Search

Objective: This study was conducted to characterize recombinant α-L-rhamnosidase from Chloroflexus aurantiacus and apply the enzyme in the production of isoquercitrin from rutin., Results: The α-L-rhamnosidase from C. aurantiacus was cloned and expressed in Escherichia coli BL21 and purified as a soluble enzyme. α-L-rhamnosidase purified from C. aurantiacus has a molecular mass of approximately 105 kDa and is predicted to exist as a homodimer with a native enzyme of 200 kDa. The purified enzyme exhibited the highest specific activity for rutin among the reported isoquercitrin producing α-L-rhamnosidases and was applied in the production of isoquercitrin from rutin. Under the optimised conditions of pH 6.0, 50 °C, 0.6 U mL -1 α-L-rhamnosidase, and 30 mM rutin, α-L-rhamnosidase from C. aurantiacus produced 30 mM isoquercitrin after 2 h with a 100% conversion yield and productivity of 15 mM h -1 ., Conclusions: We achieved a high productivity of isoquercitrin from rutin. Moreover, these results suggest that α-L-rhamnosidase from C. aurantiacus is an effective isoquercitrin producer.

Objectives: To biotransform rutin into isoquercitrin., Results: A α-L-rhamnosidase from Bifidobacterium breve was produced by using Escherichia coli BL21 for biotransformation of rutin to isoquercitrin. The enzyme was purified by Ni(2+)-NTA chromatography to yield a soluble protein with a specific activity of 56 U protein mg(-1). The maximum enzyme activities were at pH 6.5, 55 °C, 20 mM rutin, and 1.2 U enzyme ml(-1). Under optimal conditions, the half-life of the enzyme was 96 h. The K m and V max values were 2.2 mM, 56.4 μmol mg(-1) min(-1) and 2.1 mM, 57.5 μmol mg(-1) min(-1) using pNP-Rha and rutin as substrates, respectively. The kinetic behavior indicated that the recombinant α-L-rhamnosidase has good catalytic performance for producing isoquercitrin. 20 mM rutin was biotransformed into 18.25 and 19.87 mM isoquercitrin after 60 and 240 min., Conclusion: The specific biotransformation of rutin to isoquercitrin using recombinant α-L-rhamnosidase from B. breve is a feasible method for use in industrial processes.

The acetyl esterase of Trichoderma reesei belonging to carbohydrate esterase (CE) family 16 catalyzes transacylations to carbohydrate moieties of flavonoid glycosides, esculin and rutin. The enzyme recognizes as acyl donors vinyl esters of short carboxylic acids. Esculin was acylated at position 3 of the glucosyl residue in aqueous solutions saturated with vinyl acetate and vinyl propionate. The yields of esculin monoacetate and monopropionate of esculin in aqueous medium (esculin 40 mM, enzyme 40 µg/ml, 40 °C, 3 days) were 67 and 55 %, respectively. Replacement of water by 2-propanol was required for a similar acylation of rutin at 4 mM concentration. The yields of rutin monoacetate and propionate were 60 and 30 %, respectively. The results indicate that the enzyme could be used for an easy modification of solubility and hydrophobicity of glycosylated compounds, including drugs and functional food additives.

Common buckwheat (Fagopyrum esculentum Moench) is rich in phenolic compounds and may be useful for the treatment of metabolic syndrome in humans. To improve the production of rutin in buckwheat, we overexpressed the flavonol-specific transcription factor, AtMYB12 using Agrobacterium rhizogenes into hairy root culture systems. This induced the expression of flavonoid biosynthetic genes encoding phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, chalcone synthase, chalcone isomerase, flavone 3-hydroxylase, flavonoid 3'-hydroxylase, and flavonol synthase. This led to the accumulation of rutin in buckwheat hairy roots up to 0.9 mg/g dry wt. PAP1 expression, however, did not correlate with the production of rutin.

Pyrococcus furiosus β-glucosidase converted rutin to quercetin and rutinose disaccharide with a ratio of 1:1, with no glucose, L-rhamnose, and isoquercitrin, indicating that the enzyme is a β-rutinosidase. The specific activity for flavonoid glycosides followed the order of isoquercitrin > quercitrin > rutin. The conversion of rutin to quercetin was optimal at pH 5.0 and 95°C in the presence of 0.5% dimethyl sulfoxide with a half-life of 101 h, a k(cat) of 1.6 min(-1), and a K(m) of 0.3 mM. Under the improved conditions, the enzyme produced 6.5 mM quercetin from 10 mM rutin after 150 min, with a molar yield of 65% and a productivity of 2.6 mM/h. This productivity is the highest reported thus far among enzymatic transformations.