Lipase In Reverse Micelles Research Articles

Reverse micelles‐mediated transport of lipase in liquid emulsion membrane for downstream processing

This work deals with the downstream processing of lipase (EC 3.1.1.3, from Aspergillus niger) using liquid emulsion membrane (LEM) containing reverse micelles for the first time. The membrane phase consisted of surfactants [cetyltrimethylammonium bromide (CTAB) and Span 80] and cosolvents (isooctane and paraffin light oil). The various process parameters for the extraction of lipase from aqueous feed were optimized to maximize activity recovery and purification fold. The mechanism of lipase transport through LEM consisted of three steps namely solubilization of lipase in reverse micelles, transportation of reverse micelles loaded with lipase through the liquid membrane, and release of the lipase into internal aqueous phase. The results showed that the optimum conditions for activity recovery (78.6%) and purification (3.14-fold) were feed phase ionic strength 0.10 M NaCl and pH 9.0, surfactants concentration (Span 80 0.18 M and CTAB 0.1 M), volume ratio of organic phase to internal aqueous phase 0.9, ratio of membrane emulsion to feed volume 1.0, internal aqueous phase concentration 1.0 M KCl and pH 7.0, stirring speed 450 rpm, and contact time 15 min. This work indicated the feasibility of LEM for the downstream processing of lipase.

Covalently Functionalized Single-Walled Carbon Nanotubes at Reverse Micellar Interface: A Strategy to Improve Lipase Activity

The present work reports covalent functionalization of single-walled carbon nanotubes (f-SWNTs) to introduce hydrophilicity to the otherwise amphiphobic nanotubes. The charge and spacer length of the functional moiety were varied by using quaternized ethylene diamine, 6-aminocaproate, quaternized (ethylenedioxy)bis(ethylamine), and a poly(ethylene glycol) (PEG) unit (f-SWNT-1 to f-SWNT-4, respectively). These f-SWNTs with varying degrees of hydrophilicity were incorporated within cetyltrimethyl ammonium bromide (CTAB) reverse micelles to develop stable self-assembled nanohybrids. An optimum hydrophilicity on the SWNT surface led to interfacial localization of f-SWNTs resulting in the augmentation of space at the interface. A surface-active enzyme, lipase, localized at this enhanced interface of f-SWNT-containing CTAB reverse micelles exhibited significant activation (2.5-fold) compared to that in the absence of the nanoconstructs. This improvement in lipase activity was mainly due to the smooth occupancy of lipase and also presumably because of the increase in the concentrations of both substrate and the enzyme at the augmented interface. Interestingly, the f-SWNTs that activate lipase in reverse micelles deactivate the same enzyme in water. The dispersion of f-SWNTs in water and its matching integration at the interface of reverse micelles were confirmed through transmission electron microscopic (TEM) investigations. The interfacial localization of these nanoconstructs was also established from the distinct fluorescence behavior of a hydrophobic fluorescent probe, fluorescein isothiocyanate (FITC), adsorbed onto the f-SWNT surface. In concurrence with the observed lipase activity, the corresponding changes in the enzyme conformation within f-SWNTs integrated reverse micelle as well as in aqueous medium were studied by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy.

Unsaturation at the surfactant head: Influence on the activity of lipase and horseradish peroxidase in reverse micelles

Influence of unsaturation present at the surfactant head on the activity of interfacially located enzyme, lipase, and horseradish peroxidase (HRP) is investigated in cationic reverse micelles of a series of surfactants having unsaturated (allyl and pyridinium moieties) as well as analogous saturated ( n-propyl and piperidinium moieties) polar head. Lipase activity increases with n-propyl (saturated) substitution as the increase in the headgroup area ( A min) presumably provides greater space for the enzyme to attain flexible conformation and increases the local concentrations of enzyme and substrate at the interface. In contrast, activity of lipase decreases with increasing number of allyl (unsaturated) substitution though A min gradually increased. Similar trend in deactivation was observed when unsaturation is present in cyclic ring (pyridine) at the surfactant head in comparison to the saturated analogue, piperidine. Circular dichroism (CD) spectra of lipase in reverse micelles indicate that ellipticity in the far-UV region increases with increasing unsaturation. Thus, lipase probably loses its α-helix content and thereby its activity. Inhibition of biocatalyst with increasing unsaturation at the polar head of surfactant is also observed in case of HRP, an oxidoreductase enzyme.

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacteriumviscosum lipase (glycerol–ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30–40 mM. The various relevant physical parameters such as w0 (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.

Activity of acetone-treated Chromobacterium viscosum lipase in AOT reverse micelles in the presence of low molecular weight polyethylene glycol

The activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane/water reverse micelles was significantly enhanced by pretreatment with acetone, using the hydrolysis of olive oil as a model reaction. The activity of acetone treated lipase was further enhanced when low molecular weight polyethylene glycol (PEG 400) was introduced into AOT reverse micelles. To know the effects of acetone treatment on the lipase activity in simple AOT and mixed AOT/PEG 400 reverse micelles, the influence of various parameters, such as W 0 (molar ratio of water to surfactant), pH, surfactant concentration, ionic strength and reaction temperature were investigated and compared with those for native lipase in simple AOT reverse micelles. The optimal activities of treated lipase in AOT reverse micelles with and without PEG 400 were 2.0 and 1.6 times higher respectively than that of native lipase in AOT reverse micelles. A kinetic model that considers substrate adsorption equilibrium between the bulk phase of organic solvent and the micellar phase was successfully used to understand the improvement of the lipase activity. The Michaelis constant ( K m) and substrate adsorption equilibrium constant (K ad ) were obviously reduced compared with those for native lipase in AOT reverse micelles. The stability of the lipase in reverse micelles was also studied, and the values of half-life time ( t 1/2) were determined from residual activity profiles.

Pretreatment ofChromobacterium viscosum lipase with acetone increases its activity in sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles

The activity of Chromobacterium viscosum lipase for hydrolysis of olive oil in sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles was increased by pretreatment with acetone. In contrast to the untreated lipase, no sharp fall in the activity of the treated lipase at higher W0 (water to AOT molar ratio) values was observed. The fluorescence emission intensity of the treated lipase in reverse micelles was higher than that of the untreated lipase but the maximal emission wavelength (λmax) was the same for both lipases. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to better understand the activity enhancement. The Michaelis constant (Km) and substrate adsorption equilibrium constant (Kad) were reduced by lipase pretreatment with acetone whereas the maximum reaction rate (vmax) remained unaltered. Copyright © 2005 Society of Chemical Industry

Hydrolysis and synthesis reactions catalysed by Thermomyces lanuginosa lipase in the AOT/Isooctane reversed micellar system

The kinetics of hydrolysis of triolein and tributyrin and of the synthesis of ethyl-laurate by the lipase of Thermomyces lanuginosa, contained in a commercial preparation (Lipolase®), were studied in AOT/Isooctane reverse micelles. Lipolytic activity against triolein depended strongly on the water content in the system ( W 0=[H 2O]/[AOT]), in a bell-shaped manner, with a maximum at a W 0 of 15. The best conditions for enzyme activity were pH 8.0 and 37 °C. The enzyme did not show Michaelis–Menten kinetics for the hydrolysis of either triolein or tributyrin. The enzyme was unstable at temperatures of 37–60 °C, losing approximately 50% of its activity after 30 min. The catalysis of ethyl-laurate synthesis by T. lanuginosa lipase in reverse micelles was studied using factorial designs to optimize the reaction conditions. The most important variables were pH and temperature and their combined effect. The best conditions for ester synthesis were a W 0 of 10, a pH of 5.6, a molar ratio of alcohol to acid of 5.0 and a temperature of 30 °C. The specific enzymatic activity under these conditions was 220 U mg −1 and the ester yield 92% after 60 min of reaction. This high yield, obtained in a relatively short time, justifies further exploration of the potential of this system in biocatalysis.

Measurements of hydrodynamic diameter of AOT reverse micelles containing lipase in supercritical ethane and its enzymatic reaction

The enzymatic reaction by aerosol-OT (AOT)reverse micelles containing lipase in supercritical ethane was examined and is the focus of this paper. The reverse micelles were formed under various conditions at which their hydrodynamic diameters were measured by using the dynamic light scattering spectrophotometer. The reverse micelles in supercritical ethane were formed in the range of Wo (water/surfactant) less than six. The hydrodynamic diameter of the reverse micelles ranged from 2 to 5 microm. The hydrolysis reaction of triolein by the lipase in reverse micelles was also examined. The observations indicate that lipase in AOT reverse micelles in supercritical ethane showed activity. The conversion of triolein increased with the increase in size of reverse micelles and Wo, and reached its maximum near the critical temperature. Moreover, although the conversion of triolein increased with pressure, it was independent of pressure near the critical temperature.

Increased activity and stability of Candida rugosa lipase in reverse micelles formed by chemically modified AOT in isooctane

AbstractSodium bis (2‐ethylhexyl polyoxyethylene) sulfosuccinates, which can be structurally viewed as chemically modified AOT (MAOT), were prepared and successfully used to form reverse micelles in isooctane. The activity and stability of Candida rugosa lipase (glycerol‐ester hydrolase, EC 3.1.1.3) appreciably increased in the MAOT/isooctane reverse micellar system compared with the AOT/isooctane system taking the hydrolysis of olive oil as a model reaction. The best polymerization number of oxyethylene group was found to be 2.0, which corresponded to a maximum enzyme activity of twice that in the AOT/isooctane system. The half‐life of lipase in the MAOT micellar system was twice that in the AOT micellar system. The optimal operational conditions remained unchanged. The Michaelis kinetics showed that the maximum reaction rate obviously increased in the MAOT micellar system compared with that in the AOT micellar system, while the change in the Michaelis constant was insignificant.© 2001 Society of Chemical Industry

Activity and stability of lipase in Aerosol-OT/isooctane reverse micelles

The stability of Candida rugosa lipase, which catalyzes the hydrolysis reaction of olive oil in AOT/isooctane reverse micelles, decreased with the increase of ω0 (defined as the molar ratio of water to surfactant) and Aerosol-OT concentration. The addition of a non-ionic cosurfactant, tetraethylene glycol dodecyl ether (C12E4), preserved enzymatic activity. The residual activity of the lipase was 53% after 24 h, while the enzyme completely lost its activity within 6 h in the absence of C12E4 addition. The stabilizing effect of C12E4 resulted in the increase of conversion. The enhancement of the activity and stability of lipase in reverse micelles by the addition of C12E4 may contribute to increase the rigidity of the micellar matrix stabilizing the enzyme structure.

Increased activity of Chromobacterium viscosum lipase in aerosol OT reverse micelles in the presence of nonionic surfactants.

Modification of AOT reverse micellar systems with various alkyl glucosides and nonionic surfactants was attempted in order to improve the activity to Chromobacterium viscosum lipase. Tweens and Tritons, having the poly(oxyethylene) chain, have been shown to be solubilized at the oil-water interface of AOT micelles. Alkyl glucosides were also solubilized at the interface. These additives form mixed micelles with AOT and increase the concentration of the micelles. In contrast, Spans were found to be solubilized in the micro water pool of AOT micelles. Addition of nonionic surfactants decreased the hydrophobicity of the reverse micellar systems. Improvement of the lipase activity was achieved by the addition of nonionic surfactants, because of suppression of the hydrophobic and electrostatic interaction between AOT and lipase. The addition of nonionic surfactant having a poly(oxyethylene) chain has been shown to effectively increase the activity of the lipase in reverse micelles.

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.

Batchwise hydrolysis of olive oil by lipase in AOT-isooctane reverse micelles

Olive oil was almost completely hydrolyzed by lipase in reverse micelles. R value and initial water content were found to be the most important factors that determine the hydrolyzing rate and degree of hydrolysis, respectively. The hydrolysis rate and the stability of the enzyme were affected by stirring and addition of histidine or glycerol.