Kinetics of lipase deactivation in AOT/isooctane reversed micelles

The stability of lipase in AOT/isooctane reversed micellar solution was investigated. It was found that the lipase deactivated to a stable state that was not completely inactivated. The lipase residual activity after achieving the stable state in AOT/isooctane reversed micelles at 30 °C, pH 7.0, W₀=8.0 was found to be 0.15, and the first-order deactivation rate coefficient of lipase at the same conditions was regressed to be 0.75 h⁻¹. The stability of lipase was increased while oleic acid was added. Assuming the protection of oleic acid to lipase stability is due to the lipase–oleic acid complex does not decay, the kinetic model of lipase deactivation in AOT/isooctane reversed micellar solution including the influence of oleic acid was established. It was shown with the model equation that the increase in stability of the enzyme by oleic acid could be quantitatively estimated by the dissociation constant of lipase–oleic acid complex which was determined by product inhibition experiments. The model equation fit the experimental data well with an average relative deviation of 3.40%.     

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 W ₀ (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.     

Inhibition of lipase-catalyzed hydrolysis of sunflower oil in AOT/isooctane reversed micelles

The hydrolysis of sunflower oil using Candida cylindracea lipase in reversed micelles of AOT/isooctane was investigated. The inhibition caused by substrate and hydrolysis products has been found in the process of reaction. It was revealed that the extent of inhibition caused by oleic acid was higher than that caused by glycerol, and was much more serious in the case of the mixture of hydrolysis products. Moreover, with the initial addition of glycerol into the reaction mixture, the stability of lipase could be increased during the hydrolysis of sunflower oil in reversed micelles. We thank the National Natural Science Foundation of China for the financial support of this work. We also thank Prof. Xu, Jia-li for his contributions to this work.     

Kinetics, mechanism, and time course analysis of lipase-catalyzed hydrolysis of high concentration olive oil in AOT-isooctane reversed micelles

Candida rugosa lipase has been used to investigate the hydrolysis of high concentration olive oil in the AOT-isooctane reversed micellar system at W(o) = 10, pH 7.1, and 37 degrees C. Results from this work show the hydrolytic reaction obeys Michaelis-Menten kinetics up to the initial substrate concentration of 1.37M, with turnover number k(cat) and Michaelis constant K(M) of 67.1 mumol/min mg enzyme and 0.717M, respectively. A competitive inhibition by the main product, oleic acid, has been found with a dissociation constant K(I) for the complex EP* of 0.089M. The rate equation was further analyzed in the time course reaction and was found in agreement with the experimental results for lower substrate concentrations, up to 0.341M. Large deviation occurred at high substrate concentrations, which may be due to the effects of large consumption of water on kinetics, on the formation of glycerol, and on the deactivation of lipase in the hydrolysis reaction as well.     

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

Candida rugosa lipase solubilized in organic solvents in the presence of both surfactant and water could catalyze the hydrolysis of triglycerides, and kinetic analysis of the lipase-catalyzed reaction was found to be possible in this system. Among eight organic solvents tested, isooctane was most effective for the hydrolysis of olive oil in reversed micelles. Temperature effect, pH profile, K(m,app) and V(max,app) were determined. Among various chemical compounds, Cu(2+), Hg(2+), and Fe(3+) inhibited lipase severely. But the enzyme activity was restorable partially by adding histidine or glycine to the system containing these metal ions. The enzyme activity was dependent on R (molar ratio of water to surfactant) and maximum activity was obtained at R = 10.5. Upon addition of glycerol to the reversed micelles, lipase activity was affected in a different fashion depending on the R values. Stability of the lipase in reversed micelles was also dependent on R, and it was most stable at R = 5.5.     

Kinetics of lipase-catalyzed hydrolysis of palm oil in lecithin/izooctane reversed micelles

Candida rugosa lipase has been used to investigate the hydrolysis of palm oil in a lecithin/isooctane reversed micellar system. The reaction obeys Michaelis-Menten kinetics for the initial conditions. Kinetic parameters such as maximum rate and Michaelis constant (K _m) were determined for lipase-catalyzed hydrolysis in n-hexane and isooctane. According to the K _m values, the enzyme affinity towards the substrate was increased in isooctane. The maximum degree of hydrolysis was generally decreased as the initial substrate concentration was increased. This may suggest that the hydrolysis in lecithin reversed micelles should be regarded as a one-substrate first-order reversible reaction. It is shown in this study that the proposed one-substrate first-order kinetic model can serve for the precise prediction of the degree of hydrolysis for a known reaction time or vice versa, when the initial substrate concentration is less than 0.325 mol/dm³. A disagreement with this model was found when the initial substrate concentration was higher than approximately 0.3 mol/dm³. This may be due to the effects of the products on lipase activity or even to the conversion of the reversed micellar system to other systems. Received: 16 May 1997 / Received revision: 22 October 1997 / Accepted: 24 October 1997     

A steady-state fluorescence study of cutinase microencapsulated in AOT reversed micelles at optimal stability conditions

A steady-state fluorescence study of cutinase was performed to evaluate the structure of cutinase in reversed micelles of AOT with the optimised conditions assigned by factorial design. The results obtained by two independent methods are compared. At a W₀ (water to surfactant ratio) value of 2.7, and in the presence of 500 mM hexanol, the fluorescence intensity maximum (_max) remained almost constant for a period of time longer than 30.5 h and a slight red-shift from 305 to 310 nm was verified changing the W₀ value to 6. Decreasing the amount of hexanol to 100 mM, the changes in _max were more significant, especially for W₀=6 indicating a noticeable unfolding process. Structural evidence is given reinforcing the role of hexanol as a stabiliser of microencapsulated cutinase and the effect of a drastic reduction in water content.     

Effect of aprotic solvents on the enzymatic activity of lipase in AOT reverse micelles

The modification of reverse micellar systems composed of AOT, isooctane, water by the addition of aprotic solvents has been performed. The impact of this change on the activity, stability and kinetics of solubilized Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was investigated. Of seven aprotic solvents tested, dimethyl sulfoxide (DMSO) was found to be most effective. It was found that lipase activity was enhanced by optimizing some relevant parameters, such as water–AOT molar ratio (W₀), buffer pH and surfactant concentration. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to deduce some kinetic parameters (V_max, K_m and K_ad), and the values of K_m and K_ad were significantly reduced by the presence of DMSO. Higher lipase stability was found in AOT reverse micelles with DMSO compared with that in simple AOT systems with half-life of 125 and 33 days, respectively. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FT-IR) were used to elucidate the effects of DMSO on the properties of AOT reverse micelles.     

Entrapment of lipase into K-carrageenan beads and its use in hydrolysis of olive oil in biphasic system

The porcine pancrease lipase was immobilized by entrapment in the beads of K-carrageenan and cured by treatment with polyethyleneimine (PEI) in the phosphate buffer. The retention of hydrolytic activity of lipase and compressive strength of the beads were examined. The activity of free and immobilized lipase was assessed by using olive oil as the substrate. The immobilized enzyme exhibited a little shift towards acidic pH for its optimal activity and retained 50% of its activity after 5 cycles. When the enzyme concentration was kept constant and substrate concentration was varied the K_m and V_max were observed to be 0.18 × 10⁻² and 0.10, and 0.10 × 10⁻² and 0.09 respectively, for free and for entrapped enzymes. When the substrate concentration was kept constant and enzyme concentration was varied, the values of K_m and V_max were observed to be 0.19 × 10⁻⁷ and 0.41, and 0.18 × 10⁻⁷ and 0.41 for free and entrapped enzymes. Though this indicates that there is no conformational change during immobilization, it also shows that the reaction velocity depends on the concentration. Immobilized enzyme showed improved thermal and storage stability. Hydrolysis of olive oil in organic–aqueous two-phase system using fixed bed reactor was carried out and conditions were optimized. The enzyme in reactor retained 30% of its initial activity after 480 min (12 cycles).     

Tyrosinase activity in reversed micelles

The hydroxylase and oxidase activities of mushroom tyrosinase were studied in both sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane and cetyltrimethylammonium bromide (CTAB)/hexane/chloroform reversed micelles. The enzyme presented its highest activity when the water to surfactant molar ratio (W ₀) was 20 for both systems. When entrapped in the AOT reversed micelles, the enzyme activity decreased with the increase in AOT concentration at a constant W ₀, and the enzyme not only presented a higher reaction rate related to its oxidase activity but also a shorter lag period related to its hydroxylase activity. The relation between water activity and W ₀ revealed that enzyme activity in reversed micelles was more related to the size of the micelles which was determined by W ₀ and less to the water activity. Tyrosinase in CTAB reversed micelles showed potential for the analysis of o-diphenols.     

Ultrasonication enhanced hydrolytic activity of lipase in water/isooctane two-phase systems

The hydrolysis of olive oil catalyzed by Chromobacterium viscosum lipase (EC 3.1.1.3) in a water/isooctane two-phase system was carried out both under ultrasound and conventional stirring. The maximum activity of lipase in the ultrasonicated system was 1.75 times higher than that in the stirred system. The lipase activity was dependent on ultrasonic power and volume ratio of isooctane to water. The optimum reaction temperature in both systems was around 25°C. The stability of lipase at 25°C in the ultrasonicated system decreased more rapidly than that in the stirred system. In the presence of exogenous oleic acid, however the half-life of lipase in the ultrasonicated system was improved to a value, which was respectively half and twice of that in stirred systems with and without oleic acid. The maximum reaction rate (V_max) was increased by ultrasonication whereas the Michaelis constant (K_m) remained unaltered.     

Continuous glycerolysis of olive oil by Chromobacterium viscosum lipase immobilized in liposome in reversed micelles

Chromobacterium viscosum lipase which has adsorbed on liposome and solubilized in microemulsion droplets of glycerol containing a little amount of water could catalyze the glycerolysis of olive oil. Studies on the continuous glycerolysis of olive oil by the immobilized enzyme was done at 37 degrees C in continuous stirred vessel bioreactor with polysulfone membrane. The effect of the flow rate of substrate (olive oil) in isooctane on the conversion and composition of the outlet was investigated using high-performance liquid chromatography (HPLC). The conversion increased with decrease in the flow rate. And we studied the effect of water content in the glycerol-water-lipase solution on the glycerolysis reaction. The conversion to desirable products, mono- and di-olein, was improved without a substantial production of oleic acid at lower water concentrations, i.e., below 8.0% (w/v) which corresponds to a w(o) value of 0.97. At water concentration higher than 8.0% (w/v), the amount of free fatty acid was dramatically increased. Higher operational stability of the enzyme reactor, and the half-line of the enzyme continuous reaction was about 7 weeks.     

Tryptophan fluorescence studies of melanotropins in the amphiphile-water interface of reversed micelles

We report studies on the interaction of α-melanocyte stimulating hormone (α-MSH) and a synthetic analogue (MSH-I) with reverse micelles prepared from the amphiphilic sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. The tripeptide lysyl-tryptophyl- lysine and the isolated amino acid tryptophan were also investigated as simpler compounds interacting with the micelles. Tryptophan fluorescence parameters (spectral position of emission band, anisotropy, and lifetime decay) demonstrated that in the presence of reverse micelles the environment around the fluorophore is less polar and more rigid than bulk water. Those parameters are sensitive to the changes induced in the micelles by the presence of water. In large micelles having a water/amphiphile molar ratio above 10, the modifications detected by fluorescence are small and the location of the fluorophore is not affected by a further increase in the concentration of the bulk water. The results, with additional support from quenching experiments, indicated that the different compounds occupy different positions in the large reverse micelles, but in any case they are in the interface region, without dispersing into the bulk water. From decay associated spectra, conformations were identified showing different degrees of tryptophan exposition to polar and nonpolar local environments. The conformation related to the long lifetime has its tryptophan more exposed to water while that associated to the intermediate lifetime has that residue stabilized in nonpolar media. The native hormone α-MSH and the analogue MSH-I present similar conformations in dry micelles. However, in buffer and in the large hydrated micelles, differences in conformations are evident, and could be related to the different physiological activity of the peptides. Received: 4 August 1999 / Revised version: 17 December 1999 / Accepted: 4 January 2000     

Selective separation and purification of two lipases from chromobacterium viscosum using AOT reversed micelles

Selective separation and purification of two lipases form Chromobacterium viscosum were carried out by liquid-liquid extraction using a reversed micellar system. Optimum parameters for extraction were determined using a 250 mM AOT micellar solution in isooctane. Complete separation of the two lipases was achieved at pH 6.0 with a 50mM potassium phosphate buffer solution containing 50 mM KCI. By adding 2.5% by volume of ethanol to the lipase-loaded micellar solution, 85% of the extracted lipase could be recovered in a new aqueous phase, 50 mM K(2)HPO(4) with 50 mM KCl, at pH 9.0. Lipase A was purified 2.6-fold with a recovery of 86%, and lipase B by 1.5-fold with a recovery of 76%.     

Enzymatic activity of an extremely halophilic phosphatase from the Archaea Halobacterium salinarum in reversed micelles

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeon Halobacterium salinarum (previously halobium) was solubilized in reversed micelles of cetyltrimethylammonium bromide (CTAB) in cyclohexane with 1-butanol as cosurfactant. The hydrolysis reaction appears to follow Michaelis–Menten kinetics. The dependency of the maximum reaction rate (V_max) on the water content θ (% v/v) (or ω₀ value: molar ratio of water to surfactant concentrations) showed a bell-shaped curve for 0.3 M CTAB, but not for 0.2 M CTAB. The enzyme activity increased with the surfactant concentration at a constant ω₀ value (10.27). When the surfactant concentration was increased at a constant θ, the enzyme activity decreased. The enzyme was more stable in reversed micelles than in aqueous media.     

Lipase-catalyzed hydrolysis of Crambe oil in AOT-isooctane reversed micelles

Summary The hydrolysis by 1,3-specific lipases (Humicola lanuginosa, Mucor miehei, Rhizopus delemar andRhizopus javanicus) of the highly symmetric, high molecular weight triglycerides fromCrambe abyssinica (Crambe) seed oil is studied in an AOT-stabilized microemulsion system. Enzyme kinetic data shows that, of the lipases studied,Rhizopus javanicus lipases exerts the highest hydrolytic activity towards this new seed oil.     

Role of yeasts in the qualitative structuring of extra virgin olive oil

This review sought to describe the role played by some components of the microbiota of extra virgin olive oil (EVOO), particularly yeasts, in structuring the physicochemical and sensorial quality of freshly produced olive oil. Yeasts can survive during the entire storage period of the product. To date, approximately 25 yeast species isolated from oil produced in more than six countries have been identified, eight of which are classified as new species. Some yeast species improve the health qualities of oil, whereas many others improve the chemical composition and sensory characteristics based on β-glucosidase and esterase enzymes, which are involved in the hydrolysis of the bitter glucoside known as oleuropein. However, some species, which are typically favoured by the high water content in the oily matrix, such as lipase-producing yeasts, can worsen the initial chemical characteristics of EVOO oil during storage. Some physical treatments that are compatible with the EVOO production specification affect the biotic component of the oil by reducing the concentration of yeasts. The possibility of minimizing the invasive action on the biotic component of the oil by appropriately selecting the physical treatment for each oil is discussed.     

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 ₀ (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.     

A comparison of lipase-catalyzed ester hydrolysis in reverse micelles, organic solvents, and biphasic systems

The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the "anhydrous" enzyme suspension approach. (c) 1995 John Wiley & Sons, Inc.