Facile immobilization of an unstable recombinant α-L-rhamnosidase on magnetite nanoparticles for efficient naringin biotransformation.

α-L-Rhamnosidase is a significant biocatalyst that specifically cleaves terminal α-L-rhamnose group from natural flavonoid diglycosides, enabling the biocatalytic production of high-value flavonoid glucosides like prunin from naringin. Compared to its precursor naringin, prunin exhibits superior solubility and bioavailability, making this enzymatic conversion commercially valuable for the food and pharmaceutical industry. However, the industrial application of free α-L-rhamnosidases faces significant challenges, including enzyme instability, difficulty in recovery, and unsatisfactory reusability. To address these issues, this study developed an innovative magnetically recoverable biocatalyst (Fe₃O₄-Rha) through covalent immobilization of recombinant α-L-rhamnosidase from human fecal metagenome onto Fe₃O₄ nanoparticles using EDC/NHS chemistry, and the immobilization parameters were systematically optimized. The successful preparation of Fe₃O₄-Rha was verified by TEM, FTIR, TGA, and SQUID analysis. Fe₃O₄-Rha retained the catalytic property of free Rha in terms of optimal pH and temperature but exhibited superior tolerance on organic solvent especially ethanol and isopropanol. Moreover, Fe₃O₄-Rha could effectively biotransform naringin to prunin and maintained 61.34% of initial activity after 5 cycles. In a scaled-up reaction system, Fe₃O₄-Rha also efficiently converted naringin into prunin and the complete conversion was achieved within 10 h. This work successfully developed a magnetically recoverable immobilized α-L-rhamnosidase system for the efficient and reusable biotransformation of naringin, offering a promising approach for the enzymatic modification of bioactive small molecules using biological macromolecular catalysts.