Tandem use of carboxypeptidase Y reactor and displacement chromatograph for peptide synthesis.
A packed-bed enzyme reactor with immobilized carboxypeptidase Y was used in tandem with a displacement chromatograph for the preparation of N-benzoyl-L-arginyl-L-methioninamide, from N-benzoyl-L-arginine and L-methioninamide. The pumps and valves of the coupled enzyme reactor and displacement chromatograph were controlled by a microprocessor. The enzyme was immobilized on microparticulate amino-silica by glutaraldehyde and packed into a 60 X 4.6 mm I.D. column. The packed-bed reactor was used in the recirculating mode and components of the reaction mixture were subsequently separated by displacement chromatography on a 250 X 4.6 mm octadecyl-silica column using butoxyethoxyethanol as the displacer. Unreacted L-methioninamide was returned to the reaction mixture. Both the progress of the reaction and the extent of separation by displacement chromatography were monitored by high-performance liquid chromatographic analysis. The system was designed so that enzymatic peptide synthesis, separation by displacement chromatography, and column regeneration were carried out simultaneously by using two identical columns in parallel. An amount of 460 mg of N-benzoyl-L-arginyl-L-methioninamide having purity greater than 99% could be obtained in 24 h with this system. The tandem operation of the enzyme reactor and liquid chromatograph operated in the displacement mode offers a means for the synthesis and purification of peptides.
D002268:Carboxypeptidases; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004151:Dipeptides / Q000032:analysis / Q000138:chemical synthesis*; D004800:Enzymes, Immobilized
D004151:Dipeptides; D004800:Enzymes, Immobilized; C053218:N-benzoylarginyl-methioninamide; D002268:Carboxypeptidases; C022633:serine carboxypeptidase