摘要
Natural flavonoids exhibit diverse physiological effects and are used as functional factors,antioxidants and pigments in food as well as pharmaceutical and cosmetic applications.However,their poor solubility and stability,result in low bioavailability.Biomodification through deglycosylation effectively enhances their bioactivity.This study developed two surface display strategies in Escherichia coli,anchoringα-L-rhamnosidase RhaB1-ΔN to the cell surface using the anchoring protein Lpp-OmpA.Immunofluorescence and proteinase K sensitivity tests confirmed the successful construction of surface-displaying strains E.coli Lpp-OmpA-RhaB1-ΔN and E.coli Lpp-OmpA-SpyCatcher/RhaB1-ΔN-SpyTag.Enzymatic property studies revealed improved temperature and pH stability of the whole-cell catalysts with surface compared to free enzymes.After 6 catalytic cycles,E.coli Lpp-OmpA-RhaB1-ΔN retained higher activity than E.coli RhaB1-ΔN.In flavonoid hydrolysis,E.coli Lpp-OmpARhaB1-ΔN increased yields of isoquercitrin and kaempferol-3-O-glucuronide by 20.25%and 10.99%,respectively,compared with that in whole-cell catalyst with intracellular enzyme(p<0.05).In addition,it should be noted that although the enzyme expression of E.coli Lpp-OmpA-SpyCatcher/RhaB1-ΔN-SpyTag is reduced,its hydrolysis ability of flavonoids is comparable to that of E.coli RhaB1-ΔN due to the efficient catalytic ability of extracellular enzymes.This study establishes reliable whole-cell catalysts with surface-displayedα-L-rhamnosidase for efficient natural flavonoid modification,with potential applications in the food and pharmaceutical industries.
基金
supported by the National Natural Science Foundation of China(22278196)
the Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX22_3854)
the Start-up Funding from Jiangsu University of Science and Technology.
