2.5. Physical Adsorption of Lipase

Physical adsorption of lipase on the powder surface was accomplished according to the following procedure [37,38]. CALB L (2.0 mL, ~10 mg protein) was dissolved in 25 mL of phosphate buffer, 10 mM, pH = 7.0. The pH range presented by the supplier (Novozymes) as an optimal one was between 5 and 9. For that reason, a pH of 7 was selected. The powdered support (0.2 g) was placed in a 50 mm column attached to the isocratic HPLC pump, and the lipase solution was pumped through the column for 18 h at 20 °C with a flow rate 0.2 mL/min. The immobilized lipase was filtered off, washed with propan-2-ol and n-hexane, dried for 4 h at room temperature and stored at 4 °C.

In the case of the physical adsorption of lipase on the membrane, the samples with MWCO-50kDa were chosen. The sample was placed in a cross-flow reactor (Figure S8). Lipase solution (~10 mg protein) in 50 mL 0.01 M phosphate buffer pH 7.0 was circulating for 18 h at 20 °C. After immobilization the sample was washed with propan-2-ol and n-hexane, dried for 4 h at room temperature, and stored at 4 °C.

The amount of immobilized lipase was calculated as a difference of mass of protein in the enzyme solution before and after immobilization. Protein concentration was determined spectrophotometrically by Bradford method using BSA as a standard.