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 stabi...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.展开更多
Naringin is a main flavonoid in citrus industry,and it is also a bitter matter in fruit juice.In our previous work,a mutant strain can produceα-L-rhamnosidase with good hydrolysis efficiency for hesperidin other than...Naringin is a main flavonoid in citrus industry,and it is also a bitter matter in fruit juice.In our previous work,a mutant strain can produceα-L-rhamnosidase with good hydrolysis efficiency for hesperidin other than naringin.To extend its function based on our present study,we have explored the reason why the efficiency of catalysing hesperidin into hesperetin 7-O-glucoside than catalysing naringin into prunin.As a result,we found five key amino acid residues were responsible for the difference by molecular docking and molecular dynamics simulations.The terminal rhamnose of naringin is bound to the inside of the active pocket of the enzyme,and the addition of two new hydrogen bonds come out after the hydrolysis of naringin.So the active pocket began to contract,making it difficult for the terminal rhamnose to detach from it.5 key amino acid residues,THR203,GLU158,LYS276,GLN133,and ILE258 were identified,and obtained five new mutants by mutating them into ALA.Among them,the mutant strain of K276A showed higher conversion efficiency from naringin to prunin along with higher thermal stability.This work not only improved the affinity ofα-L-Rhamnosidase for catalysing bittering matter of naringin and enhances its potential application in the food and pharmaceutical industries,but also provided an effective technological strategy for improving the affinity of other enzymes.展开更多
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.
文摘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support of the Science and Technology Planning Project of Agricultural Division 9 in Xinjiang Production and Construction Corps.,P.R.China(2023JS020)the Science and Technology Planning Project in Guangdong,P.R.China(SDZX2022041,SDZX2023036 and SDZX2023033)+2 种基金the Science and Technology Innovation Strategy Special Project in Guangdong Province,P.R.China(pdjh 2023a0025)the Key R&D Program Projects in Guangdong,P.R.China(2023B0202040002 and 2024B0202010001)the‘Open Competition’Science&Technology Research Project of National Excellent Engineer Innovation Research Institute for Advanced Manufacturing Industry in Guangdong Hong Kong Macao Greater Bay Area(Foshan)(JBGS2024003).
文摘Naringin is a main flavonoid in citrus industry,and it is also a bitter matter in fruit juice.In our previous work,a mutant strain can produceα-L-rhamnosidase with good hydrolysis efficiency for hesperidin other than naringin.To extend its function based on our present study,we have explored the reason why the efficiency of catalysing hesperidin into hesperetin 7-O-glucoside than catalysing naringin into prunin.As a result,we found five key amino acid residues were responsible for the difference by molecular docking and molecular dynamics simulations.The terminal rhamnose of naringin is bound to the inside of the active pocket of the enzyme,and the addition of two new hydrogen bonds come out after the hydrolysis of naringin.So the active pocket began to contract,making it difficult for the terminal rhamnose to detach from it.5 key amino acid residues,THR203,GLU158,LYS276,GLN133,and ILE258 were identified,and obtained five new mutants by mutating them into ALA.Among them,the mutant strain of K276A showed higher conversion efficiency from naringin to prunin along with higher thermal stability.This work not only improved the affinity ofα-L-Rhamnosidase for catalysing bittering matter of naringin and enhances its potential application in the food and pharmaceutical industries,but also provided an effective technological strategy for improving the affinity of other enzymes.
