Amine-functionalization of hierarchical calcium phosphate microflowers enhances the reusability of immobilized β-galactosidase

Metal phosphate-based hierarchical microflowers have been reported for enzyme immobilization; however, they suffer from enzyme desorption due to the lack of stable interactions between enzymes and the support. Herein, polyamines were used as surface modifiers of calcium phosphate microflowers to enable covalent immobilization of β-galactosidase. Calcium phosphate microflowers showed a mean diameter of 12.6 ± 3.4 μm, pore size of 439 ± 159 nm, and sheet thickness of 196 ± 51 nm, confirming its hierarchical architecture. Microflowers were modified using non-modified and crosslinked polyallylamine variants (molar ratios of 1:0.25, 1:0.5 and 1:1), and their performance was compared to that of physically adsorbed and free β-galactosidase. Polyallylamine increased immobilized enzyme by ∼60 % compared to enzyme immobilization via adsorption on non-modified microflowers. In terms of substrate conversion, k_cat increased only when the lowest level of crosslinking was used, but the catalytic efficiency of the enzyme decreased without exception. In most cases, covalently immobilized β-galactosidase showed improved activity retention at a pH range of 5.5–8.5 and temperatures below 60 °C. Additionally, covalently immobilized β-galactosidase showed higher residual activity (10–40 % higher) and lower enzyme loss (≤10 vs 43.3 μg) compared to adsorbed β-galactosidase for 10 days under constant agitation at 20 °C. Therefore, this work demonstrates how hierarchically structured hybrid materials can be used in enzyme immobilization systems to enhance reusability and enzymatic activity performance.