| Quercetin is an important member of the polyphenolic flavonoid compounds. It exists mostly in glycosylated forms in plants. Onion waste and by-products are full of quercetin and quercetin glycosides. In the case of onion, large amount of onion waste produced within the industrial engineering are today used in animal feed, or for composting, incineration or anaerobic digestion. To maximized utilize the produced waste and by-products can not only gain extra values such as high-value compounds, but can also bring great benefits to the environment. Furthermore, large numbers of studies have shown that quercetin has greater pharmacological activity than the quercetin glycosides and the fecalases produced in the human gut are capable of hydrolyzing quercetin glycosides to yield the quercetin. Hence, it is of interest to be able to extract quercetin and quercetin glycosides from waste and a biocatalytic conversion should be applied to further improve the environmentally friendly profile.In this paper, we have evaluated the use of different thermostable β-glucosidases followed by the immobilized β-glycoside to achieve biocatalytic conversion of the quercetin glycosides to quercetin from onion waste. The hydrolysis reaction is catalyzed by enzymes rather than by chemicals or non-renewable catalysts, which demonstrates a fully "green" procedure.Onion waste is a renewable raw material, rich in different molecular species of the antioxidant quercetin. To utilize this resource, an environmentally sustainable procedure has been developed. Ethanol and water were used as environmentally friendly solvents for extraction. According to HPLC analysis, we found that quercetin in onion waste exists in four predominant forms:quercetin 3,4’-O-diglucoside (QDG), quercetin 3-O-glucoside (Q3G), quercetin 4’-O-glucoside (Q4G) and quercetin (Q). The highest content of quercetin glycosides in onion waste is Q4G, and followed by QDG and Q3G. Q exists as well. The onion waste extracted with 95%ethanol at 80℃ comes to be the best choice.We fouse on use of the thermostable β-glucosidases, Te-BglA from Thermoanaerobacter etholicus JW200, Tm-BglA from Thermotoga maritima and two mutated variant of Tm-BglA (N223S and G224T) as catalysts for deglycosylation of quercetin glycosides at high temperature. β-glucosidase has been proved to have the ability to hydrolyse quercetin glycosides, and hydrolysis of the 4’-glucosylation is clearly favoured by β-glucosidase, which causes the increment of Q3G according to the hydrolysis of QDG. We also surmise that β-glucosidase has high specificity for 4’-glucosylation. Moreover, presence of Q3G is not disturbing the deglycosylation at the 4’-position. Tm-BglA has better catalytic efficiency than Te-BglA. And two mutants, N223S and G224T are even better than Tm-BglA, which gain significantly affect substrate affinity and the effect is higher for Q3G.Using recombinant protein technology, Tt-ChBD was fused to N223S and G224T in the C-terminal as an affinity tag. After direct absorption of the protein mixture from E.coli onto chitin beads, mutants tagged with Tt-ChBD was found to specifically attach to the affinity matrix. Immobilized enzyme with N223 and G224T was used to hydrolyzed quercetin glycosides at 80℃ for 20 min per cycle. The first cycle comes to gain 3.06 mg Q from 1.0 g dry weight onion powder. QDG and Q4G were completely hydrolyzed, and only quite a few Q3G was remained.2.35 mg Q can still be collected after repeated productions of 20 times, and relative production rate was 70%. Overall, it illustrates the great usefulness of the immobilized thermostable β-glucosidase in hydrolyzing quercetin glycosides to achieve the bioconversion of quercetin glycosides to antioxidant quercetin. |
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