This paper deals with the on-line estimation of unknown kinetics for the enzymatic synthesis of ampicillin, that is carried out inside a fed-batch bioreactor. A nonlinear dynamical model of this bioprocess is obtained by using the reaction scheme and the mass balance. The dynamical kinetics is strongly nonlinear and not exactly known; therefore an online estimation strategy is developed, based on high gain approach. The tuning of the proposed high gain observers is reduced to the calibration of a single parameter. Numerical simulations are included in order to test the behavior and the performances of the proposed estimation strategy.