Lipase-catalyzed glycerolysis of olive oil in organic solvent medium: Optimization using response surface methodology

Enzymatic glycerolysis of olive oil for mono- (MG) and diglycerides (DG) synthesis was investigated. Several pure organic solvents and co-solvent mixtures were screened in a batch reaction system. The yields of MG and DG in co-solvent mixtures exceeded those of the corresponding pure organic solvents. Batch reaction conditions of the glycerolysis reaction, the lipase amount, the glycerol to oil molar ratio, the reaction time, and temperature, were studied. In these systems, the high content of reaction products, especially MG (55.8 wt%) and DG (16.4wt%) was achieved at 40 °C temperature and 0.025 g of lipase with relatively low glycerol to oil molar ratio (2: 1) within 4 h of reaction time in isopropanol/tert-butanol (1: 3) solvent mixture. Glycerolysis reaction was optimized with the assistance of response surface methodology (RSM). Optimal condition for reaction conversion was recommended as lipase amount 0.025 g, glycerol to oil molar ratio 2: 1, reaction time 4 h and temperature 40 °C.     

Lipase immobilization on ionic liquid‐modified magnetic nanoparticles: Ionic liquids controlled esters hydrolysis at oil–water interface

Ester hydrolysis at oil–water interface by lipase covalently immobilized on ionic liquid‐modified magnetic nanoparticles was investigated. Magnetic supports with a diameter of 10–15 nm were synthesized by covalent binding of ionic liquids (chain length C₄ and C₈ and anions Cl^?, BF₄^?, and PF₆^?) on the surface of Fe₃O₄ nanoparticles. Lipase was covalently immobilized on Fe₃O₄ nanoparticles using ionic liquids as the coupling reagent. Ionic liquid‐modified magnetic nanoparticle‐grafted lipase preferentially located at the oil–water interface. It has higher catalytic activity than its native counterpart. A modified Michaelis–Menten model was used to elucidate the effect of stirring rate, aqueous–organic phase ratio, total amount of enzyme, and ester chain length. The influences of these conditions on esters hydrolysis at oil–water interface were consistent with the introduction of the ionic liquids interlayer. Ionic liquids could be used to control the oil–water interfacial characteristics during lipase catalyzed hydrolysis, and thus control the behavior of immobilized lipase. © 2011 American Institute of Chemical Engineers AIChE J, 2012     

脂肪酶促乌桕脂组分甘油解及其反应机理研究

Glycerolysis of Chinese vegetable tallow （CVT） fraction was investigated using a 1,3-specific lipase from Rhizopus arrhizus as catalyst. Based upon a binary gradient HPLC with an evaporative light-scattering detector （ELSD）, the contents of free fatty acids （FFA）, monoglycerides （MG）, diglycerides（DG） and triglycerides （TG） with their positional isomers during the glycerolysis were determined. The effects of water content and the ratio of glycerol to oil on the product distribution of glycerolysis were studied. Under the optimum reactant conditions： 250 units lipase per gram oil at 37℃ with 1：2 molar ratio of oil to glycerol in a solvent-free system, after 24 h reaction, the product consisted of 7.2% TG, 25.6% MG, 56.1% DG and 4.9% FFA （all by mass）. Furthermore, the mechanism of glycerolysis was discussed in detail.     

Selective distribution of saturated fatty acids into the monoglyceride fraction during enzymatic glycerolysis

Four triglyceride fats and oils (beef tallow, lard, rapeseed oil and soybean oil) were reacted with glycerol while using lipase as the catalyst. For all fats examined, at reaction temperatures above the critical temperature (T_c), the fatty acid compositions of the monoglyceride (MG) and diglyceride (DG) fractions and of the original fat were similar. A relatively low yield of MG was obtained (20–30 wt%). When the reaction was carried out with beef tallow or lard at a temperature below the T_c (40°C), the concentration of saturated fatty acids in the MG fraction was 2 to 4 times greater than that in the DG fraction. Correspondingly, the concentration of unsaturated fatty acids in the DG fraction was more than two times greater than that in the MG fraction. At 5°C, a similar trend was observed for rapeseed oil and soybean oil. Direct analysis of partial glycerides during glycerolysis by high-temperature gas-liquid chromatography showed that below T_c the content of C16 MG increased relatively more than C18 MG. C36 DG and C54 TG were apparently resistant to glycerolysis. Preferential distribution of saturated fatty acids into the MG fraction was accompanied by a high yield of monoglyceride (45–70 wt%) and solidification of the reaction mixture. It is concluded that during glycerolysis below T_c, preferential crystallization occurs for MGs that contain a saturated fatty acid.     

Optimized Production of Biodiesel from Waste Cooking Oil by Lipase Immobilized on Magnetic Nanoparticles

Biodiesel, a non-toxic and biodegradable fuel, has recently become a major source of renewable alternative fuels. Utilization of lipase as a biocatalyst to produce biodiesel has advantages over common alkaline catalysts such as mild reaction conditions, easy product separation, and use of waste cooking oil as raw material. In this study, Pseudomonas cepacia lipase immobilized onto magnetic nanoparticles (MNP) was used for biodiesel production from waste cooking oil. The optimal dosage of lipase-bound MNP was 40% (w/w of oil) and there was little difference between stepwise addition of methanol at 12 h- and 24 h-intervals. Reaction temperature, substrate molar ratio (methanol/oil), and water content (w/w of oil) were optimized using response surface methodology (RSM). The optimal reaction conditions were 44.2 °C, substrate molar ratio of 5.2, and water content of 12.5%. The predicted and experimental molar conversions of fatty acid methyl esters (FAME) were 80% and 79%, respectively.     

A continuous process for the glycerolysis of soybean oil

A continuous process for the glycerolysis of soybean oil with pure and crude glycerol, the co-product from the transesterification of soybean oil, was investigated in a pilot plant. The process was equipped with a static and a high-shear mixer. The experimental studies explored the effects of variations in mixing intensity, temperature, reactant flow rates, and reactant stoichiometry on the formation of MG and DG. The developed process resulted in high conversion of TG to MG. The most favorable conditions were 230°C, 40 mL/min total flow, 25 min of reaction time, 2.5∶1 molar ratio of glycerol/soybean oil, and 3600 rpm for the reactions involving crude glycerol where the concentrations of MG and DG in the product were about 56 and 36 wt%, respectively. Under similar conditions, glycerolysis of pure glycerol resulted in 58% MG and 33% DG. In general, higher temperatures and mixing intensities favored the conversion of TG to MG and DG. Reaction temperature had a greater influence on the extent of the reaction than mixing. The formation of MG approached equilibrium for nearly all cases under investigation.     

Enzymatic transesterification of sunflower oil in an aqueous-oil biphasic system

A biphasic oil-aqueous system for FAME production by enzymatic catalysis was studied. The transesterification of sunflower oil with methanol was catalyzed by free or immobilized lipases from Rhizomucor miehei (Palatase 20 000 L) and Humicola insolens (Lipozyme TL 100 L). The effects of protein amount, temperature, pH, and molar ratio of methanol to sunflower oil on the enzymatic reaction using free lipase were evaluated; the best results were obtained with H. insolens, at pH 5, 40°C, and 36.8 mg of protein. By using this lipase immobilized in Hypol^® 2002 (64. 8 mg of protein) at a 6∶1 methanol/oil molar ratio and a 2∶1 volumetric oil/water phase ratio, an ester content of 96.1% and a conversion of 91.2% were achieved. The immobilized lipase could be reused, although a 30% reduction in conversion efficiency was observed after four uses.     

Production of biodiesel from palm oil using liquid core lipase encapsulated in κ-carrageenan

This work deals with the enzymatic transesterification of palm oil with methanol in a solvent-free system. Among the five lipases tested in the initial screening, lipase PS from Burkholderia cepacia resulted in the highest triglyceride conversion. Lipase PS was further investigated in a novel immobilized form by encapsulating within a biopolymer, κ-carrageenan. Using the immobilized lipase the production parameters of biodiesel from palm oil were optimized. The optimal conditions for processing 10 g of palm oil was: 30 °C, 1:7 oil/methanol molar ratio, 1 g water, 5.25 g immobilized lipase, 72 h reaction time and 23.7g relative centrifugal force. At the optimal conditions, triglyceride conversion of up to 100% could be obtained. The immobilized lipase was stable and retained 82% relative transesterification activity after five cycles. Liquid core lipase encapsulated in κ-carrageenan could be a potential immobilized catalyst for eco-friendly production of biodiesel.     

Glycerolysis of Crude Methyl Esters with Crude Glycerol from Biodiesel Production

Glycerolysis of crude fatty acid methyl esters (FAME) with crude glycerol derived from biodiesel production was performed. The reaction was accomplished at temperatures ranging between 160 and 200 °C and molar ratios of FAME to glycerol ranging between 1.5 and 3.0. Increasing the temperature improved the formation rate of monoglycerides (MG) and diglycerides (DG). However, increasing both the temperature and the molar ratio of glycerol to FAME diminished the formation of MG. Best results (43 % MG and 26 % DG in 10 min) were obtained at 200 °C using the lowest concentration of glycerol. The effects of soap and NaOH present in crude glycerol were controlled by carrying out the reaction with pure glycerol. In comparison with NaOH-catalyzed reactions, soap-catalyzed reactions resulted in a slower formation rate of products. However, soap-catalyzed reactions were less prone to secondary reactions, affording maximum yields of MG and DG, which were higher than those obtained with NaOH-catalyzed reactions at 180 and 200 °C.     

Lipase Immobilization onto the Surface of PGMA-b-PDMAEMA-grafted Magnetic Nanoparticles Prepared via Atom Transfer Radical Polymerization

A block copolymer of 2-dimethylaminoethyl methacrylate (DMAEMA) and glycidyl methacrylate (GMA) was grafted onto the surface of magnetic nanoparticles (Fe₃O₄) via atom transfer radical polymerization. The resultant PGMA-b-PDMAEMA-grafted-Fe₃O₄ magnetic nanoparticles with amino and epoxy groups were characterized by Fourier transform infrared spectroscopy, powder X-ray diffraction, thermo-gravimetric analysis, and scanning electron microscopy. Lipase from Burkholderia cepacia was successfully immobilized onto the magnetic nanoparticles by physical adsorption and covalent bonding. The immobilization capacity of the magnetic particles is 0.5 mg lipase per mg support, with an activity recovery of up to 43.1% under the optimum immobilization condition. Biochemical characterization shows that the immobilized lipase exhibits improved thermal stability, good tolerance to organic solvents with high lg P, and higher pH stability than the free lipase at pH 9.0. After six consecutive cycles, the residual activity of the immobilized lipase is still over 55% of its initial activity.     

One‐Pot Lipase Entrapment Within Silica Particles to Prepare a Stable and Reusable Biocatalyst for Transesterification

In order to enhance the reusability, Rhizomucor miehei lipase was entrapped in a single step within silica particles having an oleic acid core (RML@SiO₂). Characterization of RML@SiO₂ by scanning and transmission electron microscopy and Fourier transform infrared studies supported the lipase immobilization within silica particles. The immobilized enzyme was employed for transesterification of cottonseed oil with methanol and ethanol. Under the optimum reaction conditions of a methanol‐to‐oil molar ratio of 12:1 or ethanol‐to‐oil molar ratio of 15:1, stirring speed of 250 revolutions/min (flask radius = 3 cm), reaction temperature of 40 °C, and biocatalyst concentration of 5 wt% (with respect to oil), more than 98 % alkyl ester yield was achieved in 16 and 24 h of reaction duration in case of methanolysis and ethanolysis, respectively. The immobilized enzyme did not require any buffer solution or organic solvent for optimum activity; hence, the produced biodiesel and glycerol were free from metal ion or organic molecule contamination. The activation energies for the immobilized enzyme‐catalyzed ethanolysis and methanolysis were found to be 34.9 ± 1.6 and 19.7 ± 1.8 kJ mol^?1, respectively. The immobilized enzyme was recovered from the reaction mixture and reused in 12 successive runs without significant loss of activity. Additionally, RML@SiO₂ demonstrated better reusability as well as stability in comparison to the native enzyme as the former did not lose the activity even upon storage at room temperature (25–30 °C) over an 8‐month period.     

Stability of Immobilized Candida sp. 99–125 Lipase for Biodiesel Production

The stability of the immobilized lipase from Candida sp. 99–125 during biodiesel production was investigated. The lipase was separately incubated in the presence of various reaction components such as soybean oil, oleic acid methyl ester, n‐hexane, water, methanol, and glycerol, or the lipase was stored at 60, 80, 100 and 120 °C. Thereafter the residual lipase activity was determined by methanolysis reaction. The results showed that the lipase was rather stable in the reaction media, except for methanol and glycerol. The stability study performed in a reciprocal shaker indicated that enzyme desorption from the immobilized lipase mainly contributed to the lipase inactivation in the water system. So the methanol and glycerol contents should be controlled more precisely to avoid lipase inactivation, and the immobilization method should be improved with regard to lipase desorption.     

Modification of Magnetite Cellulose Nanoparticles via Click Reaction for use in Controlled Drug Delivery

Superparamagnetic Fe₃O₄ nanoparticles (MNPs) were functionalized by modified cellulose. The modified cellulose was synthesized through bromoacetylation of cellulose (BACell) followed by the substitution of sodium azide to form BACell-N₃. The remaining methylene bromide groups on BACell-N₃ was further reacted with the MNPs to form Fe₃O₄/Cell-N₃. Then propargyl alcohol (PA) was immobilized on the azide-terminated Fe₃O₄ nanoparticles through copper (I)-catalyzed azide-alkyne cycloaddition (click reaction) to form Fe₃O₄/Cell/TAA nanoparticles. Doxorubicin (DOX) was loaded on prepared nanoparticles and release profiles of the DOX as a model drug from the Fe₃O₄/Cell/TAA nanoparticles and its loading capacity were determined by UV–Vis absorption at λ_max 483?nm.     

Conversion of oils to monoglycerides by glycerolysis in supercritical carbon dioxide media

Glycerolysis of soybean oil was conducted in a supercritical carbon dioxide (SC-CO₂) atmosphere to produce monoglycerides (MG) in a stirred autoclave at 150–250°C, over a pressure range of 20.7–62.1 MPa, at glycerol/oil molar ratios between 15–25, and water concentrations of 0–8% (wt% of glycerol). MG, di-, triglyceride, and free fatty acid (FFA) composition of the reaction mixture as a function of time was analyzed by supercritical fluid chromatography. Glycerolysis did not occur at 150°C but proceeded to a limited extent at 200°C within 4 h reaction time; however, it did proceed rapidly at 250°C. At 250°C, MG formation decreased significantly (P<0.05) with pressure and increased with glycerol/oil ratio and water concentration. A maximum MG content of 49.2% was achieved at 250°C, 20.7 MPa, a glycerol/oil ratio of 25 and 4% water after 4 h. These conditions also resulted in the formation of 14% FFA. Conversions of other oils (peanut, corn, canola, and cottonseed) were also attempted. Soybean and cottonseed oil yielded the highest and lowest conversion to MG, respectively. Conducting this industrially important reaction in SC-CO₂ atmosphere offered numerous advantages, compared to conventional alkalicatalyzed glycerolysis, including elimination of the alkali catalyst, production of a lighter color and less odor, and ease of separation of the CO₂ from the reaction products.     

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.     

铜绿假单胞菌脂肪酶无溶剂催化棕榈油甘油解Ⅰ.单甘酯酶促合成工艺的研究

以自产铜绿假单胞菌脂肪酶为催化剂 ,通过无溶剂法棕榈油甘油解反应催化合成了单脂肪酸甘油酯。实验结果表明 :适宜加酶量为 50 0u g棕榈油 ,甘油相适宜水质量分数为w (H2 O)=3%～ 4 5% ,反应物量比宜设定为 n(甘油 )∶n(棕榈油 ) =( 2 0～ 2 5)∶1。反应器材质对单甘酯产率有影响 ,31 6L型不锈钢为适宜材质。最佳反应温度为 38～ 42℃ ,在此温度区间内适宜反应时间为 2 4h。临界反应温度为 44℃。     

Biodiesel preparation catalyzed by compound-lipase in co-solvent

Besides high cost, the most important reasons that immobilized lipases are limited in industrialization of biodiesel production are the toxicity of methanol and the adsorption of glycerol onto the surface of immobilized vector. Solvent engineering method was employed to the reaction where compound-lipase with synergistic effect, Novozym 435 and Lipozyme TL IM, catalyzed preparation of biodiesel from stillingia oil with methanol. The treatment accelerated the solubility of methanol in oil and dissolved glycerol, which helped maintain lipase activity. It is found that the yields of biodiesel in co-solvent exceeded those in the pure organic solvents. The mixture system of co-solvent with 60% acetonitrile and 40% t-butanol (v/v) was proved to be an optimal one, and RSM was used to optimize the reaction factors and the optimal conditions: methanol/oil molar ratio 6.4:1, compound-lipase 4.32% (wt/wt) and molecular sieve 5.5% (wt/wt). R²= 98.86% showed good coincidence between predicted and experimental values. There was nearly no loss inactivity of compound-lipase after being recycled for 30 times. Other oils were also investigated in the mixture system, and we got the same results, which indicated that the mixture system could be an ideal prospective medium applied to biodiesel production.     

Fe3O4 nanoparticles impregnated eggshell as a novel catalyst for enhanced biodiesel production

Biodiesel is a green fuel which can replace diesel while addressing various issues such as scarcity of hydrocarbon fuels and environmental pollution to an extent. The high production cost of biodiesel and the recovery of the catalyst after the transesterification process are the major challenges to be addressed in biodiesel production. In the present work, a cheap and promising solid base oxide catalyst was synthesized from chicken eggshell by calcination at 900 °C forming catalyst eggshells (CES) and was impregnated with the nanomagnetic material (Fe₃O₄) to obtain Fe₃O₄ loaded catalytic eggshell (CES–Fe₃O₄). Fe₃O₄ nanomaterials were synthesized by co-precipitation method and were loaded in catalytic eggshell by sonication, for better recovery of the catalyst after transesterification process. CES–Fe₃O₄ material was characterized by Thermogravimetric analysis, X-ray diffraction, Fourier transform infrared spectroscopy, a vibrating-sample magnetometer, Brunauer-Emmett-Teller, Dynamic light scattering, and Scanning electron microscopy. Biodiesel was synthesized by transesterification of Pongamia pinnata raw oil with 1:12 oil to methanol molar ratio and 2 wt% catalyst loading for 2 h at a temperature of 65 °C and yields were compared. The reusability of the catalyst was studied by the transesterification of the raw oil and its catalytic activity was found to be retained up to 7 cycles with a yield of 98%.     

Lipase immobilized on poly (vinyl alcohol) modified polysulfone membrane: application in hydrolytic activities for olive oil

The advancement of membrane research closely relates to the activities of ‘immobilization of enzymes’. The modification of polymeric membrane surfaces according to tailor-made specifications is considered an art and useful in this arena. In this study, lipase is immobilized on Polyvinyl alcohol photomodified Polysulfone (PS–PVA) membranes. The maximum immobilization (1.48 mg/cm²) for PS–PVA membranes is achieved. The amount of immobilized lipase directly relates on the PVA content on the membrane. Scanning Electron Microscope and X-ray diffraction patterns show the evidences of lipase immobilization on membranes. The hydrolytic performances of lipase immobilized PS and PS–PVA–glu membranes for olive oil are studied. The free fatty acid (FFA %) and acid value (AV) parameters are determined by titrimetic analysis (1.53 and 3.04 for PS–PVA–glu) and compared with esterification GC-mass analysis data. The K _m and V _max values are 105 mM and 0.9 mM/min for lipase immobilized on PS–PVA–glu and 153.8 mM and 0.51 mM/min for lipase on PS. The reusability feature shows the lipase immobilized on PS–PVA–glu matrix have better stability (10.7% decrease) compared to lipase immobilized on PS matrix (33.3% decrease) after five cycles.     

Continuous production of biodiesel fuel from vegetable oil using immobilized Candida antarctica lipase

Candida antarctica lipase is inactivated in a mixture of vegetable oil and more than 1∶2 molar equivalent of methanol against the total fatty acids. We have revealed that the inactivation was eliminated by three successive additions of 1∶3 molar equivalent of methanol and have developed a three-step methanolysis by which over 95% of the oil triacylglycerols (TAG) were converted to their corresponding methyl esters (ME). In this study, the lipase was not inactivated even though 2∶3 molar equivalent of methanol was present in a mixture of acylglycerols (AG) and 33% ME (AG/ME33). This finding led to a two-step methanolysis of the oil TAG: The first-step was conducted at 30°C for 12 h with shaking in a mixture of the oil, 1∶3 molar equivalent of methanol, and 4% immobilized lipase; the second-step reaction was done for 24 h after adding 2∶3 molar equivalent of methanol (36 h in total). The two-step methanolysis achieved more than 95% of conversion. When two-step reaction was repeated by transferring the immobilized lipase to a fresh substrate mixture, the enzyme could be used 70 cycles (105 d) without any decrease in the conversion. From the viewpoint of the industrial production of biodiesel fuel production, the two-step reaction was conducted using a reactor with impeller. However, the enzyme carrier was easily destroyed, and the lipase could be used only several times. Thus, we attempted flow reaction using a column packed with immobilized Candida lipase. Because the lipase packed in the column was drastically inactivated by feeding a mixture of AG/ME33 and 2∶3 molar equivalent of methanol, three-step flow reaction was performed using three columns packed with 3.0 g immobilized lipase. A mixture of vegetable oil and 1∶3 molar equivalent of methanol was fed into the first column at a constant flow rate of 6.0 mL/h. The eluate and 1∶3 molar equivalent of methanol were mixed and then fed into the second column at the same flow rate. The final step reaction was done by feeding a mixture of eluate from the second column and 1∶3 molar equivalent of methanol at the same flow rate. The ME content in the final-step eluate reached 93%, and the lipase could be used for 100 d without any decrease in the conversion.