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Isoquercitrin, a rare flavonol glycoside with wide biological activities and key synthetic intermediate for the production of enzymatically modified isoquercitrin (EMIQ), was conducted by naringinase-catalyzed conversion of rutin under ultrasound irradiation. The maximum yields were obtained to 98.35 ± 3.13% and 95.20 ± 2.52% under conventional heating and ultrasound irradiation, respectively. The optimal results under ultrasound irradiation were obtained under the following conditions: rutin concentration 0.8 g/L, naringinase concentration 3000 U/L, reaction temperature 40 °C for 20 min, which was more economical than that with conventional heating. The reaction time was reduced from 60 min to 20 min, and the apparent kinetic parameter ( V m / K m ) was increased 3.72-fold. The lower activity energy E a under ultrasonic irradiation was 0.7-fold of that in an incubator reactor, which could easily initiate the enzymatic reaction. The association saturation constant K a was 1.98-fold higher than that with conventional heating, showed a better affinity between rutin and naringinase detected by surface plasmon resonance (SPR) analysis. These results suggest that ultrasound irradiation can accelerate the enzymatic synthesis of isoquercitrin from rutin. [ABSTRACT FROM AUTHOR]

Abstract: Naringinase is an enzyme complex, expressing α-l-rhamnosidase and β-d-glucosidase activities. The impact of high pressure and temperature on naringinase activity and stability were studied, in order to assess the potential of enzyme thermostability on glycosides hydrolyses. To a better understanding of these effects on naringinase enzyme complex, they were also evaluated over α-l-rhamnosidase and β-d-glucosidase activities, using specific substrates, p-nitrophenyl α-l-rhamnopyranoside (4-NRham) and p-nitrophenyl β-d-glucopyranoside (4-NGluc), respectively. Hydrolysis rate of 4-NRham and naringin increased with pressure from 0.1 to 150MPa. The equilibrium constants for α-l-rhamnosidase and β-d-glucosidase reactions, at pressures of 0.1–200MPa and 40°C, were determined and best fitted with the model of Baliga and Whalley equation. Accordingly, reaction volumes of 93 and 64mLmol−1 were obtained for α-l-rhamnosidase and β-d-glucosidase reactions, respectively. Reaction rate constants were also determined at the same experimental conditions and well fitted to the models of Golinkin, Laidlaw, Hyne and of Burris and Laidler. A negative ΔV ≠ of −7.7±1.5 and −20.0±5.2mLmol−1 were obtained for α-l-rhamnosidase and naringinase reactions, correspondingly, which reflect the accelerating effect of pressure on the biocatalysis. Moreover, the K M, k cat and k cat/K M values, on naringin hydrolysis under atmospheric (0.1MPa) and high pressure (150MPa) conditions at different temperatures (25–80°C) were determined. A 3-fold and 4-fold increase on naringinase thermostability was observed under 150MPa at 70 and 80°C, respectively, compared to 0.1MPa experiments. In addition, a 15-fold increase of k cat/k M values from experimental conditions of 0.1MPa and 30°C to 150MPa and 70°C was observed. In fact, high pressure showed to be a powerful tool to increase stability of naringinase against thermal denaturation. In conclusion, the effect of amplification of pressure effects on reaction rates by temperature could have a pragmatic use for accelerating enzymatic reactions. [Copyright &y& Elsevier]

Abstract: The hydrolysis of naringin, a bitter flavonone glycoside, with naringinase leads to reducing sugars (rhamnose and glucose), to prunin and to the aglycone, naringenin. To overcome the low solubility of naringin in the enzymatic reaction media, the effect of different solvents was studied, in order to improve the productivity and yield of the system. The effect of increasing concentration of co-solvents on the stability of both soluble and immobilized naringinase expressing α-l-rhamnosidase and β-d-glucosidase activities was evaluated. The enzyme was immobilized onto sol–gel matrices of tetramethoxysilane and glycerol. Combining the higher naringin solubility, and the higher residual activity of both α-l-rhamnosidase and β-d-glucosidase expressed by naringinase, eight solvents were chosen for stability and activity studies: dimethyl sulfoxide, N,N-dimethylmethanamide, methanol, ethanol, acetone, tetrahydrofurane, 1,2-dimethoxyethane and 1,4-dioxane. Deactivation of soluble naringinase was analyzed according to a first-order kinetic model. For the sol–gel immobilized enzyme the two-step deactivation model, of Henley and Sadana, was adjusted. Sol–gel immobilization stables naringinase in all tested co-solvents systems. This effect was specially pronounced at higher co-solvent concentration (10%). The half-life of α-l-rhamnosidase and β-d-glucosidase expressed by naringinase even increased 21- and 59-fold, respectively, in aqueous co-solvation with tetrahydrofurane. These are high innovative and sounding results showing the protective effect of immobilization onto sol–gel (tetramethoxysilane+glycerol) matrices with naringinase in co-solvent systems, which is a great advantage for non-conventional biocatalysis. [Copyright &y& Elsevier]

Abstract: The aim of this work was the hydrolysis of naringin towards naringenin with immobilized naringinase and the evaluation of their anti-inflammatory activity. An acute local inflammation model (rat paw edema induced by λ-carrageenan) was chosen to evaluate the contribution of antioxidant properties to a possible anti-inflammatory effect. Grapefruit juice was processed with naringinase immobilized in k-carrageenan (2%) beads (≈3mm). A 95% naringin conversion in grapefruit juice was obtained with immobilized naringinase (1000mgL−1), with an activity of 19.5mgmL−1 min−1 and the formation of 215mgL−1 of naringenin. Ascorbic acid and indometacine were used as positive anti-inflammatory controls. All results were analysed using ANOVA with Dunnett''s post-test. The results show that rats (n =9) pre-treated with a solution of naringin, rats (n =9) pre-treated with a solution of naringenin (in concentrations equal as in grapefruit juice) and rats (n =9) treated with a solution of naringenin and naringin, revealed a significant reduction on edema formation, 6h after λ-carrageenan injection. Naringenin demonstrated a high in vivo anti-inflammatory activity, only 8% of paw edema (p <0.001) was observed in rats pre-treated with a solution of naringenin. Comparability studies, in rats administered orally with grapefruit juice (before and after processing), showed that enzymatic processing did not affect the anti-inflammatory properties of the juice. [Copyright &y& Elsevier]

Abstract: Naringin, the principal bitter flavonone glycoside and the primary bitter component in grapefruit juice, can be hydrolysed by naringinase (with expressed activity in alfa-l-rhamnosidase and beta-d-glucosidase) into rhamnose and prunin (one-third bitterness ratio to naringin), which can be further hydrolysed into glucose and tasteless naringenin. The main goal of this work was the modelling and optimisation of the enzymatic activity of naringinase entrapped in k-carrageenan beads, in grapefruit juice. In order to attain these goals naringinase activity, naringin conversion and naringenin formation were evaluated. In this study response surface methodology was used for modelling the naringin hydrolysis by naringinase immobilized in k-carrageenan beads as a function of temperature (9–51°C) and naringinase concentration (0.29–1.7gL−1) in grapefruit juice. Experiments were carried out following a central composite rotatable design. Naringinase activity could be described by response surfaces that enabled to fit second-order polynomials to experimental data points. The high values of R 2 (0.996) and (0.992) of the models show a close agreement between the experimental results and the theoretical values predicted by the models. The higher naringin conversion (95%) was attained with naringinase concentrations higher than 800mgL−1 and temperatures higher than 30°C. This is a promising result for a future application of enzymatic hydrolysis of naringin immobilized in k-carrageenan, in grapefruit juice industry, maintaining juice properties. [Copyright &y& Elsevier]