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     

Lipase-catalyzed esterification of citronellol with lauric acid in supercritical carbon dioxide/co-solvent media

Direct esterification of citronellol and lauric acid catalyzed by immobilized lipase B from Candida antarctica was performed in supercritical carbon dioxide with different organic solvents and ionic liquids serving as co-solvents. The highest concentration of citronellol laurate after 1 h of reaction performance (3.95 mmol/g substrates) was obtained in SC CO₂ with ethyl methylketone serving as a co-solvent. The optimal temperature and pressure for citronellol laurate synthesis in SC CO₂/EMK medium was determined to be 60 °C and 10 MPa.     

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     

Optimization of the chemoenzymatic epoxidation of soybean oil

The lipase Candida antarctica (Novozyme 435) immobilized on acrylic resin was used as an unconventional catalyst for in situ epoxidation of soybean oil. The reactions were carried out in toluene. The peracid used for converting TG double bonds to oxirane groups was formed by reaction of FFA and hydrogen peroxide. The reaction conditions were optimized by varying the lipase concentration, solvent concentration, molar ratio of hydrogen peroxide to double bond, oleic acid concentration, and reaction temperature. The kinetic study showed that 100% conversion of double bonds to epoxides can be obtained after 4 h. The addition of free acids was not required for the reaction to proceed to conversions exceeding 80%, presumably owing to generation of FFA by hydrolysis of soybean oil. The enzyme catalyst was found to deteriorate after repeated runs.     

Lipase-catalyzed randomization of fats and oils in flowing supercritical carbon dioxide

Enzymes can frequently impart more selectivity to a reaction than chemical catalysts. In addition, the use of enzymes can reduce side reactions and simplify post-reaction separation problems. In combination with an environmentally benign and safe medium, such as supercritical carbon dioxide (SC-CO₂), enzymatic catalysis makes supercritical fluids extremely attractive to the food industry. In this study, randomization of fats and oils was accomplished with an immobilized lipase in flowing SC-CO₂. Triglycerides, adsorbed onto Celite, are solubilized in CO₂ and carried over 1–10 g immobilized lipase derived from Candida antarctica. The degree of randomization and rate of triglyceride throughput could be controlled by CO₂ pressure and flow rate and quantity of enzyme used. The dropping points and solid fat indices of the resulting randomized oils were compared to oils that were randomized by conventional methods with sodium methoxide. Reversed-phase high-performance chromatography with flame-ionization detection was used to quantitate changes in triglyceride composition of various substrates, such as palm olein and high-stearate soybean oil. The resultant randomized oil mixtures have properties, e.g., solid fat index, that make them potential candidates for incorporation into traditional margarine formulations.     

Enzymatic Production of Fatty Acid Methyl Esters by Hydrolysis of Acid Oil Followed by Esterification

Acid oil, a by-product of vegetable oil refining, was enzymatically converted to fatty acid methyl esters (FAME). Acid oil contained free fatty acids (FFA), acylglycerols, and lipophilic compounds. First, acylglycerols (11 wt%) were hydrolyzed at 30 °C by 20 units Candida rugosa lipase/g-mixture with 40 wt% water. The resulting oil layer containing 92 wt% FFA was used for the next reaction, methyl esterification of FFA to FAME by immobilized Candida antarctica lipase. A mixture of 66 wt% oil layer and 34 wt% methanol (5 mol for FFA) were shaken at 30 °C with 1.0 wt% lipase. The degree of esterification reached 96% after 24 h. The resulting reaction mixture was then dehydrated and subjected to the second esterification that was conducted with 2.2 wt% methanol (5 mol for residual FFA) and 1.0 wt% immobilized lipase. The degree of esterification of residual FFA reached 44%. The degree increased successfully to 72% (total degree of esterification 99%) by conducting the reaction in the presence of 10 wt% glycerol, because water in the oil layer was attracted to the glycerol layer. Over 98% of total esterification was maintained, even though the first and the second esterification reactions were repeated every 24 h for 40 days. The enzymatic process comprising hydrolysis and methyl esterification produced an oil containing 91 wt% FAME, 1 wt% FFA, 1 wt% acylglycerols, and 7 wt% lipophilic compounds.     

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.     

Effect of co‐solvent addition on the reaction kinetics of the lipase‐catalyzed resolution of ibuprofen ester

BACKGROUND: The addition of co‐solvent is not limited to enhancing the catalytic rate, it could also assist in situ racemization in the dynamic kinetic resolution of racemic compounds by increasing the reactivity of the base catalyst employed. In the current work, reaction media with the presence of DMSO were investigated in Candida rugosa lipase (EC 3.1.1.3)‐catalyzed hydrolysis of ibuprofen ester that focuses on the thermodynamic effect, reaction stability and implication for the kinetic parameters. RESULTS: The introduction of 2% DMSO increased the reaction rate, conversion, and enantioselectivity of the Candida rugosa lipase‐mediated resolution. However, the performance of the particular enzymatic reaction was reduced when a higher DMSO concentration was added. At lower reaction temperatures, the medium with 2% DMSO exhibited an increase in enantioselectivity, which was attributed to a higher activation energy difference between the fast‐ and slow‐reacting enantiomers compared with the water‐isooctane medium. Additionally, the presence of DMSO had a significant effect on the kinetic parameters, shown by a lower value of Michaelis constant compared with that of a normal reaction without DMSO, which resulted in a fast reaction rate. Finally, inhibition due to the uncompetitive substrate inhibitor was reduced, while the non‐competitive product inhibitor consequently increased. CONCLUSION: This work has demonstrated that only 2% of DMSO can be tolerated by the free Candida rugosa lipase in the resolution of ibuprofen ester. However, it is still able to give significant positive effects on the hydrolysis rate, kinetic parameters and enantioselectivity as well as reaction stability. © 2012 Society of Chemical Industry     

Lipase-catalyzed hydrolysis of palm oil

The hydrolysis of palm oil, palm olein and palm stearin, soybean oil, corn oil and peanut oil by the commercial lipase fromCandida rugosa (formerly known asC. cylindracea) was studied. The optimal conditions for the hydrolysis of palm oil by the lipase were established. The lipase fromC. rugosa exhibits an optimal activity at 37 C and at pH 7.5. The optimal oil to hexane ratio is 1 g of oil to 0.5 ml hexane. The rate of hydrolysis of palm oil by the lipase is linear on a logarithmic scale. Under the same conditions, palm oil and palm olein were hydrolyzed at the same rate, whereas palm stearin was hydrolyzed much more slowly.     

Fully Enzymatic Resolution of Chiral Amines: Acylation and Deacylation in the Presence of Candida antarctica Lipase B

A fully enzymatic methodology for the resolution of chiral amines has been demonstrated. Candida antarctica lipase B (CaLB)‐catalyzed acylation with N‐methyl‐ and N‐phenylglycine, as well as analogues having the general formula R¹ X CH₂CO₂R² (R¹=Me, Ph; X=O, S) afforded the corresponding enantioenriched amides, which were subsequently enzymatically hydrolyzed. Surprisingly, CaLB also proved to be the catalyst of choice for this latter step. The heteroatom in the acyl donor profoundly influences both the enzymatic acylation and deacylation; the O‐substituted reagents performed best with regard to enantioselectivity as well as reaction rate in synthesis and hydrolysis.     

Activation of lipase by sol–gel coating with hydrophobic alkyl‐substituted silicates in supercritical carbon dioxide

BACKGROUND: Sol–gel entrapment of lipases is usually performed in an aqueous solution. A novel method of sol–gel coating of lipase in supercritical carbon dioxide (SC‐CO₂) is proposed. RESULTS: Crude lipase powder (Rhizopus oryzae) coated with hydrophobic silicates, derived from dimethyldimethoxysilane and tetramethoxysilane in SC‐CO₂ at 35 °C and 15 MPa, exhibited 5–7 times higher esterification activity than that prepared via an aqueous sol–gel route. Lipase immobilized in a methyl‐substituted silica monolith was also highly activated by sol–gel coating using the same silica precursors in SC‐CO₂. CONCLUSION: Sol– gel coating in SC‐CO₂, of lipases in powder and immobilized forms with hydrophobic alkyl‐substituted silicates provides an efficient tool for the enhancement of enzymatic activities in non‐aqueous media. Copyright © 2009 Society of Chemical Industry     

Characterization of Candida rugosa Lipase Immobilized on Poly(N‐methylolacrylamide) and Its Application in Butyl Butyrate Synthesis

Candida rugosa lipase was immobilized on poly(N‐methylolacrylamide) by physical adsorption. The biocatalyst performance (immobilized lipase) was evaluated in both aqueous (hydrolysis) and organic (butyl butyrate synthesis) media. In the first case, a comparative study between free and immobilized derivatives was provided in terms of pH, temperature and thermal stability following the olive oil hydrolysis, establishing new optimum values. In the second case, the influence of temperature, biocatalyst concentration and acid/alcohol molar ratio was simultaneously studied according to a 2³ full experimental design. The highest molar conversion (96 %), volumetric productivity (1.73 g L^–1 h^–1) and specific esterification activity (1.00 μM mg^–1 min^–1) were obtained when working at the lowest level of temperature and butyric acid in excess. Under these conditions, repeated batch use of the immobilized enzyme was performed and half‐life time (t_1/2) was found to be 145 h.     

Studies on the enzymatic synthesis of lipophilic derivatives of natural antioxidants

The esterification of some natural antioxidants such as cinnamic acid derivatives and ascorbic acid in non-aqueous media, catalyzed by immobilized lipases from Candida antarctica and Rhizomucor miehei, was investigated. The alcohol chain length affected the rate of esterification of cinnamic acids by both lipases. Higher reaction rates were observed when the esterification was carried out with medium- or long-chain alcohols. The rate also depended on aromatic acid structure. The reactivity of the carboxylic function of the cinnamic acids was affected by electron-donating substituents in the aromatic ring. Higher yields were observed for the esterification of p-hydroxyphenylacetic acid (97%) catalyzed by C. antarctica lipase and for the esterification of cinnamic acid (59%) catalyzed by R. miehei lipase. Candida antarctica lipase was more suitable for producing ascorbic acid fatty esters, catalyzing with a relatively high yield (up to 65% within 24 h) the regioselective esterification of ascorbic acid with various fatty acids in 2-methyl-2-propanol. The reaction rate and yield depended on the fatty acid chain length and on the molar ratio of reactants. All ascorbic acid fatty esters produced by this procedure exhibited a significant antioxidant activity in a micellar substrate composed of linoleic acid.     

Lipase-Catalyzed Production of Biodiesel by Hydrolysis of Waste Cooking Oil Followed by Esterification of Free Fatty Acids

Biodiesel is conventionally produced by alkaline‐catalyzed transesterification, which requires high‐purity oils. However, low‐quality oils can be used as feedstocks for the production of biodiesel by enzyme‐catalyzed reactions. The use of enzymes has several advantages, such as the absence of saponification side reactions, production of high‐purity glycerol co‐product, and low‐cost downstream processing. In this work, biodiesel was produced from lipase‐catalyzed hydrolysis of waste cooking oil (WCO) followed by esterification of the hydrolyzed WCO (HWCO). The hydrolysis of acylglycerols was carried out at 30 °C in salt‐free water (WCO/water ratio of 1:4, v/v) and the esterification of HWCO was carried out at 40 °C with ethanol in a solvent‐free medium (HWCO/ethanol molar ratio of 1:7). The hydrolysis and esterification steps were carried out using immobilized Thermomyces lanuginosus lipase (TLL/WCO ratio of 1:5.6, w/w) and immobilized Candida antarctica lipase B (10 wt%, CALB/HWCO) as biocatalysts, respectively. The hydrolysis of acylglycerols was almost complete after 12 h (ca. 94 %), and in the esterification step, the conversion was around 90 % after 6 h. The purified biodiesel had 91.8 wt% of fatty acid ethyl esters, 0.53 wt% of acylglycerols, 0.003 wt% of free glycerol, viscosity of 4.59 cP, and acid value of 10.88 mg KOH/g. Reuse hydrolysis and esterification assays showed that the immobilized enzymes could be recycled five times in 10‐h batches, under the conditions described above. TLL was greatly inactivated under the assay conditions, whereas CALB remained fully active. The results showed that WCO is a promising feedstock for use in the production of biodiesel.     

γ-Linolenic acid-rich triacylglycerols derived from borage oil via lipase-catalyzed reactions

γ-Linolenic acid (GLA), a precursor of arachidonic acid, possesses physiological functions of modulating immune and inflammatory response. Highly purified GLA is desired both as a medicine and as an ingredient of cosmetics. In this work, urea fractionation and lipase-catalyzed reactions were employed for the enrichment of GLA in borage oil. GLA content in free fatty acids from saponified borage oil can be increased from 23.6 to 94% by the method of urea fractionation. Partial hydrolysis of borage oil catalyzed by immobilized Candida rugosa lipase raises GLA content in the unhydrolyzed acylglycerols from 23.6 to 52.1%. The IM-60 catalyzed acidolysis reaction between the GLA-rich free fatty acid and the unhydrolyzed acylglycerols increases the GLA content in the acylglycerols from 52.1 to 75%. The acylglycerols in the reaction product contains ca. 90% triacylglycerol. The effects of temperature, water content, substrate weight ratio, and organic solvents on the GLA content in the acylglycerols were examined.     

The incorporation of n-3 polyunsaturated fatty acids into acylglycerols of borage oil via lipase-catalyzed reactions

The purpose of this work was to add n-3 polyunsaturated fatty acids (PUFA) into the acylglycerols of borage oil. The acidolysis reaction between borage oil and n-3 PUFA was carried out with lipase (Lipozyme IM-60) in organic solvent. The effects of temperature, solvent, and water content on the reaction product were investigated. For the acidolysis reaction between acylglycerols (product of the selective hydrolysis of borage oil, catalyzed by immobilized Candida rugosa lipase) and n-3 PUFA, the total content of n-3 and n-6 PUFA in acylglycerols was 72.8% after a reaction time of 18 h. The contents of γ-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid were 26.5, 19.8, and 18.1%, respectively. By properly controlling the reaction time, acylglycerols with ca. 70–72% PUFA and a ratio of n-3 PUFA to n-6 PUFA from 0–1.09 can be obtained.     

The Influence of Supercritical Carbon Dioxide (SC‐CO2) on Electrolytes and Hydrogenation of Soybean Oil

The electrochemical hydrogenation of soybean oil with supercritical carbon dioxide (SC‐CO₂) has been studied to seek ways for substantial reduction of the trans fatty acids (TFA). The solubility of CO₂ in electrolytes and the conductivity of electrolytes were investigated using a self‐made electrochemical hydrogenation reactor. The optimum hydrogenation parameters were assessed. Both the solubility of CO₂ in electrolytes and the conductivity of electrolytes increased with increasing CO₂ pressure. When the pressure reached a critical point of CO₂, the solubility of CO₂ expressed as a mole fraction was 0.42 in cathode electrolyte and 0.1 in anode electrolyte. At 8 MPa, the conductivity of electrolytes was 1.5 times higher than that at 2 MPa. When the pressure was higher than the critical point of CO₂, the solubility of CO₂ in electrolytes and the conductivity of electrolytes reached a stable value. The optimum condition for electrochemical hydrogenation of soybean oil in SC‐CO₂ were reaction pressure (8 MPa), reaction temperature (48 °C), current (125 mA), agitation speed (300 rpm), and reaction time (8 h). Fatty acid profile, iodine value, and TFA content were evaluated at the optimum parameters. This investigation showed that the electrochemical hydrogenation of soybean oil in SC‐CO₂ was improved. The reaction time was shortened by 4 h, and TFA content was reduced by 35.8% compared to traditional hydrogenation process.     

Enzymatic glycerolysis and transesterification of vegetable oil for enhanced production of feruloylated glycerols

Novel functional groups can be introduced into vegetable oils using enzymes, resulting in value-added products. The transesterification kinetics of ethyl ferulate with MAG, DAG, and TAG were examined. Transesterification was catalyzed by immobilized Candida antarctica lipase B in solventless batch and packed-bed reactors. Initial reaction rates with TAG were slightly sensitive to water activity, whereas rates with MAG and DAG were water activity independent. Transesterification was also three-to sixfold faster with MAG and DAG. These observations indicate that the reaction is rate limited by the acyl acceptor, and that oils with free hydroxyl groups are preferred acyl acceptors in comparison with TAG, which must undergo partial hydrolysis before becoming reactive.     

Production of structured TAG rich in 1,3-dicapryloyl-2-γ-linolenoyl glycerol from borage oil

γ-Linolenic acid (GLA) has the physiological functions of modulating immune and inflammatory responses. We produced structured TAG rich in 1,3-dicapryloyl-2-γ-linolenoyl glycerol (CGC) from GLA-rich oil (GLA45 oil; GLA content, 45.4 wt%), which was prepared by hydrolysis of borage oil with Candida rugosa lipase having weak activity on GLA. A mixture of GLA45 oil/caprylic acid (CA) (1∶2, w/w) was continuously fed into a fixed-bed bioreactor (18×180 mm) packed with 15 g immobilized Rhizopus oryzae lipase at 30°C, and a flow rate of 4 g/h. The acidolysis proceeded efficiently, and a significant decrease of lipase activity was not observed in full-time operation for 1 mon. GLA45 oil contained 10.2 mol% MAG and 27.2 mol% DAG. However, the reaction converted the partial acylglycerols to structured TAG and tricaprylin and produced 44.5 mol% CGC based on the content of total acylglycerols. Not only FFA in the reaction mixture but also part of the tricaprylin and partial acylglycerols were removed by molecular distillation. The distillation resulted in an increase of the CGC content in the purified product to 52.6 mol%. The results showed that CGC-rich structured TAG can efficiently be produced by a two-step process comprising selective hydrolysis of borage oil using C. rugosa lipase (first step) and acidolysis of the resulting GLA-rich oil with CA using immobilized R. oryzae lipase (second step).     

Selectivity of lipases: isolation of fatty acids from castor,coriander, and meadowfoam oils

The lipase‐catalyzed hydrolysis of castor, coriander, and meadowfoam oils was studied in a two‐phase water/oil system. The lipases from Candida rugosa and Pseudomonas cepacia released all fatty acids from the triglycerides randomly, with the exception of castor oil. In the latter case, the P. cepacia lipase discriminated against ricinoleic acid. The lipase from Geotrichum candidum discriminated against unsaturated acids having the double bond located at the Δ‐6 (petroselinic acid in coriander oil) and Δ‐5 (meadowfoam oil) position or with a hydroxy substituent (ricinoleic acid). The expression of the selectivities of the G. candidum lipase was most pronounced in lipase‐catalyzed esterification reactions, which was exploited as part of a two‐step process to prepare highly concentrated fractions of the acids. In the first step the oils were hydrolyzed to their respective free fatty acids, in the second step a selective lipase was used to catalyze esterification of the acids with 1‐butanol. This resulted in an enrichment of the targeted acids to approximately 95—98% in the unesterified acid fractions compared to the 70—90% content in the starting acid fractions.