Present communication is concerned with the application of monolayer engineering, in particular of 'protective plate' technique, for the fabrication of alternate-layer assemblies based on enzyme penicillin G acylase. Several structures are compared with each other. The deposited films are tested to determine the values of enzymatic activity and the level of protein detachment in aqueous solutions. As the result, the deposition procedure is found, which enables to obtain biocatalytic media with enhanced performances. The biocatalyst efficiency is proved by three independent techniques including direct yield determination with HPLC. The advantage of applied method of enzyme immobilization with respect to other techniques is demonstrated.