Screening of mutants, enzymatic properties, and scale-up fermentation for catalysis of naringin using recombinant α-L-rhamnosidase

Abstract

Background Prunin is scarce in nature and suffers from low extraction efficiency, and industrial-scale production of prunin and L-rhamnose remains technically inadequate. Therefore, developing an α-L-rhamnosidase capable of efficient and specific naringin hydrolysis is of great practical significance. Methods An α-L-rhamnosidase gene from Aspergillus nidulans was expressed recombinantly in E. coli Rosetta (DE3). Expression conditions were optimized, and random mutagenesis coupled with high-throughput screening was performed to obtain a beneficial mutant (R11). Enzyme properties were characterized, and fed-batch fermentation in a 2-L bioreactor was carried out for scale-up. Results The purified recombinant enzyme (120.7 kDa) showed optimal activity at 75 °C and pH 5.0, with a Km of 5.23 mM toward pNPR. A key mutant, R11, exhibited 22.2% higher relative activity than the wild type. After tank fermentation, enzyme activities of both wild-type and R11 were increased approximately two-fold, and the conversion rate of naringin to prunin by R11 was 2.77-fold higher than that of the unoptimized wild-type control. Conclusion The recombinant α-L-rhamnosidase possesses broad temperature adaptability and excellent catalytic properties. This study provides a novel integration of random mutagenesis and bioreactor scale-up for efficient naringin-to-prunin bioconversion, laying a theoretical foundation for industrial production.