Exploring the possibility of using a thermostable mutant of β-glucosidase for rapid hydrolysis of quercetin glucosides in hot water

Abstract

The antioxidant quercetin was extracted from yellow onion waste and converted to its aglycone form by a combination of subcritical water extraction and enzymatic hydrolysis. The hydrolytic step was catalysed by a double residue (N221S, P342L) mutant of the thermostable β-glucosidase (TnBgl1A), isolated from the thermophile Thermotoga neapolitana and cloned and produced in E. coli. The activity of wt TnBgl1A was shown to be dependent on the position of the glucosylation on the quercetin backbone, favouring hydrolysis of quercetin-4′-glucoside over quercetin-3-glucoside. The mutated variant of the enzyme harboured a mutation in the +2 sub-site (N221S) and showed increased catalytic efficiency in quercetin-3-glucoside hydrolysis and also to a certain extent hydrolysis of quercetin-4′-glucoside. The mutated enzyme was used directly in yellow onion extracts, prepared by subcritical water extraction, resulting in complete hydrolysis of the glucosylated flavonoids quercetin-3,4′-diglucoside, quercetin-4′-glucoside, quercetin-3-glucoside, isorhamnetin-4′-glucoside and isorhamnetin-3,4′-diglucoside. To complete hydrolysis within five minutes, 3 mg of TnBgl1A_N221S was used per gramme of onion (dry weight). A life cycle assessment was done to compare the environmental impact of the new method with a conventional solid–liquid extraction-and-hydrolysis method utilising aqueous methanol and hydrochloric acid. Comparison of the methods showed that the new method is preferable regarding primary energy consumption and global warming potential. Another advantage of this method is that handling of toxic chemicals (methanol and HCl) is avoided. This shows that combined subcritical water extraction/enzyme hydrolysis is both a fast and sustainable method to obtain quercetin from onion waste.