Gelatin is a low value water-soluble protein which has the ability to form gels and its use with the addition of plasticisers has been studied in the development of biofilms. Many studies show the use of gelatin in high concentrations agent immobilisation successfully, however, most of these do not show the Bloom value of the gelatin used. In this study, lipase immobilisation in gelatin of different Bloom values with the addition of hydrophilic plasticisers, glycerol and mannitol was investigated. Mannitol showed higher efficiency for lipase immobilisation in more uniform pore structures. The immobilisation stability was affected by the pH, temperature and agitation conditions, and the matrices E8-280-M (corresponding to experiment 8, 280 Bloom gelatin and mannitol) and E9/10-280-M (corresponding to experiment 9 and 10, 280 Bloom gelatin and mannitol) were the most stable. In the hydrolysis of olive oil, the enzymatic activity of free and immobilised lipases was studied in relation to temperature, pH and time, and 42 degrees C was found to be the optimal temperature for both forms of lipases. In terms of activity, for free lipase, the optimum pH was 7.0, for lipase immobilised in E8-280-M it was between 5.5 and 7.5 and for lipase immobilised in E9/10-280-M the optimum range was 6.5 and 7.5. Ten reuse cycles were possible, and by the 5th cycle the lipase immobilised in E8-280-M showed 77.66% of the initial activity. The highest retention of activity during storage was observed for the lipase immobilised at 25 degrees C in the E8-280-M matrix.