A original strategy to obtain intramolecular crosslinking is discussed. This strategy consisted of three consecutive steps to direct the reaction to the formation of intramolecular crosslinks : (a) enzyme are partially modified with the bifunctional reagent in a very controlled fashion; (b) the excess of reagent is removed; and (c) the modified enzyme is incubated long-term to allow a crosslinking reaction without the competition of additional single-point modifications. In this way, we were able to obtain interesting stabilizations of immobilized derivatives of penicillin G acylase using glutaraldehyde as a crosslinking reagent under very controlled conditions.