Abstract
The α-L-rhamnosidase (rha1) gene was homologously expressed in Aspergillus niger strains CCTCC 206047 and CCTCC 206047ΔpyrG, using hygromycin B and auxotrophic as selection markers. The engineered A. niger strains RHA001-1 and RHA003-1 were screened, yielding α-L-rhamnosidase activities of 20.81 ± 0.56 U/mL and 15.35 ± 0.87 U/mL, respectively. The copy numbers of the rha1 gene in strains RHA001-1 and RHA003-1 were found to be 18 and 14, respectively. Correlation analysis between copy number and enzyme activity in the A. niger strains revealed that α-L-rhamnosidase activity increased with the copy number of the rha1 gene. Recombinant α-L-rhamnosidase was utilized for the enzymatic debittering of Ougan juice, and its process conditions were optimized. Furthermore, the primary bitter substance neohesperidin (2.22 g/L) in Ougan juice was converted into hesperetin 7-O-glucoside (1.47 g/L) and hesperidin (0.143 g/L). This study presents a novel approach for the production of food-grade α-L-rhamnosidase and establishes a technical foundation for its application in the beverage industry.