In the present study, penicillin G acylase (PGA), an enzyme belonging to the family of hydrolases, has been investigated as chiral selector in CE using the partial filling technique. Owing to the strong disposition of PGA to be adsorbed by the inner capillary permanently coated capillaries were used to diminish both the protein-wall interwall, actions and the EOF. In particular, the silica surface of the capillary was chemically coated by an antiadhesive and an hydrophilic layer of pullulan, a high-molecular-mass homopolysaccharide. The coating procedure consisted in the silanization with glycidoxypropyltrimethoxysilane and the subsequent coupling of the hydroxyl groups of pullulan onto the silanized capillary. Using this approach, a significant EOF suppression was obtained within a wide pH range (pH 3.0-9.0); this result was very important in order to find the suitable conditions for the application of partial filling technique. The optimization of partial filling was carried out by considering the effects of different experimental conditions (buffer pH, PGA concentration, and loading duration), on the migration time and enantioresolution of rac-ketoprofen. Under the selected conditions as: 100 mM sodium phosphate buffer (pH 5.5) containing 240 mu M of PGA (partial filling of 120 s at a pressure of 50 mbar), a series of acidic compounds resulted to be enantioresolved in about 10 min. The long-term stability of the proposed coating was evaluated; more than 100 injections were performed without significant loss of reproducibility.