Preparation of polyhydroxyalkanoate-based magnetic microspheres for carbonyl reductase purification and immobilization

Abstract

A polyhydroxyalkanoate (PHA) magnetic microsphere was designed for one-step purification and immobilization of a novel carbonyl reductase (RLSR5) from recombinant Escherichia coli lysate. The hydrophobic core of this microsphere was composed of a highly biocompatible polymer, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), in which magnetic Fe₃O₄ particles were embedded during solvent evaporation. The hydrophilic shell of the fusion protein formed by PHA particle-binding protein (PhaP) and RLSR5 (PR) was expressed in recombinant E. coli. The magnetic core of Fe₃O₄@PHBHHx directly purified the hydrophilic shell from the E. coli lysate, and the two self-assembled to form Fe₃O₄@PHBHHx-PR through hydrophobic and hydrophilic interactions, eliminating the separation of the fusion protein. The microstructure, magnetic properties, morphology, size, and dispersion of Fe₃O₄@PHBHHx-PR were investigated by XRD, VSM, SEM, TEM, elemental mapping and DLS. It was found that Fe₃O₄@PHBHHx-PR correctly assembled, with a well dispersed spherical structure at the nanoscale and superparamagnetism properties. The amount of RLSR5 immobilized on PHA microspheres reached 121.9 mg/g. The Fe₃O₄@PHBHHx-PR was employed to synthesize (R)-tolvaptan with 99 % enantiomeric excess and 97 % bioconversion efficiency, and the catalyst maintained 78.6 % activity after 10 recovery cycles. These PHA magnetic microspheres are versatile carriers for enzyme immobilization and demonstrate improved stability and reusability of the free enzyme.

Keywords: Asymmetric reduction; Enzyme immobilization; Magnetic separation; PHA microspheres; Reusability.