We studied enzymatic adipyl-7-ADCA hydrolysis as a new process for the production of 7-aminodeacetoxycephalosporanic acid (7-ADCA), one of the building blocks for cephalosporin antibiotics like cephalexin and cefadroxil. Adipyl-7-ADCA hydrolysis carried out with immobilised glutaryl acylase was considerably enhanced by addition of phenylglycine amide, the side-chain donor used for cephalexin synthesis; unlike reactions carried out with free enzyme. The rate enhancing effect was not specifically related to phenylglycine amide; we found a linear relationship between the reaction rate and the buffering capacity of the added substance. These observations can be explained by a pH-gradient in the immobilised enzyme, the pH inside the particle being lower (corresponding to low enzyme activity) than outside. It was concluded that the buffer reduced the pH-gradient inside the biocatalyst, and therewith, caused the reaction rate enhancing effects. Further, chloride ions decreased the reaction rate strongly, while sodium, magnesium, sulphate, and potassium did not influence the reaction rate much. For an actual process, it is important to use a buffer that is appropriate for the reaction-pH. In that way the amount of enzyme required in a process can be reduced considerably, in our case a factor of three was found.