Lipase-catalyzed Hydrolysis Of Olive Oil Research Articles

Study on Lipase-Catalyzed Hydrolysis of Olive Oil at Oil-Water Interface

Abstract Olive oil was selected as the oil substrate and hydrolyzed by Candida sp. 99–125 lipase. The hydrolysis rate of olive oil was used as an indicator. Based on the single factor experiment, the effects of dosage of Candida sp. 99–125 lipase, reacting temperature, pH value and water-oil ratio were investigated. Box-Behnken center combination and response surface methodology were utilized to optimize the hydrolysis rate. The results showed that the significant differences of each single factor on lipase hydrolysis of olive oil on the oil-water interface were different. pH value is the first significance factor, and the significance of water oil ratio on lipase hydrolysis of olive oil is second only to pH value. Finally, the mechanism of Candida sp. 99–125 lipase hydrolyzing olive oil at the oil-water interface was discussed.

Kinetics of lipase-catalyzed hydrolysis of olive oil in AOT/isooctane reversed micelles

The kinetics of lipase-catalyzed hydrolysis of olive oil in AOT/isooctane reversed micellar media was studied. It was shown that the deactivation of lipase had a great influence on the reaction kinetics. Based on whether the enzyme deactivation and influences of both product and substrate on enzyme stability were included or not, four different kinetic models were established. The simulating results demonstrated that the kinetic model, which including product inhibition, enzyme deactivation and the improvements of lipase stability by both product and substrate, fit the experimental data best with an overall relative error of 4.68%.

A kinetic model for enzymatic reactions in reverse micellar systems involving water-insoluble substrates and enzyme activators

The activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane reverse micelles was significantly increased by the addition of short chain polyethylene glycols (PEGs) or methoxypolyethylene glycols (MPEGs) for the hydrolysis of olive oil. To understand enzyme activity in the presence of PEG 400 or MPEG 550 molecules, a kinetic model was proposed. The validity of this model was verified by experimental data on the lipase-catalyzed hydrolysis of olive oil in AOT/isooctane reverse micellar systems, in which PEG 400 or MPEG 550 had been added. The large value of the equilibrium constant (kD) for enzyme activation indicated that the affinity between C viscosum lipase and PEG 400 or MPEG 550 molecules was very strong. The Michaelis constant (Km) predicted by the proposed model explained enzymatic reactions more exactly than that by the previously published model. Copyright © 2003 Society of Chemical Industry

Lipase-catalyzed hydrolysis of olive oil in chemically-modified AOT/isooctane reverse micelles in a hollow fiber membrane reactor

The hydrolysis of olive oil catalyzed by Candida rugosa lipase in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane and the synthetic sodium bis(2-ethylhexyl polyoxyethylene)sulfosuccinate (MAOT)/isooctane reverse micellar systems was investigated in a polysulfone hollow fiber membrane reactor with recycle of the reaction mixture. Lipase was completely retained by the membrane while olive oil and oleic acid freely passed through. The retention of reverse micelles depended on W0 (molar ratio of water to surfactant). At an olive oil concentration of 0.23 mol l−1 the final substrate conversion in the MAOT micellar system was about 1.4 times of that in the AOT micellar system.

Selection of hydrophobic membranes in the lipase-catalyzed hydrolysis of olive oil

A hydrophobic membrane (HVHP, polyvinylidene difluoride) was selected out of HVHP, PTHK and PTGC (polysulfone) membranes to immobilize Candida rugosa lipase by physical adsorption in the hydrolysis of olive oil in a stirred diffusion cell. A previous model that assumed the Michaelis–Menten kinetics and Langmuir adsorption isotherm for the adsorbed lipase was used to interpret the variation of initial hydrolysis rates with enzyme and substrate concentrations. Replacing the aqueous phase by a fresh buffer, with or without containing partially deactivated lipases, during the reaction did not affect the enzyme activity for the adsorbed lipase. Moreover, the same enzyme performance was obtained when a fresh and a regenerated membrane was used as the carrier in the membrane reactor.

Kinetics of lipase-catalyzed hydrolysis of olive oil

Lipase-catalyzed hydrolysis of olive oil has been studied in the absence of added emulsifier. The kinetic analysis of the lipase-catalyzed hydrolysis reaction was found to be possible in this system. The amount of fatty acids produced was linearly proportional to the enzyme concentration of 0.1 mg protein ml −1. The specific enzyme activity was 2500 units mg −1 enzyme, at 37 °C in 25 mM phosphate buffer, pH 7.0, at 50% (v/v) olive oil concentration. The hydrolytic reaction obeys Michaelis-Menten kinetics with V max,app and K m,app of 3470 units mg −1 enzyme and 16.7% (v/v), respectively. Maximum activity was obtained at pH 7.0, at low buffer ionic strength, 25 mM and 37 °C. The enzyme was found to be inhibited by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and o-phthaldialdehyde in a biphasic manner. These results suggested that carboxyl groups and lysyl and cysteinyl residues might be involved in the catalytic site or substratebinding site of this enzyme.

Characteristics and kinetics of lipase-catalyzed hydrolysis of olive oil in a reverse micellar system

The hydrolysis rates of olive oil catalyzed by lipase were measured in a reverse micellar system using sodium bis(2-ethylhexyl) sulfosuccinate (AOT) under various conditions. The maximum activity of lipase in the reverse micelles was obtained between pH 6.3 and 7.3. The dependence of the activity on the water content, Wo, was influenced by the concentrations of both AOT and lipase. The activity at Wo = 7 decreased with increasing lipase concentration when the AOT concentration was below 100 mol/m3. The stability of the lipase in the micelles decreased with increases in the value of Wo and in the concentration of AOT. The hydrolysis reaction catalyzed by lipase in the reverse micelles was interpreted by a reaction model based on the interfacial reaction between lipase adsorbed at the interface and the substrate in the organic phase. The rate is controlled by the desorption step of free fatty acids into the organic solution. The desorption rate constants of fatty acids produced were independent of the AOT concentration at a fixed Wo, and were of nearly the same order of magnitude as that obtained in the emulsion system.

Application of a recycle dialysis system in a reversed micellar reactor.

A recycle dialysis stirred cell has been successfully used for integrating the reaction and product recovery of the lipase-catalyzed hydrolysis of olive oil in bis(2-ethylhexyl) sodium sulfosuccinate (AOT)-iso-octane reversed micelles. The resistance of membrane to reversed micelles was monitored by the water content and found to be 95.9% rejection after 24 h and 93.4% rejection after 48 h. The resistance of the membrane to free surfactants, by monitoring the UV absorption, was found to be 98.5% rejection after 10 h and 97.3% rejection after 24 h. Mathematical formulations involving the enzymatic reaction coupled with mass transfer were developed for predicting the performance of a membrane reactor. Theoretical predictions in terms of time course of oleic acid concentrations were found to be in agreement with the experimental results.

Mathematical modelling and simulation of a recycle dialysis membrane reactor in a reversed micellar system.

A mathematical framework was developed for the evaluation of a recycle dialysis membrane reactor (RDMR). The lipase-catalyzed hydrolysis of olive oil in an AOT-iso-octane reversed micellar system was employed as a model. Three specific operational strategies have been considered, namely batch, fed-batch, and fed-batch--bleed. Simulation shows the conversion of substrate to be strongly dependent on efficient use of the substrate, since the permeability coefficients of both substrate and product are quite similar. Sensitivity analyses were performed to assess the influences of various parameters (membrane area, substrate feed rate, solvent bleed rate and permeability) on the performance of the reactor in different modes of operation. The analyses presented are useful to assist the optimization of the operational strategy used for the RDMR system.

Flow-through pH-stat method for lipase activity

A new method for lipase activity which combines the simplicity and rapidity of continuous pH-stat methods with the flexibility in choice of conditions (pH, temperature, etc.) of manual methods is described. The lipase-catalyzed hydrolysis of olive oil and titration of fatty acid products can be carried out separately in two small continuously stirred reactors in series. The response is linear with enzyme dilution.