This work designed and prepared a novel type of carrier for immobilization of penicillin G acylase (PGA), and then the performances of the immobilized PGA were studied in detail. The process is presented as follows: First, dopamine (DA), an adhesive biological secondary metabolite, was adopted to form a polydopamine (PDA) coating on the surface of Fe3O4nanoparticles (NPs) prepared by the inverse microemulsion method to generate Fe3O4@PDA NPs through in-situ polymerization. After that, the obtained Fe3O4@PDA NPs were modified by reversible addition fragmentation chain transfer (RAFT) reagent containing carboxyl groups on its surface. Then, taking N,N diethyl acrylamide (DEA) as the temperature-sensitive monomer, [Formula: see text]-hydroxyethyl methacrylate (HEMA) as the hydrophilic monomer, glycidyl methacrylate (GMA) as the target monomer and methyl methacrylate (MMA) as the monomer controlling the distance between targets, through the “Grafting from” strategy and the RAFT polymerization method, the magnetic temperature-sensitive polymer composite, Fe3O4@PDA-g-PDEA-b-PHEMA-b-P(MMA-co-GMA) NPs with different feeding ratios of MMA/GMA, was prepared to realize the immobilization of penicillin G acylase (PGA). Based on samples prepared at each stage of the carrier, the structure and performances were characterized by Fourier Infrared Spectroscopy (FTIR), Transmission Electron Microscope (TEM), X-ray Diffraction (XRD), and Vibration Specimen Magnetometer (VSM), respectively. Besides, the content of RAFT graft Fe3O4@PDA was also quantitatively characterized by ICP, and the molecular weight of the polymer was characterized by mass spectrometry. Result showed that the target space influenced enzyme load capacity (ELC), enzyme activity (EA) and enzyme activity recovery rate (EAR) at a large degree, and there was no positive relationship between ELC to EA and EAR. Last, performances of the immobilized PGA, including relative enzyme activity ([Formula: see text]) of immobilized PGA, were 83.9% after storing for 90 days, and 90.3% of the initial activities were still retained after 12 times of repeated use, and the catalytic stability (temperature, pH) and Michaelis–Menten const [Formula: see text] were investigated with free PGA as control if operation was allowed.
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