Parameters relevant to the thermodynamically controlled synthesis of cephalothin utilizing highly active stabilized penicillin G acylase derivatives were studied. These included solubility/stability of substrates, enzyme derivative activity/stability, reaction course and synthetic yields. These parameters were altered by varying the pH, dimethylformamide concentration and temperature. Simultaneous optimization of the selected parameters could not be achieved with a single set of conditions. However, continuous adjustment of conditions throughout the reaction course allowed each parameter to be optimized (dynamic reaction design). This strategy works by optimizing those parameters that are critical to the overall reaction at a given point, whilst leaving others sub-optimal when their contribution to the total is minimal. This strategy has achieved a 90% transformation of antibiotic nucleus to cephalothin at a final concentration of 20 g/l, high enzyme and reactant stability, with a reaction period of 3 h (using 1 ml of derivative/40 ml of reaction solution).