A single-pot enzymatic synthesis of Z-CCK5 (4-8) is presented in this work, employing Z-Gly-Trp-OBzl as acyl donor, under kinetic control. The first goal of the work is the development of a synthetic strategy allowing the use of the same medium for two reactions catalyzed by immobilized alpha-chymotrypsin, discriminating between simultaneous and consecutive addition systems. The second goal is the maximization of the pentapeptide yield as a function of the molar excess of both nucleophiles employed. A maximum yield of 36% was obtained, and the addition strategy as well as the optimal initial concentrations of substrates have been determined.