The effect of stirring and seeding on the AcPheLeuNH(2) synthesis and crystallization in a reversed micellar system

The present work describes the enzymatic synthesis and simultaneous crystal lization of the dipeptide AcPheLeuNH(2) by alpha-chymotrypsin In a reversed micellar system of tetradecyltrimethylammonium bromide (TTAB)/heptane/octa no/carbonate buffer. The low solubility of the dipeptide in the micellar so lution led to the formation and growth of needle-like crystals during the s ynthesis as soon as supersaturation was achieved. The crystallization proce ss then followed a typical pattern, proceeding in three phases: nucleation, de-supersaturation, and re-equilibrium of saturation. Crystallization was followed by visual observation with an optical microscope, and the increase of crystal number and size was confirmed. Experiments showed chat the supe rsaturation concentration decreases with the addition of AcPheLeuNH(2) seed s, before the reaction, and also with a decrease of the stirring speed. It was also observed that the increase of both seed concentration and stirring advances the start of crystallization, so that the dipeptide is more quick ly removed from solution. The consequent decrease in its loss through hydro lysis causes an increase in its yield. Both stirring and seeding could cons titute important generic strategies for promoting crystallization of more s oluble dipeptides during their synthesis in similar reversed micellar syste ms. (C) 2000 Elsevier Science Inc. All rights reserved.