Improvement of the stability and activity of immobilized trypsin on modified Fe3O4 magnetic nanoparticles for hydrolysis of bovine serum albumin and its application in the bovine milk

• Fe3O4 NPs was synthesized by coprecipitation method and modified by gallic acid (MNPs-GA).
• Trypsin enzyme was immobilized onto MNPs-GA.
• Immobilization enhanced free trypsin activity.
• A possible mechanism of trypsin immobilization onto Fe3O4 NPs was proposed.
• Hydrolysis of BSA by immobilized trypsin was successfully performed.

Trypsin (EC 3.4.21.4) was successfully immobilized on the surface of Fe3O4 magnetic nanoparticles that had been pre-treated with gallic acid (GA). Measurements of protein load by using Bradford assay and the trypsin-catalyzed hydrolysis of Nα-Benzoyl-dl-arginine 4-nitroanilide hydrochloride (BApNA) were made for the immobilized enzyme. By using magnetic nanoparticles, which provides easy separation and decent support material for enzyme immobilization with high surface area to volume ratio, and by employing biocompatible material gallic acid, immobilized enzyme system was synthesized along with improving trypsin activity and stability. Immobilized trypsin (TR) was more stable than the free one and demonstrated higher enzymatic activity at elevated temperatures (45–55 °C) and in the alkaline pH region (6–10.5). Fe3O4 NPs–GA–TR retained 92% of its initial activity after 120 days of storage at 4 °C in sodium phosphate buffer (0.1 M, pH 7.5), whereas the free trypsin maintained about 64% of its initial activity during the same storage period. In addition, activity of the immobilized trypsin was preserved 54.5% of its initial activity after eight times successive reuse. The Michaelis–Menten kinetic constant (Km) and maximum reaction velocity (Vmax) for free trypsin were 5.1 mM and 23 mM/min, respectively, whereas Km and Vmax values of immobilized trypsin were 7.88 mM and 18.3 mM/min, respectively. The performance of the immobilized trypsin was demonstrated by carrying out the hydrolysis of bovine serum albumin (BSA) within 1 h, and the assay was performed by using liquid chromatography–mass spectrometry (LC–MS/MS) technique. The hydrolysis of bovine milk as a real food was investigated by immobilized trypsin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).