Peptide Synthesis Using Enzyme as Synthetic Catalyst-Synthesis of New Water-Soluble Ester Substrates and Enzyme Immobilization-

Abstract

Two problems which are currently urged to be solved in enzymatic synthesis of peptid are described. One is low solubility of substrate which reduces the efficiency of enzymatic reaction, especially in the case of continuous flow system of immobilized enzyme. The other is an enzyme immobilization with high enzyme activity which brings economical profit by repeating use.
The water-soluble polyethylene glycol esters, [1], [4], [5], [6] in Table 1, reacted with leucinamide in the presence of ce-chymotrypsin to give the corresponding amides which were perceived by the precipitation of insoluble products. The ester [7] also gave CET by enzymatic displacement with 7-ACA. The water-soluble esters having suitable poly(oxyethylene)chain length is considered to be applicable to another field of enzymatic synthesis as substrates.
The five kinds of immobilized protease carriers were prepared and the efficiency was tested in the coupling reaction of porcine des-alanyl(B30)-insulin(p-DAI) with Thr-OBu^t which is the key step in converting porcine insulin into human insulin (Fig.2). Carrier-5 (poly (glutamic acid)) was found to be good for immobilizing Achromobacter protease I with high activity. Immobilized Achromobacter protease I on carrier-5 (Poly-Glu-A in Table 4) showed 74 per cent yield in the coupling step. Protease activities immobilized on carrier-1, -2, -3, and -4 were not so high as to be used in the coverting 'processes.