Enzyme-catalysed synthesis of calcium phosphates

A biomimetic method is described for the precipitation of nanosized calcium phosphates using the alkaline phosphatase (EC 3.1.3.1), which is responsible for hydrolysis of organic and inorganic phosphates in vivo. Buffered solutions containing glycerol-2-phosphate and CaCl₂ in addition to MgCl₂ and the respective enzyme were prepared for calcium phosphate precipitation. The phosphate group of glycerol-2-phosphate was cleaved through enzymatic hydrolysis. The local inorganic phosphate concentration increased resulting in the precipitation of nanosized calcium phosphates phases (Ca–P phase) composed of calcium deficient hydroxyapatite (CDHA) and hydroxyapatite (HA). At high Ca²⁺-concentration and large enzyme amounts mixed phases of HA/CDHA with an increasing quantity of HA were favoured. Under basic conditions (pH > 9) formation of HA was observed, whereas at neutral pH of 7.5 CDHA was primarily formed. The assignment of Ca–P phases was accomplished by FT-IR and Raman-spectroscopy in addition to X-ray diffractometry. The Ca–P materials exhibited BET surface areas of 173 m²/g. SEM-micrographs of the Ca–P powders showed globular-shaped agglomerates of Ca–P particles. The size of the Ca–P crystallite ranged from 9 nm to 25 nm according to transmission electron microscopy (TEM), where round-shaped, platelike and fibrelike crystallites were found. All crystallites showed diffuse ring patterns in electron diffraction confirming the nanosize of the precipitate. Using the developed technique, it was possible to synthesise 100 g of bonelike Ca–P materials in 1 day using 15 L batches with optimised parameters.