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, 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.     

Characterization of lipase in reversed micelles formulated with cibacron blue F-3GA modified span 85

Sorbitan trioleate (Span 85) modified by Cibacron Blue F-3GA (CB) was prepared and used as an affinity surfactant to formulate a reversed micellar system for Candida rugosa lipase (CRL) solubilization. The system was characterized and evaluated by employing CRL-catalyzed hydrolysis of olive oil as a model reaction. The micellar hydrodynamic radius results reflected, to some extent, the redistribution of surfactant and water after enzyme addition, and the correlation between surfactant formulation, water content (W0), micellar size, and enzyme activity. An adequate modification density of CB was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited a different activity behavior versus W0. The optimal pH and temperature of the encapsulated lipase remained unchanged, but the apparent activity was significantly higher than that of the native enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant was found to decrease with increasing surfactant concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0.     

An ultrafiltration membrane bioreactor for the lipolysis of olive oil in reversed micellar media

The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of dioctyl sodium sulfosuccinate (AOT) in isooctane was investigated in an ultrafiltration ceramic membrane reactor of tubular type, operating in a batch mode. Water concentration was found to be a critical parameter in the enzyme kinetics and hydrolysis yield of the reaction. The size of micelles, recirculation rate, and substrate concentration were found to be the major factors affecting the separation process. A correlation that enables the prediction of final conversion degrees in this bioreactor from the initial reaction conditions was established. (c) 1993 Wiley & Sons, Inc.     

Enzymatic Biodiesel Production from Sunflower Oil by Candida antarctica Lipase in a Solvent-free System

Methanolysis (transesterification with methanol) of sunflower oil by lipase from Candida antarctica (Novozym 435) in a solvent-free system has been studied. Stepwise as well as continuous methanol feeding was applied to avoid strong substrate inhibition. Glycerol was found to cause strong product inhibition on the enzymatic reaction, therefore glycerol removal by dialysis was investigated using a flat sheet membrane module.     

Enzymatic Biodiesel Production from Sunflower Oil by Candida antarctica Lipase in a Solvent-free System

Methanolysis (transesterification with methanol) of sunflower oil by lipase from Candida antarctica (Novozym 435) in a solvent-free system has been studied. Stepwise as well as continuous methanol feeding was applied to avoid strong substrate inhibition. Glycerol was found to cause strong product inhibition on the enzymatic reaction, therefore glycerol removal by dialysis was investigated using a flat sheet membrane module.     

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

Summary 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.     

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     

Effect of various additives on enzymatic hydrolysis of castor oil

Hydrolysis of castor oil using lipase enzyme is carried out in a batch reactor at room temperature (35–40 °C). In order to reduce the cost of enzyme catalyzed reaction, water in oil emulsion and a 3:1 ratio of oil to water is selected. The concentration of enzyme in the reaction mixture is optimized. The effect of various additives like solvent and salt which can enhance the rate of reaction is studied. It is found that the glycerol has no effect on the hydrolysis of oil. The reusability of the lipase enzyme has also been tested. The yield of enzymatic hydrolysis of castor oil is compared with those of coconut oil and olive oil.     

Ester hydrolyses in reversed micelles using lipase

Lipases are enzymes which require a favourable reaction system for efficient catalysis of their hydrophobic substrates and reversed micellar environment is one such medium which offers many advantages. Hydrolytic studies of esters of paranitrophenol and glycerol using imidazole and four fungal lipases are studied in AOT/isooctane reversed micelles. The effect of water and surfactant concentration on the hydrolysis of rice bran oil is investigated and the overall potential of the reversed micellar system for hydrolytic reactions is assessed.     

Studies on localization and regulation of lipase production by Aspergillus niger

The lipase from strain BTL of Aspergillus niger was studied. The enzyme, which was mainly extracellular, was produced at elevated activity levels under optimum growth conditions. De novo biosynthesis of lipase occurred only in the presence of lipids and was completely repressed by glucose and glycerol. The reaction products, oleic acid and glycerol, showed differed inhibition patterns during triolein hydrolysis. The enzyme exhibited high specificity towards middle chain triglycerides and was possibly activated by double bonds in the fatty acid chain. It exhibited a marked stability against organic solvents.     

Solvent-free glycerolysis of sunflower oil and anchovy oil catalyzed by a 1,3-specific lipase

Solvent-free glycerolysis of sunflower oil catalyzed with lipase D Amano 100 gave the highest partial acylglycerols content at 40°C using an oil:glycerol molar ratio of 1:2 and 500 Units lipase/ g oil. After 6 h, the partial acylglycerols content of the reaction mixture was 53% (w/w). Glycerolysis of anchovy oil catalyzed under the same conditions gave a partial acylglycerols content of 47% (w/w) after 24 h.     

Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase

Acid oil, which is a by-product in vegetable oil refining, mainly contains free fatty acids (FFAs) and acylglycerols, and is a candidate of materials for production of biodiesel fuel. A mixture (acid oil model) of refined FFAs and vegetable oil was recently reported to be converted to fatty acid methyl esters (FAMEs) at >98% conversion by a two-step reaction system comprising methyl esterification of FFAs and methanolysis of acylglycerols using immobilized Candida antarctica lipase. The two-step system was thus applied to conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel. Under similar conditions that were determined by using acid oil model, however, the lipase was unstable and was not durable for repeated use. The inactivation of the lipase was successfully avoided by addition of excess amounts of methanol (MeOH) in the first-step reaction, and by addition of vegetable oil and glycerol in the second-step reaction. Hence, the first-step reaction was conducted by shaking a mixture of 66 wt% acid oil (77.9 wt% FFAs, 10.8 wt% acylglycerols) and 34 wt% MeOH with 1 wt% immobilized lipase, to convert FFAs to their methyl esters. The second-step reaction was performed by shaking a mixture of 52.3 wt% dehydrated first-step product (79.7 wt% FAMEs, 9.7 wt% acylglycerols), 42.2 wt% rapeseed oil, and 5.5 wt% MeOH using 6 wt% immobilized lipase in the presence of additional 10 wt% glycerol, to convert acylglycerols to FAMEs. The resulting product was composed of 91.1 wt% FAMEs, 0.6 wt% FFAs, 0.8 wt% triacylglycerols, 2.3 wt% diacylglycerols, and 5.2 wt% other compounds. Even though each step of reaction was repeated every 24 h by transferring the immobilized lipase to the fresh substrate mixture, the composition was maintained for >100 cycles.     

Properties of a membrane-bound triglyceride lipase of rapeseed (Brassica napus L.) cotyledons

The properties of the alkaline lipase activity (EC 3.1.1.3) that was recovered almost completely from a microsomal membrane fraction of 4-d-old rapeseed (Brassica napus L.) cotyledons were studied employing a titrimetric test procedure. The apparent K_M was 6.5 mmol l^-1, with emulgated sunflower oil as the substrate. The products of triglyceride hydrolysis in vitro were glycerol, free fatty acids, and minor amounts of mono- and diglycerides. Maximum lipase activity depended on the preincubation of the lipolytic membrane fraction in 0.15 mol l^-1 NaCl and on the presence of at least 0.1 mol l^-1 NaCl in the test mixture. Desoxycholate and up to 0.1 mol l^-1 CaCl₂ also activated the enzyme while EDTA and detergents such as trito x-100, digitonin, tween 85, and sodium dodecylsulfate were inhibitory. The rapeseed lipase displayed a conspicuous substrate selectivity among different plant triglycerides; the activity was inversely correlated with the oleic acid content of the oils. Water-soluble triacetin and the phospholipid lecithin were not hydrolyzed. Increasing amounts of free fatty acids reduced lipase activity; erucic acid, a major component of rapeseed oil, exhibited the strongest effect, suggesting a possible role in the regulation of lipase activity in vivo. The data demonstrate that the lipolytic membrane fraction houses a triglyceride lipase with properties similar to other plant and animal lipases. It can both qualitatively and quantitatively account for the fat degradation in rapeseed cotyledons. The evidence that provides further reason to acknowledge the membranous appendices of the spherosomes as the intracellular site of lipolysis is discussed.     

Specificity of a Tween-Lipase in Hydrolysis of Olive Oil

A specific lipase for the hydrolysis of Tween present in a Sclerotinia lipase preparation was fractionated by various extraction procedures, ion-change resin treatment and dialysis. The activity of the Tween-lipase on olive oil was significant in Increasing the hydrolysis of the olive oil by combination with various other lipases. Also, it seemed to develop with the change in the state of emulsion of the reaction mixture during hydrolysis.     

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%.     

Novozym 435-catalyzed 1,3-diacylglycerol preparation via esterification in t-butanol system

1,3-Diacylglycerol (1,3-DAG) oil has beneficial effects on suppressing the accumulation of body fat and preventing the increase of body weight. So, more and more attention has been paid to enzyme-mediated 1,3-DAG production in recent years due to its mild reaction condition and safe products. In this work, t-butanol was adopted as the reaction medium for lipase-catalyzed esterification for 1,3-DAG preparation. In t-butanol system, the harmful effects on lipase caused by glycerol could be eliminated completely, so the high enzymatic activity was maintained and the stability of the lipase could be improved significantly. Under the optimum conditions (60 °C, 1.00 g Novozym 435, 2.5:1 molar ratio of oleic acid to glycerol (10.0 g oleic acid and 1.3 g glycerol) and 6.0 g t-butanol), 1,3-DAG concentration of 40% was achieved and Novozym 435 can be used 100 times. A simplified model based on Ping-Pong Bi-Bi with substrate competitive inhibition by glycerol was found to fit the initial rate data and the kinetics parameters were evaluated by nonlinear regression analysis.     

Interfacial reaction dynamics and acyl-enzyme mechanism for lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters

The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.     

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.