Kinetic mechanism and equilibrium thermodynamic of lipase catalyzed synthesis of rutin flavonoid ester / Nurul Nadiah Abd Razak

Flavonoids are natural compounds in plant with wide spectrum of healthbeneficial activities. They exhibit a variety of physico-chemical properties and biological activities but they are normally characterized by low solubility and stability. In order to improve upon these limitations, the enzymatic acylation of these molecules with fatty acids is seen as a selective, specific and mild route. In this study, the synthesis of flavonoid esters catalyzed by lipase was investigated. Three different flavonoid structures from different classes were selected to be studied viz. rutin (flavonols), naringin (flavanones) and catechin (flavanols). The effects of these flavonoid structures on the esterification were investigated with lauric acid as an acyl donor. The results showed that rutin was the most reactive substrate. Conversion yields were at 56 % and 47 % for rutin and naringin, respectively. For an aglycone flavonoid, such as catechin, no glycoside ester formation was observed. Based on the conversion yield and solubility consideration, rutin was selected for further studies. For different chain length fatty acid (C12–C16), no significant difference was observed in terms of yield irrespective of fatty acids used. Full factorial experimental design (FFD) was used to study the effects of different process parameters towards the synthesis of rutin laurate. The effects of three selected operating variables viz. lauric acid concentration (M), temperature (ºC) and enzyme loading (g) were significant in all cases (P < 0.05). The factors studied showed temperature has the strongest influence on the ester yield followed by lauric acid concentration and enzyme loading. In thermo-kinetic investigation, analysis indicated that lipase-mediated esterification exhibited Ping-Pong Bi-Bi mechanism with no apparent inhibition by both substrates. The apparent kinetic of the esterification reaction followed a first-order behavior. The magnitude of glycoside ester formation and the apparent first-order rate constant, k1’ value increased with temperature. Activation energy, Ea for the esterification was calculated at 37 kJ mol−1. The esterification process was endothermic with the enthalpy, H and entropy, S values calculated at +51 kJ mol-1 and +113 J mol-1 K-1, respectively. Based on the value of Gibbs free energy change, G the esterification reaction under the conditions studied was predicted to be non-spontaneous below 175 °C but spontaneous at higher temperatures. The study also confirmed, through 13C-NMR analysis, the exact location of rutin esterification. The investigated process biochemistry highlighted important behavior concerning the biosynthesis of functionalized flavonoid with implication for reactor design.behavior. The magnitude of glycoside ester formation and the apparent first-order rate constant, k1’ value increased with temperature. Activation energy, Ea for the esterification was calculated at 37 kJ mol−1. The esterification process was endothermic with the enthalpy, H and entropy, S values calculated at +51 kJ mol-1 and +113 J mol-1 K-1, respectively. Based on the value of Gibbs free energy change, G the esterification reaction under the conditions studied was predicted to be non-spontaneous below 175 °C but spontaneous at higher temperatures. The study also confirmed, through 13C-NMR analysis, the exact location of rutin esterification. The investigated process biochemistry highlighted important behavior concerning the biosynthesis of functionalized flavonoid with implication for reactor design

Hydrodynamics and kinetics of lipase-catalysed synthesis of diacylglycerols (dag) in batch and continuous flow systems

Les diacylglycérols (DAG) ont été largement utilisés dans les industries alimentaires, médicales, cosmétiques et pharmaceutiques grâce à leurs propriétés exceptionnelles en tant qu'émulsifiants, stabilisants et additifs nutritionnels pour l'homme. Comme alternative à l'utilisation de catalyseurs chimiques, cette étude a examiné la synthèse de dioléates de glycérol (GDO) par estérification du glycérol avec l'acide oléique par la Candida antartica lipase B (CALB 1) dans les systèmes batch et à flux continu. La première section de ce travail a examiné les effets de différents systèmes de solvants sur la conversion, le rendement et la sélectivité des oléates de glycérol. Les propriétés rhéologiques des systèmes sans solvant, ou utilisant comme solvants l’acétone, le tert-butanol ou un mélange, ont montré un comportement d'écoulement newtonien, et leurs viscosités ont diminué à des températures élevées. Par rapport aux monooléates de glycérol (GMO) et au trioléate de glycérol (GTO), la synthèse de GDO a conduit à un rendement et à une conversion plus élevées (70 %) avec une sélectivité de 54 %, à 40 ºC en 2 h dans un mélange acétone:tert-butanol. La deuxième section se concentre sur l'optimisation et la cinétique de la synthèse de GDO catalysée par les lipases en utilisant un réacteur discontinu. Une conversion élevée de l'acide oléique de 73% et une sélectivité du GDO de 77% ont été obtenues dans un mélange de acétone:tert-butanol 70:30 (v/v) à 45°C et avec une quantité d’enzyme de 0,15g. A 45°C, le modèle cinétique de Ping-Pong Bi-Bi, sans inhibition du substrat, donne le meilleur ajustement avec des valeurs de paramètres de Vmax = 1.21 M h-¹, K Gm = 1.81 M et K OAm = 6.81 M, pour les concentrations de glycérol et d'acide oléique entre 0.31 et 1.32 mol L-1 et 1.88 à 2.13 mol L-¹, respectivement. La dernière section s’intéresse à la synthèse enzymatique de GDO dans un système à flux continu. La conversion maximale de l'acide oléique et la sélectivité du GDO ont atteint 85% et 74% respectivement, à 77 min de temps de passage (2x10-5 L min-¹) avec un rapport molaire 1,6:1 d'acide oléique:glycérol à une température constante de 45 ºC. Les études hydrodynamiques ont montré que la réaction d'estérification est cinétiquement contrôlée et non affectée par le transfert de matière externe et interne. En utilisant le modèle de Lilly-Hornby pour l'évaluation cinétique, les valeurs de Km augmentent avec le débit, tandis que Vmax semble être indépendant du débit. Les tests de réutilisation ont révélé que l'activité de la lipase immobilisée reste la même après 9 cycles de réaction successive. Après 11 jours de fonctionnement, la stabilité de la lipase dans le milliréacteur à lit fixe continu n'a diminué que de 5 à 7 %, ce qui indique une opérabilité et une recyclabilité satisfaisantes. Les résultats globaux ont mis en évidence tout l’intérêt de l’utilisation de la lipase dans la synthèse du DAG dans des réacteurs discontinus et des réacteurs à flux continu. Ce travail a montré que la conversion de l'acide oléique et la sélectivité en GDO atteintes étaient de 73-85 % et 74-77 %, respectivement. Le temps de réaction optimal pour la synthèse de GDO dans un réacteur discontinu est de 2 h et peut être réduit à 1,3 h dans un réacteur à flux continu. Contrairement à l'approche du système discontinu, la lipase peut être facilement éliminée et efficacement recyclée de la colonne du réacteur à flux sans subir de changements de conversion appréciables. En utilisant les avantages de l'intensification des procédés, les résultats obtenus à partir des travaux pourraient être appliqués à la conception de procédés futurs et à l’extrapolation de la synthèse du DAG à des fins de commercialisation.Diacylglycerols (DAG) have been extensively used in food, medicine, cosmetic and pharmaceutical industries due to their outstanding properties as emulsifiers, stabilizers and nutritional benefits to human. To circumvent the use of chemical catalysts, this study investigated the synthesis of glycerol dioleates (GDO) through esterification of glycerol with oleic acid by Candida antartica lipase B (CALB 1) in the batch and continuous flow systems. The first section of this work examined the effects of different solvent systems on conversion, yield, and selectivity of glycerol oleates. The rheological properties of solventless, acetone, tert-butanol, and blended solvents systems exhibited Newtonian flow behavior, and their viscosities decreased at elevated temperatures. As compared to glycerol monooleates (GMO) and glycerol trioleate (GTO), GDO synthesis showed the highest yield (70%) and selectivity (54%) achieved at 40ºC within 2 h in the mixed solvents of acetone:tert-butanol at an enzyme load of 0.15 g. The second section focused on the optimization and kinetics of lipase-catalyzed synthesis of GDO using a batch reactor. A high oleic acid conversion of 73% and GDO selectivity of 77% were obtained in acetone: tert-butanol 70:30 (v/v) mixture at 45°C and an enzyme load of 0.15 g. At 45°C, the Ping-Pong Bi–Bi kinetic model with no substrate inhibition gives the best fit with parameter values of Vmax = 1.21 M h-¹, K Gm = 1.81 M and K OAm = 6.81 M, for glycerol and oleic acid concentrations between 0.31 to 1.32 M and 1.88 to 2.13 M, respectively. The final section studied the enzymatic synthesis of GDO under a continuous flow system. The maximum oleic acid conversion and GDO selectivity were achieved at 85% and 74% respectively, at 77 min of residence time (2x10-5 L min-¹) with 1.6:1 molar ratio of oleic acid:glycerol at a fixed temperature of 45ºC. The GDO content started to decrease after the water content reached 5%. Hydrodynamic studies showed that the esterification reaction is kinetically controlled and unaffected by external and internal mass transfer. Employing Lilly–Hornby model for kinetic evaluation, Km values increased with increasing flow rates, whereas, Vmax appeared to be flow rate independent. Reusability tests revealed that the activity of immobilized lipase remained the same after 9 successive reaction cycles. This was accomplished by using the same enzyme repeatedly. After 11 days of storage, the stability of the lipase in the continuous packed bed millireactor decreased only 5 to 7%, indicating satisfying operational results and recyclability. The overall findings highlighted the successful application of lipase in the synthesis of DAG in batch reactor and continuous flow reactors. It was discovered that oleic acid conversion and GDO selectivity were attained at 73-85% and 74-77%, respectively. The optimum reaction time for GDO synthesis in a batch reactor was 2 h and decreased to 1.3 h in a continuous flow reactor. In contrast to the batch system approach, lipase enzyme can be recycled effectively without suffering any appreciable conversion changes. Utilising the benefits of process intensification, the results obtained from work could be applied for future process design and up scaling of DAG for commercialization purpose

Kinetics and hydrodynamics of Candida antartica lipase-catalyzed synthesis of glycerol dioleate (GDO) in a continuous flow packed-bed millireactor

Diacylglycerols (DAG) have been widely used in many industries due to their remarkable capabilities as emulsifiers and stabilisers. However, developing a sustainable and an effective synthesis method for DAG remains a challenge. Continuous flow bio-reactor is recognized to be more productive, controllable, and reliable instrument for developing green and intensified processes. In this work, a continuous flow packed bed millireactor was employed for the synthesis of glycerol dioelate (GDO) catalyzed by immobilized lipase namely Candida antartica. Experiments were carried out to evaluate the kinetic parameters as well as to assess the internal and external mass transfer limitations. Using one-factor-at-a-time variables method, maximum oleic acid conversion and GDO selectivity were achieved at 85% and 74% respectively, at 0.15 g of lipase, 77 min of residence time with 1.6:1 molar ratio of oleic acid/glycerol. Hydrodynamic studies showed that the esterification reaction is kinetically controlled and unaffected by external and internal mass transfer. Employing Lilly–Hornby model for kinetic evaluation, K m values increased with increasing flow rates, whereas, V max appeared to be flow rate independent. Reusability tests revealed that the activity of immobilized lipase remained the same after 9 successive reaction cycles. At 11 days of operation, the stability of the lipase in the continuous packed bed millireactor decreased only 5–7%, indicating satisfying operational results and recyclability. This work may promote the enzymatic engineering synthesis of DAG, facilitating the creation of a cleaner and safer process. It has the potential to broaden the application of enzymes in continuous flow micro or millireactors