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. 2011:2011:692618.
doi: 10.4061/2011/692618. Epub 2011 Sep 15.

Enzymatic Synthesis of the Flavone Glucosides, Prunin and Isoquercetin, and the Aglycones, Naringenin and Quercetin, with Selective α-L-Rhamnosidase and β-D-Glucosidase Activities of Naringinase

Hélder Vila-Real  1 , António J Alfaia, M Rosário Bronze, António R T Calado, Maria H L Ribeiro
Affiliations

Enzymatic Synthesis of the Flavone Glucosides, Prunin and Isoquercetin, and the Aglycones, Naringenin and Quercetin, with Selective α-L-Rhamnosidase and β-D-Glucosidase Activities of Naringinase

Hélder Vila-Real et al. Enzyme Res. 2011.

Abstract

The production of flavonoid glycosides by removing rhamnose from rutinosides can be accomplished through enzymatic catalysis. Naringinase is an enzyme complex, expressing both α-L-rhamnosidase and β-D-glucosidase activities, with application in glycosides hydrolysis. To produce monoglycosylated flavonoids with naringinase, the expression of β-D-glucosidase activity is not desirable leading to the need of expensive methods for α-L-rhamnosidase purification. Therefore, the main purpose of this study was the inactivation of β-D-glucosidase activity expressed by naringinase keeping α-L-rhamnosidase with a high retention activity. Response surface methodology (RSM) was used to evaluate the effects of temperature and pH on β-D-glucosidase inactivation. A selective inactivation of β-D-glucosidase activity of naringinase was achieved at 81.5°C and pH 3.9, keeping a very high residual activity of α-L-rhamnosidase (78%). This was a crucial achievement towards an easy and cheap production method of very expensive flavonoids, like prunin and isoquercetin starting from naringin and rutin, respectively.

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Figures

Figure 1
Figure 1
Scheme of the production of flavonoid glucosides and aglycones starting from its rutinosides precursors using an enzymatic approach (α-L-rhamnosidase and β-D-glucosidase activities of naringinase).
Figure 2
Figure 2
pH profile of 4-NGP and 4-NRP hydrolysis using naringinase in sodium citrate buffer 20 mM (mean value ± standard error).
Figure 3
Figure 3
Thermal inactivation under combined temperature and pH conditions of β-D-glucosidase (a) and α-L-rhamnosidase (b) activities of naringinase.
Figure 4
Figure 4
Response surfaces fitted to the experimental data points corresponding to (a) α-L-rhamnosidase residual activity and (b) to first deactivation rate coefficients (k 1) for (b.1) α-L-rhamnosidase (rhase) and (b.2) β-D-glucosidase (gase) activities of naringinase, as a function of temperature (T) and pH.
Figure 5
Figure 5
Inactivation kinetics of β-D-glucosidase and α-L-rhamnosidase activities of naringinase at 81.5°C and pH 3.9.
Figure 6
Figure 6
TLC of (a) naringin (Rf = 0.62), (b) prunin (Rf = 0.58), (c) naringenin (Rf = 0.30), (d) rutin (Rf = 0.55), (e) isoquercetin (Rf = 0.53), and (f) quercetin (Rf = 0.30). All of compounds were visible with UV light (254 nm). After acid revelation + heating (150°C), naringin, prunin, and naringenin showed orange spots and rutin, quercetin, and isoquercetin, yellow.
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