In this study, the poly (sodium 4-styrenesulfonate)-mediated calcium carbonate magnetic microspheres were prepared by a simple spontaneous biomimetic mineralization method to immobilize kallikrein. The immobilized kallikrein can be easily separated from the reaction mixture by an external magnet. The structural properties of the materials were studied by several characterization techniques. Subsequently, the immobilized kallikrein enzyme activity and stability were studied. Moreover, the inhibitory potency of ten natural compounds on kallikrein was evaluated by combining with capillary electrophoresis analysis. The immobilized kallikrein exhibited optimum pH and temperature at 7.5 and 75°C, respectively. After seven successive cycles, the immobilized kallikrein retained 82% of its initial activity. Furthermore, the Michaelis–Menten constant (Km) and the half-maximal inhibitory concentration (IC50) of serine protease inhibitor on the immobilized kallikrein were determined to be 3.30 mM and 67.04 μM, respectively. In addition, berberine, evodine, coptisine, and jatrorrhizine among the ten natural compounds showed good inhibitory activity on the immobilized kallikrein, and their % of inhibition were (43.1 ± 1.7), (34.4 ± 1.9), (38.0 ± 1.7), and (28.5 ± 1.0)%, respectively. These results indicate that the biomimetic mineralization synthetic magnetic microsphere is an efficient method for kallikrein immobilization.
