Synthetic complexes were formed by interaction between acid phosphatase and either tannic acid or natural allophanic clay, and used as model systems to simulate enzymatic reactions occurring in heterogeneous environment. The presence of Mn and Mo on the kinetic constants of the immobilized phosphatase was also tested. Complexes were prepared at 30 °C by interaction of acid phosphatase and tannic acid or allophanic clay in the presence and absence of Mn or Mo. The effect of the addition order of the metal (micronutrient in soils) to the clay–enzyme complexes was evaluated, as well. acid phosphatase immobilized on tannic acid and mineral clay showed variable activity levels. When phosphatase was immobilized on tannic acid, a recovery of about 51% of the initial enzymatic activity and a decrease of 17% of its catalytic efficiency was measured. The presence of Mn and Mo decreased the Vmax of phosphatase–tannic complexes as compared with that of the free phosphatase. All the added phosphatase molecules were immobilized onto the clay, when it was used as the immobilizing support. Phosphatase–clay complexes showed an increase of both enzymatic activity (higher Vmax value) and substrate affinity (lower Km values) as compared to the free enzyme, resulting in an increase by about 65% of the catalytic efficiency. The presence of Mn added at the same time with enzyme and clay decreased the enzymatic activity of the immobilized enzyme. However, when Mn was applied after the interaction of the enzyme with the clay, no significant effects on the residual activity of the immobilized enzyme were observed. Conversely, the order of the addition of Mo to the clay–enzyme complexes strongly influenced the activity and the kinetic behaviour of the immobilized enzyme. Moreover, Mo had higher inhibitory effects than Mn on phosphatase immobilized on both supports. The overall results seem to suggest that the immobilization of acid phosphatase on clay not only preserved but also enhanced the activity of the enzyme as compared with organic matter.
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