António J. Alfaia

α-Rhamnosidase and β-glucosidase expressed by naringinase immobilized on new ionic liquid sol–gel matrices: Activity and stability studies

Novel ionic liquid (IL) sol-gel materials development, for enzyme immobilization, was the goal of this work. The deglycosylation of natural glycosides were performed with α-l-rhamnosidase and β-d-glucosidase activities expressed by naringinase. To attain that goal ILs with different structures were incorporated in TMOS/Glycerol sol-gel matrices and used on naringinase immobilization. The most striking feature of ILs incorporation on TMOS/Glycerol matrices was the positive impact on the enzyme activity and stability, which were evaluated in fifty consecutive runs. The efficiency of α-rhamnosidase expressed by naringinase TMOS/Glycerol@ILs matrices increased with cation hydrophobicity as follows: [OMIM]>[BMIM]>[EMIM]>[C(2)OHMIM]>[BIM] and [OMIM]≈[E(2)-MPy]≫[E(3)-MPy]. Regarding the imidazolium family, the hydrophobic nature of the cation resulted in higher α-rhamnosidase efficiencies: [BMIM]BF(4)≫[C(2)OHMIM]BF(4)≫[BIM]BF(4). Small differences in the IL cation structure resulted in important differences in the enzyme activity and stability, namely [E(3)-MPy] and [E(2)-MPy] allowed an impressive difference in the α-rhamnosidase activity and stability of almost 150%. The hydrophobic nature of the anion influenced positively α-rhamnosidase activity and stability. In the BMIM series the more hydrophobic anions (PF(6)(-), BF(4)(-) and Tf(2)N(-)) led to higher activities than TFA. SEM analysis showed that the matrices are shaped lens with a film structure which varies within the lens, depending on the presence and the nature of the IL. The kinetics parameters, using naringin and prunin as substrates, were evaluated with free and naringinase encapsulated, respectively on TMOS/Glycerol@[OMIM][Tf(2)N] and TMOS/Glycerol@[C(2)OHMIM][PF(6)] and on TMOS/Glycerol. An improved stability and efficiency of α-l-rhamnosidase and β-glucosidase expressed by encapsulated naringinase on TMOS/Glycerol@[OMIM][Tf(2)N] and TMOS/Glycerol@[C(2)OHMIM][PF(6)] were achieved. In addition to these advantageous, with ILs as sol-gel templates, environmental friendly processes can be implemented.

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

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.

Quaternization Reaction of Aromatic Heterocyclic Imines in Methanol − A Case of Strong Anti-Reactivity Selectivity Principle with Isoselective Temperature

Accurate second-order rate constants were measured at 5 °C intervals in the temperature range 20−60 °C for the Menshutkin reaction of 1-methylbenzimidazole, 2-amino-1-methylbenzimidazole and N,N-dimethylaniline with iodomethane and iodoethane in methanol. In every case a good linearity in the Eyring plots was observed. Values for the activation enthalpy and entropy are reported. Analysis in terms of Exner’s redefinition of the reactivity−selectivity principle (RSP) identified the present reaction series as a case of strong anti-RSP for selectivity towards the substrate. This case is shown to represent an isoselective relationship with the isoselective temperature lower than the experimental temperatures (βis = −52 °C). The isokinetic relationship does not hold in the reaction series with a fixed substrate. These findings suggest an early transition state in the Menshutkin reaction of polyfunctional aromatic imines.

High pressure studies on hesperitin production with hesperidinase free and immobilized in calcium alginate beads

The use of high pressure for the enzymatic synthesis of pharmacologically interesting molecules is a very important tool. Hesperidin and hesperitin exhibit anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic properties and prevent bone loss. However, hesperidin has a low bioavailability compared with hesperitin, due to the rutinoside moiety attached to the flavonoid. The aim of this work was the enzymatic production of hesperitin from hesperidin (soluble and insoluble) with hesperidinase free and immobilized in Ca-alginate beads, under high pressure conditions. The work was focused on the optimization of enzyme activity, studying the effects: pressure (50–150 MPa), temperature (35–75 °C), concentration of substrate (100–800 mg/L), and immobilization of hesperidinase. An 18-fold increase in hesperidinase residual activity was observed under high pressure conditions of 100 MPa compared to 0.1 MPa. A higher specificity of the hydrolytic reaction under high pressure (100 MPa) with a two-and three-fold increase in the ratio K cat/K M (specificity constant) at 55 °C and 75 °C was observed. A two-fold increase in the maximum activity at 100 MPa was observed with immobilized hesperinase compared to 0.1 MPa. In the second reutilization, almost a four-fold increase was obtained under high pressure conditions in comparison to atmospheric pressure.

High pressure: a tool to improve the enzymatic production of glycosides

Naringinase, an enzyme complex that expresses α -l-rhamnosidase and β -d-glucosidase activities in native state, can be used to deglycosylate natural glycosides. The selective inactivation of one of these activities will allow the biosynthesis of different bioactive compounds in a simple, effective and cheap way. In this work, pressure and temperature were the tools used to selectively inactivate the activities expressed by naringinase. The main goal was the identification of pressure–temperature conditions to acquire conditions for the maximization of enzymatic hydrolysis of substrates with different numbers of glycosidic residues. α -l-Rhamnosidase was 32-fold more resistant against inactivation at 250 MPa than at atmospheric pressure. The best pressure condition to reduce β -d-glucosidase inactivation at 75°C was 173 MPa, while in the case of α -l-rhamnosidase inactivation at 85°C, it was above 250 MPa. Moreover, a selective inactivation of β -d-glucosidase activity of naringinase was attained, allowing an easy and cheap method with which to produce prunin and other expensive glycosides. The present work highlights the effect of high pressure on enzyme protection against thermal inactivation, demonstrating its potential as a powerful tool in biosynthesis.