and recirculating packed‐bed reactors for peptide synthesis. Packed‐bed reactors (40 × 4.6 mm, 60 × 4.6 mm) were used to catalyze the synthesis of 170 mg N‐benzoyl‐L‐arginyl‐L‐methioninamide, 380 mg N‐benzoyl‐L‐arginyl‐L‐methionyl‐L‐leucinamide, and 200 mg N‐benzoyl‐L‐arginyl‐L‐methionyl‐L‐leucyl‐L‐phenylalaninamide in 8, 3, and 1 hour, respectively, as intermediates in the synthesis of L‐methionyl‐L‐leucyl‐L‐phenylalanine. No inactivation of the immobilized enzyme was observed during the course of the reactions. The N‐benzoyl‐L‐arginyl group served to increase the water solubility of the peptides and was removed by immobilized trypsin at the end of synthesis to obtain the final product. While the first two syntheses were conducted with aqueous reaction mixtures, the synthesis of N‐benzoyl‐L‐arginyl‐L‐methionyl‐L‐leucyl‐L‐phenylalaninamide was carried out in a reaction mixture containing dimethylformamide to avoid precipitation of the product. HPLC and amino acid analysis confirmed the high purity and amino acid composition of the final product.