Effects of Regioselectivity and Lipid Class Specificity of Lipases on Transesterification,Exemplified by Biodiesel Production

Lipase-catalyzed ethanolysis of triolein was studied as a model for biodiesel production. Four lipases were immobilized on porous polypropylene, and ethanolysis reactions were carried out in methyl t-butyl ether. The reaction products were analyzed using gas chromatography. Three of the four lipases studied were efficient in the conversion of triolein to 2-monoolein, but slow in the final step of producing glycerol. However, Candida antarctica lipase B was slow in the conversion of triolein, but more efficient in the subsequent two steps than the other lipases. The 1,3-selectivity of the lipases was less pronounced for the monooleins than for triolein. Silica gel was investigated as a catalyst for acyl migration, showing an increase in biodiesel yield with three of the lipases, but a reduction in yield when C. antarctica lipase B was used. The highest biodiesel yield (96 %) was obtained with a combination of Rhizopus arrhizus lipase and C. antarctica lipase B.     

Lipase-catalyzed esterification of 2-monoricinolein for 1,2(2,3)-diricinolein synthesis

The purpose of this investigation was to develop conditions for producing 2-monoricinoleoyl DAG. We used lipase-catalyzed hydrolysis of triricinolein to obtain 2-monoricinolein and thereafter synthesized 1,2(2,3)-diricinolein through esterification of 2-monoricinolein, using ricinoleic acid as the acyl donor. Five different 1,3-specific immobilized lipases were tested for the initial methanolysis reaction: Candida antarctica type B, Rhizomucor miehei, Rhizopus oryzae (ROL), Thermomyces lanuginosus, and Aspergillus niger. For the second esterification reaction, we investigated these five lipases plus Pseudomonas cepacia, Penicillium roquefortii, Candida rugosa, and Pseudomonas fluorescence. Toluene and diisopropyl ether (DIPE) were examined as reaction media at a water activity of 0.11. ROL in DIPE gave the highest yield of 2-monoricinolein from triricinolein, 78% after 3 h of reaction. The isolated 2-monoricinolein was esterified with ricinoleic acid for synthesis of 1,2(2,3)-diricinolein. ROL in DIPE gave the highest yield of 1,2(2,3)-diricinolein, 58% after 1 h of reaction, and NMR analysis showed that the purity was 97.2%. This methodology can used for synthesizing radiolabeled 1,2(2,3)-diricinolein to study lipid biosynthesis in castor and other oilseeds.     

FA selectivity of lipases in acyl-transfer reactions with acetate esters of polyols in organic media

FA reaction selectivity of Burkholderia cepacia, Rhizomucor miehei, and Candida antarctica fraction B lipases was compared between acyl-transfer and esterification reactions. Multicompetitive reaction mixtures containing a series of n-chain FA (a C₄–C₁₈ series; and a C_18∶x series, where X=0-3 double bonds) and a single acetate ester co-substrate [triacetin, 1,2-propanediol (1,2-PD)diacetate, and 1,3-PD diacetate] were studied in tert-butyl methyl ether at an a _w of 0.69. For B. cepacia lipase, FA optima for C₈, C₁₆, and C_18∶2 were observed in all reactions with 1.0- to 5.9-fold differences in FA selectivity. For R. miehei lipase, an optimum for C₈ FA was observed in all reactions with 1.2- to 6.7-fold differences in FA selectivity. For C. antarctica lipase, FA optima for C₈/C₁₀ were observed in all reactions with 1.0- to 2.8-fold differences in FA selectivity. FA selectivities were broadly modulated upon changing from free polyol to acetate ester co-substrates for B. cepacia and R miehei lipases, whereas FA selectivity modulations were more specific upon this change in reaction configuration for C. antarctica B lipase. For all lipases, reactivity toward unsaturated C_18∶x FA was enhanced in acyl-transfer relative to esterification reactions with these polyol co-substrates.     

Synergism of microwave irradiation and enzyme catalysis in synthesis of isoniazid

Isoniazid is a useful antitubercular drug widely employed in combination therapy with rifampicin. The synthesis of isoniazid from ethyl isonicotinate and hydrazine hydrate was studied in non‐aqueous media via lipase‐catalyzed hydrazinolysis under both conventional heating and microwave irradiation by using different supported lipases. Among three different commercial lipases used, namely Novozym 435 (Candida antarctica lipase), Lipozyme RM IM (Rhizomucor miehei lipase) and Lipozyme TL IM (Thermomyces lanuginosus lipase), Novozym 435 was found to be the most effective, with conversion of 54% for equimolar concentrations at 50 °C in 4 h. The rate of reaction as well as final conversion increased synergistically under microwave irradiation in comparison with conventional heating, which showed 36.4% conversion, even after 24 h, for the control experiment. Effects of various process parameters such as speed of agitation, catalyst loading, substrate concentration, product concentration and temperature were studied. A kinetic model is also described. Copyright © 2007 Society of Chemical Industry     

High‐pressure enzymatic hydrolysis of oil

The hydrolysis of sunflower and soybean oil, catalyzed by two enzymes, non‐immobilized Candida rugosa and immobilized Candida antarctica lipase, was performed at atmospheric and high‐pressure. The results showed that at atmospheric pressure between 40 °C and 60 °C initial reaction rates were influenced by the temperature variation, as expected. Due to favorable physico‐chemical properties of dense gases as reaction media, hydrolysis of soybean oil was performed in non‐conventional solvents: in supercritical (SC) CO₂ and near‐critical propane. In SC CO₂ the activity of non‐immobilized Candida rugosa lipase decreased while the reaction rates of hydrolysis catalyzed by immobilized Candida antarctica lipase were 1.5‐fold higher than at atmospheric pressure. However, the reaction rates for the hydrolyses catalyzed by both lipases, were much higher in propane than at atmospheric pressure.     

Regioselective acylation of flavonoids catalyzed by lipase in low toxicity media

Flavonoid fatty esters were prepared by acylation of flavonoids (rutin and naringin) by fatty acids (C8, C10, C12), catalyzed by immobilized lipase from Candida antarctica in various solvent systems. The reaction parameters affecting the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity (a_w) of the system, as well as the acyl donor concentration have been investigated. At optimum reaction conditions, the conversion of flavonoids was 50—60% in tert‐butanol at a_w less than 0.11. In all cases studied, only flavonoid monoester was identified, which indicates that this lipase‐catalyzed esterification is regioselective.     

Lipase-Catalyzed Synthesis of Medium-Long-Medium Type Structured Lipids Using Tricaprylin and Trilinolenin as Substrate Models

The synthesis of medium-long-medium type structured lipids (SL) by the interesterification of tricaprylin (TC) and trilinolenin (TLN), using selected commercial lipases from Rhizomucor miehei (Lipozyme RM IM) and Candida antarctica (Novozym 435) was investigated. Although the bioconversion yield (BY) for Lipozyme RM IM (24.7 %) was close to that for Novozym 435 (24.0 %), the initial enzyme activity was 6.3 μmol CLnC/g enzyme/min and 1.6 μmol CLnC/g enzyme/min, respectively. Lipozyme RM IM was subsequently selected for further investigation. The structural analyses of SL indicated that the major products were 1,3-dicapryl-2-linolenyl glycerol (CLnC) and 1(3)-capryl-2,3(1)-dilinolenyl glycerol (CLnLn). In order to optimize the BY, selected parameters were investigated. The experimental results showed that using hexane as the reaction medium, at an initial water activity (a _w ) of 0.06, 10 mg solid enzyme/mL, substrate molar ratio of TC to TLN of 6:1 and a reaction time of 9 h, resulted in the highest BY (73.2 %). Using the optimized conditions, the effects of TLN concentration and other selective parameters, including the denaturation of the enzyme, controlling the a _w and the addition of silica gel, on the mass productivity (P _M), enzymatic productivity (P _E) and volumetric productivity (P _V ) of the interesterification reaction, were also investigated.     

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.     

Lipase-catalyzed methanolysis of triricinolein in organic solvent to produce 1,2(2,3)-diricinolein

The objective of this study was to find the optimal parameters for lipase-catalyzed methanolysis of triricinolein to produce 1,2(2,3)-diricinolein. Four different immobilized lipases were tested, Candida antarctica type B (CALB), Rhizomucor miehei (RML), Pseudomonas cepacia (PCL), and Penicillium roquefortii (PRL). n-Hexane and diisopropyl ether (DIPE) were examined as reaction media at three different water activities (a _w), 0.11, 0.53, and 0.97. The consumption of triricinolein and the formation of 1,2(2,3)-diricinolein, methyl ricinoleate, and ricinoleic acid were followed for up to 48 h. PRL gave the highest yield of 1,2(2,3)-diricinolein. Moreover, this lipase showed the highest specificity for the studied reaction, i.e., high selectivity for the reaction with triricinolein but low for 1,2(2,3)-diricinolein. Recoveries of 93 and 88% DAG were obtained using PRL in DIPE at a _w of 0.11 and 0.53, respectively. Further, NMR studies showed that a higher purity of the 1,2(2,3)-isomer vs. the 1,3-isomer was achieved at higher a _w (88% at a _w=0.53), compared to lower a _w (71% at a _w=0.11). The DAG obtained was acylated by the DAG acyltransferase from Arabidopsis thaliana. Therefore, this enzymatic product is a useful enzyme substrate for lipid biosynthesis. Accordingly, the use of PRL in DIPE at a _w 0.53 is considered optimal for the synthesis of 1,2(2,3)-diricinolein from triricinolein.     

Lipase-catalyzed incorporation of conjugated linoleic acid into tricaprylin

Three commercially available immobilized lipases, Novozym 435 from Candida antarctica, Lipozyme IM from Rhizomucor miehei, and Lipase PS-C from Pseudomonas cepacia, were used as biocatalysts for the interesterification of conjugated linoleic acid (CLA) ethyl ester and tricaprylin. The reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The effects of molar ratio, enzyme load, incubation time, and temperature on CLA incorporation were investigated. Novozym 435, as compared to Lipozyme IM and Lipase PC-C, showed the highest degree of CLA incorporation into tricaprylin. By hydrolysis with pancreatic lipase, it was found that Lipozyme IM and Lipase PS-C exhibited high selectivity for the sn-1,3 position of the triacylglycerol early in the interesterification, with small extents of incorporation of CLA into the sn-2 position, probably due to acyl migration, at later reaction times. A small extent of sn-1,3 selectivity during interesterification by Novozym 435 was observed.     

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     

Characteristics of Crosslinking Polymers Play Major Roles in Improving the Stability and Catalytic Properties of Immobilized Thermomyces lanuginosus Lipase

This study aimed to improve the stability and catalytic properties of Thermomyces lanuginosus lipase (TLL) adsorbed on a hydrophobic support. At the optimized conditions (pH 5 and 25 °C without any additions), the Sips isotherm model effectively fitted the equilibrium adsorption data, indicating a monolayer and the homogenous distribution of immobilized lipase molecules. To preserve the high specific activity of adsorbed lipase, the immobilized lipase (IL) with a moderate loading amount (approximately 40% surface coverage) was selected. Polyethylenimine (PEI) and chitosan (CS) were successfully applied as bridging units to in situ crosslink the immobilized lipase molecules in IL. At the low polymer concentration (0.5%, w/w) and with 1 h incubation, insignificant changes in average pore size were detected. Short-chain PEI and CS (MW ≤ 2 kDa) efficiently improved the lipase stability, i.e., the lipase loss decreased from 40% to <2%. Notably, CS performed much better than PEI in maintaining lipase activity. IL crosslinked with CS-2 kDa showed a two- to three-fold higher rate when hydrolyzing p-nitrophenyl butyrate and a two-fold increase in the catalytic efficiency in the esterification of hexanoic acid with butanol. These in situ crosslinking strategies offer good potential for modulating the catalytic properties of TLL for a specific reaction.     

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.     

Production of Human Milk Fat Substitutes Catalyzed by a Heterologous Rhizopus oryzae Lipase and Commercial Lipases

This study aims to produce human milk fat substitutes by an acidolysis reaction between lard and the free fatty acids (FFA) from a fish oil concentrate rich in docosahexaenoic acid, in solvent-free media. The immobilized commercial lipases from (1) Rhizomucor miehei (Lipozyme RM IM), (2) Thermomyces lanuginosa (Lipozyme TL IM) and (3) Candida antarctica (Novozym 435) were tested as biocatalyst. Also, the heterologous Rhizopus oryzae lipase (rROL), immobilized in Accurel^® MP 1000, was tested as a feasible alternative to the commercial lipases. After 24 h of reaction at 50 °C, similar incorporations of polyunsaturated fatty acids (c.a. 17 mol%) were attained with Novozym 435, Lipozyme RM IM and rROL. The lowest incorporation was achieved with Lipozyme TL IM (7.2 mol%). Modeling acidolysis catalyzed by rROL and optimization of reaction conditions were performed by response surface methodology, as a function of the molar ratio FFA/lard and the temperature. The highest acidolysis activity was achieved at 40 °C at a molar ratio of 3:1, decreasing with both temperature and molar ratio. Operational stability studies for rROL in seven consecutive 24-h batches were carried out. After the fourth batch, the biocatalyst retained about 55 % of the original activity (half-life of 112 h).     

Concentration of Docosahexaenoic Acid (DHA) by Selective Alcoholysis Catalyzed by Lipases

The aim of this work was to obtain acylglycerols from tuna oil (23 % weight DHA) rich in docosahexaenoic acid (DHA) by selective ethanolysis, catalyzed by lipases. First, seven immobilized lipases were tested and the best DHA concentration and recovery in the acylglycerol fraction were attained with Lipozyme TL IM^® from Thermomyces lanuginosus, Lipozyme RM IM from Rhizomucor miehei, and lipase from Thermomyces lanuginosus immobilized on Immobead 150. As it is the cheapest, Lipozyme TL IM^® was selected to optimize the reaction conditions. The influence of temperature, reaction time, and ethanol/oil and lipase/oil ratios were studied. Under the optimized conditions (35 °C, ethanol/oil molar ratio 2.3, lipase/oil ratio 5 % weight and 48 h) and for a 56 % conversion, acylglycerols were obtained with a 45 % DHA concentration and 90 % recovery. In these optimized conditions the reaction was scaled up to 766 g of tuna oil and carried out in a batch stirred tank reactor, with the lipase contained in a cartridge filter attached to the stirring rod. The results were similar to those obtained on the smaller scale. The DHA-enriched acylglycerols were separated from the ethyl esters by evaporation of the latter in a short-path vacuum distiller, where the influence of distillation temperature was studied. At 170 °C DHA-rich acylglycerols (44 % DHA) were recovered in the residue with 94.5 % purity and 72 % recovery.     

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.     

Lipase-Catalysed Synthesis of Fatty Acid Modified Chitobiose in a Low-Water Organic Solvent: Effect of Water Distribution in a Multiphase Reaction System

Lipase-catalysed condensation of chitobiose and myristic acid in low-water acetone media was investigated. A product identified as monomyristoyl chitobiose was obtained using immobilized lipase B from Candida antarctica (Novozym 435). The product yield was significantly affected by reaction conditions, such as the initial water concentration in acetone, the initial water content of immobilized lipases, and the amount of added molecular sieves. The product yield varied in the range 0–10%. The effects of the reaction conditions are discussed in relation to the quantitative distribution of water in the reaction system, that is, water adsorbed onto immobilized enzymes, water adsorbed onto molecular sieves, and free water dissolved in the organic solvent.     

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.     

Lipase-catalyzed esterification of lactic acid with straight-chain alcohols

Enzymatic synthesis of esters of lactic acid and straight-chain alcohols with different chain lengths (C₆–C₁₈) were investigated in batch reactions with hexadecanol (C₁₆) as the model alcohol. Cyclohexane was the best solvent for higher ester yields, and the best biocatalyst was the immobilized Candida antarctica lipase B (Novozym 435) as well as the textile-immobilized Candida sp. lipase. A method was established to obtain ester yields in the range of 71 to 82% for the different alcohols, and the most favorable conditions for the esterification reaction using Novozym 435 were an equimolar ratio of lactic acid to alcohol, each at a concentration of 120 mM each; a 50°C reaction temperature; 190 rpm shaking speed; and the addition of 100 mg molecular sieves (4 Å) for drying. The ester yield increased with increasing lipase load, and a yield of 79.2% could be obtained after 24 h of reaction at 20 wt% of Novozym 435. The immobilized Candida sp. lipase prepared in the laboratory also could be used to produce esters of lactic acid and straight-chain alcohols, but it had a much lower activity than Novozym 435 with a temperature optimum of 40°C.     

Enzymatic interesterification of tallow-sunflower oil mixtures

In an effort to improve the physical and/or thermal characteristics of solid fats, the enzymatic interesterification of tallow and butterfat with high-oleic sunflower oil and soybean oil was investigated. The two simultaneously occurring reactions, interesterification and hydrolysis, were followed by high-performance liquid chromatography of altered glycerides and by gas-liquid chromatography of liberated free fatty acids. The enzymes used in these studies were immobilized lipases that included either a 1,3-acyl-selective lipase or acis-9-C₁₈-selective lipase. The degree of hydrolysis of the fat/oil mixtures was dependent upon the initial water content of the reaction medium. The extent of the interesterification reaction was dependent on the amount of enzyme employed but not on the reaction temperature over the range of 50–70°C. Changes in melting characteristics of the interesterified glyceride mixtures were followed by differential scanning calorimetry of the residual mixed glycerides after removal of free fatty acids. Interesterification of the glyceride mixes with the two types of enzymes allowed for either a decrease or increase in the solid fat content of the initial glyceride mix.