Alkaline phosphatase (ALP) was sucessfully incorporated into the halloysite (HAL) nanotubes, as confirmed by FTIR-ATR and XRD analyses. The loading efficiency (LE) of ALP was found to be 13.5%, while its encapsulation efficiency (EE) was estimated to be around 27%, as determined using the Bradford test. The influence of the immobilization in HAL on the enzyme activity was measured using standard ALP activity assay. Immobilized ALP effectively induced the bomineralization process, as showed by SEM, EDS, and XRD studies. As a result, calcium phosphate was produced in the form of hydroxyapatite cauliflower-like structures, with a slight content of calcium hydroxide. Interestingly, the encapsulation of ALP guest molecules in the HAL nanotubes considerably increased its thermal stability, most probably due to the heat sink effect. The activity of HAL-bound ALP was also found to be pH-independent in the wide range of pH values (3-10) due to the amphoteric nature of the aluminum oxide lining the HAL nanotube internal surface. Due to an increased resistance to the unfavorable conditions, which are often encountered during scaffold preparation or sterilization, ALP-HAL nanocomposite material may constitute an attractive bioactive component of the scaffolds for bone regeneration.
Keywords: Alkaline phosphatase; Biomineralization; Enzymatic activity; Halloysite; Nanoclay; Thermal stability.
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