Oil-water interfacial activation of lipase for interesterification of triglyceride and fatty acid

Lipase usually has little interesterification activity in organic solvents, probably owing to the absence of an oil-water interface. Lipases were processed in a two-phase hydrocarbon-water system that had an oil-water interface. Crude lipase (from Rhizopus japonicus) in a buffer and a small volume of aliphatic hydrocarbon as an oil phase were mixed and then lyophilized to remove the aqueous and oil phases. The interfacially processed lipase has a remarkable interesterification activity in n-hexane compared to crude native lipases. We postulate that this activation is caused by the oil-water interface, i.e., the interface between hydrocarbon and water makes the lipase lid open and enables the lipase to work effectively in n-hexane. Several different hydrocarbons were investigated as an oil phase, and n-tetradecane was found to be the best for interesterification. Activated lipase was successfully inactivated in a water suspension without an oil-water interface, and the inactivated lipase could be reactivated. We demonstrated that the oil (hydrocarbon)-water interface induced reversible activation to lipase for interesterification. This work was presented at the Biocatalysis Symposium in April 2000, held at the 91st Annual Meeting and Expo of the American Oil Chemists’ Society, San Diego, CA     

Structured lipids: Lipase-catalyzed interesterification of tricaproin and trilinolein

Structured lipids were synthesized by interesterification of trilinolein and tricaproin with sn-1,3-specific (IM 60) and nonspecific (SP 435) lipases. The interesterification reaction was performed by incubating a 1:2 mole ratio of trilinolein and tricaproin in 3 mL hexane at 45°C for the IM 60 lipase from Rhizomucor miehei, and at 55°C for the SP 435 lipase from Candida antarctica. Reaction products were analyzed by reverse-phase high-performance liquid chromatography with an evaporative light-scattering detector. The fatty acids at the sn-2 position were identified after pancreatic lipase hydrolysis and analysis with a gas chromatograph. IM 60 lipase produced 53,5 mol% dicaproyllinolein (total carbon number = C33) and 22.2% monocaproyldilinolein (C45). SP 435 lipase produced 41% C33 and 18% C45. When caproic acid was used in place of tricaproin as the acyl donor, the IM 60 lipase produced 62.9% C33. The effects of variation in mole ratio, temperature, added water, solvent polarity, and time course on the interesterification reaction were also investigated. In the absence of organic solvent, IM 60 lipase produced 52.3% C33.     

Lipase/acyltransferase‐catalysed interesterification of fat blends containing n‐3 polyunsaturated fatty acids

The lipase/acyltransferase from Candida parapsilosis is an original biocatalyst that preferentially catalyses alcoholysis over hydrolysis in biphasic aqueous/organic media. In this study, the performance of the immobilised biocatalyst in the interesterification in solvent‐free media of fat blends rich in n‐3 polyunsaturated fatty acids (n‐3 PUFA) was investigated. The interesterification activity of this biocatalyst at a water activity (a_w) of 0.97 was similar to that of commercial immobilised lipases at a_w values lower than 0.5. Thus, the biocatalyst was further used at an a_w of 0.97. Response surface modelling of interesterification was carried out as a function of medium formulation, reaction temperature (55–75 °C) and time (30–120 min). Reaction media were blends of palm stearin (PS), palm kernel oil and triacylglycerols (TAG) rich in n‐3 PUFA (“EPAX 4510TG”; EPAX AS, Norway). The best results in terms of decrease in solid fat content were observed for longer reaction time (>80 min), lower temperature (55–65 °C), higher “EPAX 4510TG” content and lower PS concentration. Reactions at higher temperature led to final interesterified fat blends with lower free fatty acid contents. TAG with high equivalent carbon number (ECN) were consumed while acylglycerols of lower ECN were produced.     

Sugar ester-modified lipase for the esterification of fatty acids and long-chain alcohols

The esterification reaction of a long-chain fatty acid and a fatty alcohol with a surfactant-modified lipase in a microaqueousn-hexane system was studied. Various lipases from different sources were first modified with a surfactant of the sugar ester type to improve their dispersibility in apolar organic solvents. This enzyme modification technique converted inactive crude lipases to highly active biocatalysts for the esterification of long-chain fatty acids and fatty alcohols in a microaqueous n-hexane system. The hydrophilic-lipophilic balance value and chainlength of the fatty acid residue of the fatty acid sugar ester, used for modifying the lipases, significantly influenced the amount of precipitated lipase that was dissolved in the aqueous solution, the protein content of the lipase-surfactant complex and its esterification activity.     

Evaluation of immobilized modified lipase: Aqueous preparation and reaction studies in n-hexane

Lipase Saiken 100 (Rhizopus japonicus) and its immobilized form displayed very poor activity (hydrolysis and interesterification) in microaqueous n-hexane solutions. Enzyme modification by the addition of stearic acid or sorbitan monostearate significantly improved activity. A ceramic carrier (SM-10) was used to immobilize modified lipase Saiken (stearic acid, sorbitan monostearate, and lecithin) and was found to further enhance hydrolysis and interesterification rates in n-hexane. In addition, the biocatalysts were re-used for four consecutive batch reactions with no significant shortfall in activity. Reaction rates were also greatly affected by the total reaction water content. Careful control of the biocatalyst water content prior to use and additional reaction water were required to optimize activity and minimize hydrolytic diglyceride byproducts. Hydrolysis and interesterification reaction rates were favored with immobilized biocatalyst water contents of 6.25 and 0.43 wt% with additional reaction water contents of 600 and 20 mg/L, respectively.     

Invert emulsion as a medium for fungal lipase activity

An extracellular lipase from the fungusPythium ultimum was active in an invert [water-in-oil] emulsion consisting of 4% water emulsified into edible oils with taurocholic acid as the surfactant. The pH range for optimum lipolytic activity was 7.5–8.5, and the optimum temperature for activity was 45°C. Specific activity of the purified lipase was 919.5 μmol/min/mg protein in the invert emulsion. Water content of the invert emulsion influenced activity of the lipase differently, depending on the substrate. The rate of olive oil hydrolysis with thePythium lipase decreased with time, possibly due to inactivation of the enzyme and inhibition by free fatty acid products of the reaction. Total hydrolysis of olive oil by thePythium lipase was compared with that by lipases fromCandida rugosa andRhizopus arrhizus in the invert emulsion. Hydrolysis essentially ceased within 24 h or less for the lipases from each source. However, the addition of aqueous solution at 8 h from the beginning of incubation stimulated hydrolysis byC. rugosa andR. arrhizus lipases by 1.8-fold and 2.5-fold, respectively, but not by theP. ultimum lipase, over corresponding controls after 48 h.     

Surfactant modification of lipases for lipid interesterification and hydrolysis reactions

Commercial grade lipases from Rhizopus japonicus, R. delemar, and Rhizomucor miehei were modified by surfactant (sorbitan monostearate), and their protein recovery and interesterification, and hydrolysis activities were investigated. By repeating the lipase modification processes three times, total protein recoveries of 17–35% could be obtained. The original lipases had no interesterification activities at all; however, all modified lipases in the first process had significant interesterification activities. In the hydrolysis reactions, all modified lipases obtained from the first process showed about three times higher specific activities than the original lipases. The modified lipases obtained from the second and third processes had lower specific interesterification and hydrolysis activities than the lipases from the first one. These results suggest that the surfactant modification process is effective not only for interesterification but also lipase purification.     

Activity and stability of lipases from different sources in supercritical carbon dioxide and near‐critical propane

The stability and activity of lipases from Pseudomonas fluorescens, Rhizopus javanicus, Rhizopus niveus, porcine pancreas and Candida rugosa in a non‐solvent system at atmospheric pressure, in supercritical carbon dioxide (SC CO₂), and near‐critical propane at 100 bar and 40 °C were studied. Esterification of n‐butyric acid with ethanol and isoamyl alcohol was used as a model system. In supercritical carbon dioxide there was a great loss in activity of the examined lipases. Decreased relative activity of lipases in SC CO₂ was attributed to the interactions between CO₂ and the enzyme. The second reason for this effect was the differences in water partitioning between the enzyme and its surroundings. In contrast, the use of near‐critical propane improved the activity of lipases in the comparison to the non‐solvent system by four‐ (porcine pancreas lipase) to nine‐times (Rhizopus javanicus lipase). The use of near‐critical propane also improved the thermal stability of porcine pancreas lipase compared with the non‐solvent system. The calculated deactivation constant for esterification between butyric acid and isoamyl alcohol, catalyzed by porcine pancreas lipase, showed that there was more than twice as much inactive as active enzyme in the non‐solvent system studied whereas the ratio in propane was 1. © 2001 Society of Chemical Industry     

Enzymatic transesterification of triolein and stearic acid and solid fat content of their products

Two systems were investigated and compared as models for making margarine-type fats. Two immobilized lipases, IM60 from Rhizomucor miehei and SP435 from Candida antarctica, were used to catalyze the transesterification of triolein with stearic acid and stearic acid methyl ester, respectively, in n-hexane. The optimal reaction temperature for both enzymes was 55°C at a mole ratio of triolein to acyl donor of 1:2. Equilibria were reached at 18 h for IM60 and 24 h for SP435. Analysis of the overall yield and incorporation of fatty acid at the sn-2 position indicated that the triacylglycerol products contained 38.4 and 16.2% 18:0 for acidolysis and 34.2 and 11.3% for interesterification reactions, respectively, at the 2-position. With SP435, the softest fat was produced after 18 h of incubation, and the hardest after 30 h. For IM60 system, 18 h of incubation gave the most plastic fat.     

Interesterification of butterfat by commercial microbial lipases in a cosurfactant-free microemulsion system

Lipase-catalyzed interesterification of butterfat was carried out in a cosurfactant-free microemulsion system containing mixtures of Span 60 and Tween 60 (ICI Specialty Chemicals Altemix Inc., Brantford, Ontario, Canada) as surfactants. Four commercial lipases were used—Lipozyme 10,000L (Novo Nordisk, Copenhagen, Denmark) and N, D and MPA (Amano Pharmaceutical Co. Ltd., Nagoya, Japan). Stereospecific analyses of fractionated selected high-molecular weight triacylglycerols were performed by enzymatic deacylation with commercial pancreatic lipase, random generation ofrac-1,2-diacylglycerols by Grignard degradation, synthesis ofrac-phosphatidylcholines and a stereospecific release ofsn-1,2 diacylglycerols by phospholipase A₂. The results showed that the hydrolytic affinity of commercial lipases demonstrated an acyl-group specificity toward lower-molecular weight fatty acids C4–C14∶0. Stereospecific analyses of fatty acids of interesterified selected triacylglycerols of butterfat catalyzed by lipase N demonstrated a 46% increase in the proportion of C18∶1cis Δ⁹ at thesn-2 position, whereas those catalyzed by lipases MAP, D and Lipozyme 10,000L were enriched with C16∶0 at the same position by 21, 35 and 41%, respectively.     

Synthesis of cocoa butter equivalent by lipase-catalyzed interesterification in supercritical carbon dioxide

With supercritical carbon dixoide as a reaction medium, the syntheses of cocoa butter equivalent by interesterification with various lipases were investigated. The study showed that among those five lipases tested, lipase IM-20 from Mucor miehei was the most effective and specific in synthesizing this cocoa butter equivalent product by interesterification. The yields of cocoa butter equivalent are affected by pressure, substrate oil composition, solubility and co-solvent. The best reaction conditions were: reaction pressure at 1500 psi, triglyceride with high content of POP (P, palmitate; O, oleate) and POO, reaction medium with 5.0% water, and reaction temperature at 50°C. The major component of cocoa butter, POS (S, stearate), can be increased by 6.0% by adding a small amount of carbon dioxide. The yield and melting point of the purified cocoa butter equivalent are 53.0% and 34.3°C, respectively.     

Enzymatic Synthesis of Biodiesel from Transesterification Reactions of Vegetable Oils and Short Chain Alcohols

Biodiesel synthesis by alcoholysis of three vegetable oils (soybean, sunflower and rice bran) catalyzed by three commercial lipases (Novozym 435, Lipozyme TL-IM and Lipozyme RM-IM), and the optimization of the enzymes stability over repeated batches is described. The effects of the molar ratio of alcohol to oil and the reaction temperature with methanol, ethanol, propanol and butanol were also studied. All three enzymes displayed similar reaction kinetics with all three oils and no significant differences were observed. However, each lipase displayed the highest alcoholysis activity with a different alcohol. Novozym 435 presented higher activity in methanolysis, at a 5:1 methanol:oil molar ratio; Lipozyme TL-IM presented higher activity in ethanolysis, at a 7:1 ethanol:oil molar ratio; and Lipozyme RM-IM presented higher activity in butanolysis, at a 9:1 butanol:oil molar ratio. The optimal temperature was in the range of 30–35 °C for all lipases. The assessment of enzyme stability over repeated batches was carried out by washing the immobilized enzymes with different solvents (n-hexane, water, ethanol, or propanol) after each batch. When washing with n-hexane, approximately 90% of the enzyme activity remained after seven synthesis cycles.     

Lipase-catalyzed synthesis of structured triacylglycerides from 1,3-diacylglycerides

A new method for the lipase-catalyzed synthesis of structured TAG (ST) is described. First, sn1,3-dilaurin or-dicaprylin were enzymatically synthesized using different published methods. Next, these were esterified at the sn2-position with oleic acid or its vinyl ester using different lipases. Key to successful enzymatic synthesis of ST was the choice of a lipase with appropriate FA specificity, i.e., one that does not act on the FA already present in the sn1,3-DAG, but that at the same time exhibits high selectivity and activity toward the FA to be introduced. Reactions were performed in the presence of organic solvents or in solvent-free systems under reduced pressure. With this strategy, mixed ST containing the desired compounds 1,3-dicaprylol-2-oleyl-glycerol or 1,3-dilauroyl-2-oleyl-glycerol (CyOCy or LaOLa) were obtained at 87 and 78 mol% yield, respectively, using immobilized lipases from Burkholderia cepacia (Amano PS-D) in n-hexane at 60°C. However, regiospecific analysis with porcine pancreatic lipase indicated that in CyOCy, 25.7% caprylic acid and in LaOLa 11.1% lauric acid were located at the sn2-position. Oleic acid vinyl ester was a better acyl donor than oleic acid. Esterification of sn1,3-DAG and free oleic acid gave very low yield (<20%) of ST in a solvent system and moderate yield (>50%) in a solvent-free system under reduced pressure.     

Synthesis of (Z)-3-hexen-1-yl butyrate in hexane and solvent-free medium using Mucor miehei and Candida antarctica lipases

(Z)-3-Hexen-1-yl butyrate is an important flavor and fragrance compound as it represents the model of a natural herbaceous (green) note. Two immobilized lipases from Mucor miehei (Lipozym IM) and from Candida antarctica (Novozym 435) were investigated for their use in the synthesis of (Z)-3-hexen-1-yl butyrate by direct esterification in n-hexane. To determine optimal conditions for esterification, we examined the following parameters: temperature, amount of lipase, acid/alcohol ratio, and absence of solvent. In n-hexane, bioconversion yields reached 95 (after 4 h) and 92% (after 6 h) for, respectively, Lipozym IM [17 (w/w reactants)] and Novozym 435 [2% (w/w reactants)]. In the absence of solvent, at 60°C, Novozym 435-catalyzed esterification afforded the title compound in 80% yield. Up to 250 g (in hexane) and 160 g (without solvent) of ester were easily prepared, in a single operation, at a laboratory scale, in few hours, using 2% (w/w reactants) lipase.     

Interesterification and hydrolysis catalyzed by fatty acid‐modified lipases

Native lipases often exhibit poor interesterification activity. We previously developed a fatty acid modification method to improve the activity of lipases. In this study, we applied this fatty acid modification method to several lipases and evaluated their interesterification and hydrolytic activities. The resulting interesterification activity was strongly dependent on the modifying fatty acid used. Of the saturated fatty acids tested, stearic acid modification substantially improved the interesterification activity of three lipases. Hydrolytic activity was affected slightly by the modifying fatty acid used. Substrate specificity of the modified lipase with triglycerides was also investigated and it was found that fatty acid modification changed the substrate specificity of some lipases.     

Synthesis of structured triglycerides from peanut oil with immobilized lipase

Structured triglycerides (ST) that contain medium- and long-chain fatty acids were synthesized by lipase-catalyzed interesterification between tricaprylin and peanut oil. To select appropriate enzymes, we investigated nine commercial lipase preparations for their ability to hydrolyze pure triglycerides as well as natural oils. Three microbial lipases from Rhizomucor miehei (RML), Candida sp. (CSL), and Chromobacterium viscosum (CVL) gave good results, and immobilized preparations were used in the interesterification. RML gave the highest yields of ST (73%, 40°C), although its hydrolytic activity toward triolein was low. As the temperature was raised to 50°C, the yield of ST increased to 79%. After 120 h reaction time, remaining activities were high for CSL (71%), moderate for CVL (48%), and low for RML (20%). Parts of this paper were presented as a poster at the Biochemical Engineering Conference IX, May 1995, Davos, Switzerland.     

Acylation of glucose catalysed by lipases in supercritical carbon dioxide

The acylation of glucose with lauric acid in a reaction catalysed by two Candida lipases and a Mucor miehei lipase in supercritical carbon dioxide (SCCO₂) was investigated. A linear dependence of the reaction rate on enzyme concentration was observed. Studies on the effect of temperature on enzyme activity showed that Candida antarctica lipase remains stable at temperatures as high as 70°C. Non-immobilised Candida rugosa lipase was found to have a temperature optimum at 60°C. The acylation reaction rate depended on the initial water activity of both substrates and enzyme; the optimum was 0·75 for Candida antarctica lipase, 0·53 for Candida rugosa lipase, and between 0·3 and 0·5 for Mucor miehei lipase. Candida rugosa lipase was most active at a molar ratio of sugar: acyl donor of 1: 3, while the optimum ratio was found to increase to 1: 6 when the reaction was catalysed by Candida antarctica and Mucor miehei lipases. © 1998 SCI     

Biocatalytic Properties of Lipase from Walnut Seed (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Lipase (E.C. 3.1.1.3) from walnut seed was purified 28.6-fold with 31% yield using Sephadex G-100 gel chromatography. Olive oil served as good substrate for the enzyme. The optimum pH and temperature were 9.0 and 70 °C, respectively. The lipase was stable between 30 and 80 °C for 5 min. K _m and V _max values were determined as 48 mM and 23.06 × 10⁻³ U/min mg for triolein as substrate. Lipase activity was slightly reduced by Cu²⁺, Ca²⁺, Hg²⁺, Mn²⁺, and Ni²⁺ ions, while Mg²⁺ and Zn²⁺ had no effects. Anionic surfactant sodium dodecyl sulfate stimulated lipase activity while non-ionic surfactants Tween-80 and Triton X-100 had negligible effects on enzymatic activity. The enzyme activity was not affected by 50 mM urea and thioacetamide. Potassium ferricyanide, n-bromosuccinamide and potassium cyanide reduced the enzyme activity. The enzyme showed a good stability in organic solvents, the best result being in n-hexane (113% residual activity). The activity of dialysate was maintained approximately 80% for 1 year at −20 °C.     

Triglyceride selectivity of immobilized <Emphasis Type="Italic">Thermomyces lanuginosa</Emphasis> lipase in interesterification

The triglyceride (fatty acid) selectivity of an immobilized lipase from Thermomyces lanuginosa (Lipozyme TL IM) was investigated in lipase-catalyzed interesterification reactions between two nono-acid TG in n-hexane. Tristearin (tri-C18∶0) was used as a reference in a series of TG with saturated FA from tri-C4∶0 to tri-C20∶0, except for tri-C6∶0, and in a series of unsaturated FA from tri-C18∶1 to tri-C18∶3. The quantification was performed by HPLC, and different methods of selectivity evaluation were used. None of the methods used showed any significant differences between the performances of the lipase on the different TG, indicating that Lipozyme TL IM is nonselective toward FA or TG in the system used. A response surface design was used to investigate the influence of water activities (a _w ) and reaction temperatures on the reactivity of Lipozyme TL IM with a system of tripalmitin (tri-C16∶0) and trilaurin (tri-C12∶0) in n-hexane. An increase in temperature (40 to 60°C) was found to affect the reactivity of the lipase significantly. The reactivity of Lipozyme TL IM was unaffected by the change in a _w from 0.1130 to 0.5289. An increase in a _w only led to an increase in FFA formation.     

Synthesis of 2‐monoglycerides by alcoholysis of palm oil and tuna oil using immobilized lipases

Commercial immobilized lipases were used for the synthesis of 2‐monoglycerides (2‐MG) by alcoholysis of palm and tuna oils with ethanol in organic solvents. Several parameters were studied, i.e., the type of immobilized lipases, water activity, type of solvents and temperatures. The optimum conditions for alcoholysis of tuna oil were at a water activity of 0.43 and a temperature of 60 °C in methyl‐tert‐butyl ether for ～12 h. Although immobilized lipase preparations from Pseudomonas sp. and Candida antarctica fraction B are not 1, 3‐regiospecific enzymes, they were considered to be more suitable for the production of 2‐MG by the alcoholysis of tuna oil than the 1, 3‐regiospecific lipases (Lipozyme RM IM from Rhizomucor miehei and lipase D from Rhizopus delemar). With Pseudomonas sp. lipase a yield of up to 81% 2‐MG containing 80% PUFA (poly‐unsaturated fatty acids) from tuna oil was achieved. The optimum conditions for alcoholysis of palm oil were similar as these of tuna oil alcoholysis. However, lipase D immobilized on Accurel EP100 was used as catalyst at 40 °C with shorter reaction times (<12 h). This lead to a yield of ～60% 2‐MG containing 55.0‐55.7% oleic acid and 18.7‐21.0% linoleic acid.