BACKGROUND: A totally new approach has been applied for mathematical modeling of the enzyme activity/pH relationship, for quantification and distribution of enzyme activity in and out of carrier pores. This is a very simple and elegant method for determination of the distribution of enzyme molecules on the surface and inside the particles, simply through measurement of enzyme activity at different pH values. RESULTS: Amyloglucosidase (AG) from Aspergillus niger was covalently immobilized onto poly(GMA-co-EGDMA) by the glutaraldehyde and periodate methods. Mathematical modeling of the pH optima for two types of covalently immobilized AG resulted in higher enzyme amounts on the surface within periodate immobilizate (67%) in comparison with glutaraldehyde immobilizate (53%). These values are modified to 64.25% for periodate immobilizate and 49.95% for glutaraldehyde immobilizate when diffusion effects are taken into account. CONCLUSION: The mathematical model applied enabled observation of the difference between the two types of coupling agents and different immobilization procedures throughout quantification of the immobilized enzyme on the matrix surface and inside pores. Copyright (c) 2012 Society of Chemical Industry