Interesterification kinetics of triglycerides and fatty acids with modified lipase inn-hexane

The kinetics of lipase-catalyzed interesterification of triglycerides and fatty acids in organic media was studied. First, the lipase Saiken 100,Rhizopus japonicus, was modified by surfactant to form an enzyme precipitate in aqueous solution, which was well dispersed in organic solvents. This modified lipase catalyzed the interesterification of tripalmitin and stearic acid. The enzyme has 1,3-positional specificity and does not distinguish between stearic and palmitic acids. The kinetic model developed to describe the interesterification reaction system is based on mass balance of two consecutive second-order reversible reactions. The reaction rate constant, k, was determined by solving the differential rate equations of the reaction system and by expressing the value of k as a function of concentrations of the substrates with time. The model gave satisfactory results. The best value of the specific reaction rate constant k* that fits all experimental data was 1.2 · 10⁻⁵ [L²/(mmol · mg biocatalyst · h)] under the reaction conditions in this study.     

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.     

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.     

Functional display of Rhizomucor miehei lipase on surface of Saccharomyces cerevisiae with higher activity and its practical properties

BACKGROUND: A display system, which can translate DNA to functional peptides or proteins, is used as a new protein expression system. In this system, peptides or proteins are displayed on the cell surface as a fusion form with some anchoring proteins. Yeast cells displaying lipases on their cell‐surface could be used as whole‐cell biocatalysts. This research focuses on the functional display of Rhizomucor miehei lipase (RML) on the surface of Saccharomyces cerevisiae with higher activity. RESULTS: The lipases (RML) from R.miehei 3.4960 were of active form. The RML‐α‐agglutinin fusion proteins produced were not secreted into the culture media and were mostly immobilized on the yeast cells. Cell surface displayed lipase showed the highest activity at 45 °C and pH 8.0. CONCLUSION: The gene encoding RML from R.miehei 3.4960 can be functionally expressed on the cell surface of S. cerevisiae MT8‐1 using a glycosylphosphatidylinositol (GPI) anchor with higher activity. Copyright © 2007 Society of Chemical Industry     

Selection of surfactant-modified lipases for interesterification of triglyceride and fatty acid

Interesterification of tripalmitin and stearic acid inn-hexane was investigated with surfactant-modified lipases. Various kinds of lipases and surfactants were screened for high interesterification activity of the modified lipases. The modified-lipase hydrophile-lipophile balance value and fatty acid group of the surfactants. The modified lipase obtained fromRhizopus japonicus with sorbitan monostearate as surfactant had the highest activity in then-hexane system. The interesterification activity of the selected modified lipase in molten substrates at 75°C without solvents was the same as that in then-hexane system at 40°C.     

Enantioselective esterification reaction using immobilized Candida rugosa lipase on poly(N‐vinyl‐2‐pyrrolidone‐co‐styrene) hydrogel

Lipase from Candida rugosa was immobilized on poly(N‐vinyl‐2‐pyrrolidone‐co‐styrene) hydrogel (poly‐(VP‐co‐ST)) with ethylene dimethacrylate and α,α'‐azoisobutyronitrile, which act as crosslinker and initiator, respectively. Three different compositions of monomers were used, namely VP(%):ST(%), 10:90, 50:50, and 70:30 (wt(%)/wt(%)). The immobilized lipases were used in the enantioselective esterification of (R,S)‐2‐(4‐chlorophenoxy)‐propanoic acid with n‐tetradecanol. The optimum reaction condition of the enantioselective esterification for the native lipase and the poly(VP‐co‐ST) hydrogel immobilized lipases was determined with respect to temperature, solvents, and initial water activity (a_w). The optimum temperature obtained was 40°C, with the poly(VP‐co‐ST) hydrogel immobilized lipase VP(%)/ST(%):10:90 showing the highest enantiomeric excess. In the solvent effect studies, the best solvents for high enantioselectivity were chloroform and carbon tetrachloride. In the a_w studies, optimum α_w for NL, VP(%):ST(%), 10:90, and 50:50 was 0.328, while for VP(%):ST(%), 70:30, it was 0.55. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3381–3386, 2004     

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.     

Modification of Fats by Lipase Interesterification I: Changes in Glyceride Structure

Blends of refined cottonseed oil and fully hydrogenated soybean oil were subjected to interesterification reactions catalyzed by Rhizomucor miehei and Candida antartica lipases in a solvent free system. The interesterification decreased the levels of triunsaturated and trisaturated triglycerides and increased the amounts of mono- and disaturated triglycerides in the blends. The triglyceride products obtained by the two enzyme preparations were similar in composition but differed in the proportions: levels of high melting glycerides were higher in the products obtained with C. antartica lipase. The amounts of hydrolysis products formed depended on both the choice of enzyme and the substrate composition. The content of 1,3-diglycerides formed exceeded that of 1,2-diglycerides and low levels of monoglycerides were formed during the reactions.     

Comparison of acyl donors for lipase-catalyzed production of 1,3-dicapryloyl-2-eicosapentaenoylglycerol

Synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol (CEC) catalyzed by Lipozyme IM (immobilized Rhizomucor miehei lipase) was performed by interesterification of trieicosapentaenoylglycerol (EEE) with caprylic acid (CA) (acidolysis) and EEE with ethyl caprylate (EtC) (interesterification). Both methods involved two steps: (i) transesterification at an optimized water content and temperature for the high yield conversion of the substrate to CEC, 1-capryloyl-2-eicosapentaenoylglycerol (CEOH) and 2-eicosapentaenoylglycerol (OHEOH), and (ii) reesterification of CEOH and OHEOH to CEC by water removal under reduced pressure. Interesterification had clear advantages over acidolysis. The reaction rates for interesterification were higher and the reaction times shorter. The final yield of CEC by interesterification was higher, and the extent of acyl migration, indicated by the tricapryloylglycerol content, was lower. The disadvantage of the higher price of EtC used for interesterification (approximately 10 times higher than the price of CA) was overcome by synthesizing it directly in the same reaction vessel prior to the interesterification step. EtC was rapidly synthesized by esterification of CA with ethanol in high yield (92% obtained in 2.5 h). The amount of water added to the reaction mixture and the reaction temperature influenced the yields of CEC, CEOH, and OHEOH in the transesterification step for both interesterification and acidolysis methods. The regioisomeric purity of CEC was 100% for both methods at temperatures of 40°C or less. The highest yield of CEC (81%) was obtained for the interesterification of EEE with EtC, formed directly in the same reaction vessel, at a CA/EEE molar ratio of 20∶1 and 30°C.     

Lipase activity and fatty acid typoselectivities of plant extracts in hydrolysis and interesterification

Lipase fatty acid typoselectivities of Euphorbia characias latex and commercially available crude preparation of bromelain were determined in the hydrolysis of homogeneous triacylglycerols (TAG) and natural TAG mixtures. Their activities were compared to a commercially available crude preparation of papain. Under optimal lipolysis conditions at pH 8.0 and 10 min of incubation time, maximal activities were observed at 45, 55, and 50°C, respectively, for E. characias latex, crude bromelain, and crude papain. Commercially available crude preparations of bromelain exhibited very poor hydrolysis activity. Latex from E. characias, which contained 340 mg of dried material per milliliter of fresh latex, exhibited a high lipase activity and a short-chain fatty acid preference in the hydrolysis of homogeneous TAG. For all substrates, it showed a better activity than crude papain. Lipase fatty acid typoselectivities of crude bromelain and crude papain also were studied in interesterification reactions of tributyrin with a series of homogeneous TAG. Experiments showed that crude bromelain [water activity (A _w )∶ 0.21] had no activity in interesterification. Regarding reactions with crude papain (A _w ∶ 0.55), yields of newly formed TAG decreased with increasing chain length of TAG, except for the reaction with trimargarin. For interesterification of tributyrin with unsaturated TAG, triolein reacted faster than polyunsaturated TAG. During these interesterification reactions, the proportion of new TAG with two butyroyl residues was higher than new TAG with only one butyroyl residue. This phenomenon was more pronounced for reactions with long-chain TAG.     

Enzymatic preparation of enantiomerically pure sn-2,3-diacylglycerols: A stereoselective ethanolysis approach

Stereoselective ethanolysis of monoacid TAG by immobilized Rhizomucor miehei lipase (RML) was studied for preparation of optically pure sn-2,3-DAG. Trioctanoylglycerol (TO) was used as a model substrate. The enantiomeric purity of the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very high (percent enantiomeric excess >99%) when an excess of ethanol was used. The result indicated that RML was highly stereoselective toward the sn-1 position of TO under conditions of excess ethanol. The stereoselectivity of RML depended on the amount of ethanol. The larger the amount of ethanol was, the higher the stereoselectivity became. After optimizing the parameters such as reactant molar ratio, water content, and temperature, (ethanol/TO molar ratio =31∶1 and water content =7.5 wt% of the reactants at 25°C), optically pure sn-2,3-DO was obtained at 61.1 mol% in the glyceride fraction in 20 min. The above conditions were further applied for ethanolysis of monoacid TAG with different acyl groups such as tridecanoylglycerol (C10∶0), tridodecanoylglycerol (C12∶0), tritetradecanoylglycerol (C14∶0) and trioctadecenoylglycerol [triolein, (C18∶1)]. The yields and enantiomeric purities of 1,2(2,3)-DAG were dramatically reduced when TAG with FA longer than decanoic acid were used.     

Lipase-catalyzed synthesis of monoacylglycerides by glycerolysis of campher tree seed oil and cocoa-butter

The lipase-catalyzed glycerolysis of Campher tree seed oil and Cocoa-butter in a solid-phase system was studied. Lipases from Pseudomonas cepacia (PCL) and Chromobacterium viscosum (CVL) were considered as suitable for the synthesis of monoacylglycerols (MAG). The glycerolysis of Campher tree seed oil was performed initially at 25°C followed by cooling to 7°C. This resulted in 86% (PCL) and 90% (CVL) MAG. Maximum concentration of MAG from Cocoa-butter was 89% with both lipases, when the reaction was performed at 25°C. Isolation of MAG was performed by extraction with chloroform at 4°C followed by purification via silica gel chromatography.     

Triglyceride interesterification by lipases. 1. Cocoa butter equivalents from a fraction of palm oil

Twelve commercially available triacylglycerol lipase preparations were screened for their suitability as catalysts in the interesterification of palm oil mid fraction and ethyl stearate to form a cocoa butter equivalent. Five fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipase preparations was tested with sunflower seed oil and was independent of their interesterification activity. The operational stability of three of the preparations most suited for production of cocoa butter equivalents was examined. The amount of a commercial lipase preparation loaded onto a support was surveyed for optimum short-term catalytic activity. The influence of solvent concentration on the reaction rate and the purity of the product was examined at two temperatures. The optimum solvent concentration at 40°C was 1–1.5 grams of solvent/gram of substrate; at 60°C, the rate of interesterification diminished and the purity of the product decreased with increasing amounts of solvent. Four of the commercial lipase preparations found to be suitable interesterification catalysts were immobilized on five supports and their ability to catalyze the interesterification of a triglyceride and palmitic acid or ethyl palmitate was measured. The choice of support and substrate form (esterified or free fatty acid) greatly affected the catalytic activity. Some preparations were more affected by the choice of support, others by the form of the substrate. No preparation yielded maximum activity on all supports, and no support was found which produced an immobilized enzyme preparation of high activity with every commercial lipase preparation. Caution is advised in transferring observations about the suitability of a support from tests on one commerical enzyme preparation to others; individual testing is required.     

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.     

Application of palm olein in the production of zero‐trans Iranian vanaspati through enzymatic interesterification

The utilization of palm olein in the production of zero‐trans Iranian vanaspati through enzymatic interesterification was studied. Vanaspati fat was made from ternary blends of palm olein (POL), low‐erucic acid rapeseed oil (RSO) and sunflower oil (SFO) through direct interesterification of the blends or by blending interesterified POL with RSO and SFO. The slip melting point (SMP), the solid fat content (SFC) at 10–40 °C, the carbon number (CN) triacylglycerol (TAG) composition, the induction period (IP) of oxidation at 120 °C (IP₁₂₀) and the IP of crystallization at 20 °C of the final products and non‐interesterified blends were evaluated. Results indicated that all the final products had higher SMP, SFC, IP of crystallization and CN 48 TAG (trisaturated TAG), and lower IP₁₂₀, than their non‐interesterified blends. However, SMP, SFC, IP₁₂₀, IP of crystallization and CN 48 TAG were higher for fats prepared by blending interesterified POL with RSO and SFO. A comparison between the SFC at 20–30 °C of the final products and those of a commercial low‐trans Iranian vanaspati showed that the least saturated fatty acid content necessary to achieve a zero‐trans fat suitable for use as Iranian vanaspati was 37.2% for directly interesterified blends and 28.8% for fats prepared by blending interesterified POL with liquid oils.     

Enzymatic interesterification of palm stearin and palm kernel olein

Palm stearin (POs) and palm kernel olein (PKOo) blends were modified by enzymatic interesterification (IE) to achieve the physical properties of margarine fats. POs and PKOo are both products of the palm oil industry that presently have limited use. Rhizomucor miehei lipase (Lipozyme IM 60) was used to catalyze the interesterification of oil blends at 60°C. The progress of interesterification was monitored by following changes in triacylglyceride composition. At 60°C interesterification can be completed in 5 h. Degrees of hydrolysis obtained through IE for all blends were decreased from 2.9 to 2.0 by use of dry molecular sieves. The solid fat contents of POs/PKOo 30:70 and 70:30 interesterified blends were 9.6 and 18.1 at 20°C, and 0 and 4.1 at 35°C, respectively. The slip melting point (SMP) of POs/PKOo 30:70 was 40.0°C before interesterification and 29.9°C after IE. For POs/PKOs 70:30, SMP was 47.7 before and 37.5°C after IE. These thermal characteristics of interesterified POs/PKOo blend ratios from 30:70 to 70:30 were comparable to those of commercial margarines. Results showed that IE was effective in producing solid fats with less than 0.5% trans.     

Modification of Stearidonic Acid Soybean Oil by Immobilized Rhizomucor miehei Lipase to Incorporate Caprylic Acid

Immobilized sn-1,3 specific Rhizomucor miehei lipase (RML) was used to catalyze the incorporation of caprylic acid (C8:0) into high stearidonic acid (SDA, C18:4ω3) soybean oil (SDASO) to form structured lipids (SL). The effects of type of biocatalyst (Celite-, octyl-Sepharose-, and Duolite-immobilized RML) and reaction temperature (30, 40, 50, and 60 °C) on acidolysis and acyl migration were studied. Celite-immobilized RML (C-RML) at 50 °C maximized C8:0 incorporation and minimized acyl migration compared to other treatments. Optimal levels of substrate molar ratio (C8:0 to SDASO), incubation time, and enzyme load for SL synthesis by C-RML at 50 °C was determined by response surface methodology to be 6:1, 24 h, and 20 % weight of substrates, respectively. This optimum treatment was scaled-up in hexane or solvent-free reaction media using SDASO or an SDA-enriched acylglycerol mixture as substrate. This yielded various SL with C8:0 contents ranging from 17.0 to 32.5 mol% and SDA contents ranging from 20.6 to 42.3 mol%. When digested, these SL may deliver C8:0 for quick energy and SDA for heart health making them potentially valuable for medical and nutraceutical applications.     

Utilization of high-melting palm stearin in lipase-catalyzed interesterification with liquid oils

Palm stearin with a melting point (m.p.) of 49.8°C was fractionated from acetone to produce a low-melting palm stearin (m.p.=35°C) and a higher-melting palm stearin (HMPS, m.p.=58°C) fraction. HMPS was modified by interesterification with 60% (by weight) of individual liquid oils from sunflower, soybean, and rice bran by means of Mucor miehei lipase. The interesterified products were evaluated for m.p., solid fat content, and carbon number glyceride composition. When HMPS was interesterified individually with sunflower, soybean or rice bran at the 60% level, the m.p. of the interesterified products were 37.5, 38.9, and 39.6°C, respectively. The solid fat content of the interesterified products were 30–35 at 10°C, 17–19 at 20°C, and 6–10 at 30°C, respectively. The carbon number glyceride compositions also changed significantly. C₄₈ and C₅₄ glycerides decreased remarkably with a corresponding increase of the C₅₀ and C₅₂ glycerides. All these interesterified products were suitable for use as trans acid-free and polyunsaturated fatty acid-rich shortening and margarine fat bases.     

Limit of the solid fat content modification of butter

Three ways have been undertaken to modify solid fat content of butter oil: (i) interesterification, (ii) adjunction of high-melting glycerides and (iii) joint effect of adjunction of high-melting glycerides and interesterification. A solvent-free interesterification, carried out with 1,3-specific lipase fromMucor miehei, resulted in an increase of the solid fat content (SFC) by about 114% after 48 h of interesterification. The changes in triglyceride composition induced by this method were followed by quantitative determination of triglycerides of different equivalent carbon number (ECN) and different theoretical carbon number. The major changes in the triglyceride composition occurred mainly in the concentration of three groups of triglycerides with the same ECN (ECN=38). Adding high-melting glycerides trimyristin (MMM) and tripalmitin (PPP) led to an increase of the SFC measured at 20°C as these proportions increased in the mixture. The joint effect of the addition of MMM or PPP and interesterification was quite significant, mainly for triglycerides that included myristic and palmitic acids. As far as the increase of SFC is concerned, the effect of interesterification decreases when both substrate amounts increase.     

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

The aim of this work was to investigate the catalytic functions of a new immobilized Thermomyces lanuginosa lipase in interesterification and to optimize the conditions of interesterification for the production of human milk fat substitutes (HMFS) containing n−3 PUFA by response surface methodology (RSM). Thermomyces lanuginosa lipase had an activity similar to that of immobilized Rhizomucor miehei lipase (Lipozyme RM IM) in the glycerolysis of sunflower oil, but the former had higher activity at a low reaction temperature (5°C). Thermomyces lanuginosa lipase was found to have much lower catalytic activity than Lipozyme RM IM in the acidolysis of sunflower oil with caprylic acid. However, the activity of T. lanuginosa lipase was only slightly lower than that of Lipozyme RM IM in the ester-ester exchange between tripalmitin (PPP) and the ethyl esters of EPA and DHA (EE). For this reason, the new immobilized T. lanuginosa lipase was used to produce HMFS from PPP by interesterification with EE. The optimization of major parameters was conducted with the assistante molar ratio of 5 (EE/PPP), a lipase load of 20 wt% (on substrates), and a reaction time of 20 h, with acyl incorporation up to 42%. The model generated significantly represented real relationships between the response (incorporation) and reaction parameters.