Related Experiment Video
Updated: Aug 5, 2026
Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
Published on: August 8, 2016
Screening of mutants, enzymatic properties, and scale-up fermentation for catalysis of naringin using recombinant
Chuncheng Wang1, LingShan Liang1, Bei Ni1
1School of Base Medical Sciences, Guangdong Pharmaceutical University, Guangzhou, China.
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.
