Enzymatic acyl modification of phosphatidylcholine using immobilized lipase and phospholipase A2

Ethanol‐soluble (ES) lecithin mainly contains phosphatidylcholine (PC). The incorporation of caprylic acid into PC using immobilized phospholipase A₂ (PLA₂) and lipase was investigated. The Rhizomucor meihei lipase and the porcine pancreatic PLA₂ were immobilized on the hydrophobic resin Diaion HP‐20 and the modification was carried out in hexane as solvent. HPTLC with densitometer technique was successfully used for monitoring the production of structured phospholipids (PL) (ML‐type PC, MM‐type PC, and lysophosphatidylcholine; L: long‐chain fatty acid, M: medium‐chain fatty acid). The various parameters such as the effects of reaction temperature, enzyme loading, and the effect of molar proportion of substrate were studied in order to determine the optimum reaction conditions for the acidolysis reaction. The optimal operating conditions for the PLA₂‐catalyzed reaction were obtained as 50°C temperature, 50% (wt/wt of substrate) enzyme loading, and a 1:12 molar proportion of PC/caprylic acid. For the lipase‐catalyzed reaction, the optimized temperature was the same as for PLA₂, but the enzyme loading and molar proportion were slightly lower, i.e., 40 % w/w of substrate and 1:9 PC/caprylic acid, respectively. The effects of these parameters on the production of structured PL were compared. Under these optimal conditions, the ML‐type PC content was higher in the PLA₂‐catalyzed reaction, i.e., 45.29 mol%, and in the lipase‐catalyzed reaction it was 38.74 mol%.     

Synthesis of structured phosphatidylcholine containing punicic acid by the lipase-catalyzed transesterification with pomegranate seed oil

Incorporation of conjugated linolenic acid (punicic acid; 18:3, 9c,11t,13c) into egg-yolk phosphatidylcholine (PC) in the lipase-catalyzed transesterification process was the aim of this work. Pomegranate seed oil (PSO) containing over 77% of punicic acid was used as an acyl donor and three commercially available immobilized lipases were examined as biocatalysts. The effects of enzyme load, reaction time and molar ratio of substrates (PC:PSO) on the incorporation of punicic acid into the modified PC were tested. In all experiments the best results were obtained using lipase B from Candida antarctica. In optimum conditions (20% of enzyme load; 1:3 PC:PSO molar ratio; 72 h), the incorporation of punicic acid into the sn-1 position of PC was 56%. Additionally, the total content of polyunsaturated fatty acids in modified PC was almost 50%.     

Production of specific-structured lipids by enzymatic interesterification in a pilot continuous enzyme bed reactor

Production of specific-structured lipids (interesterified lipids with a specific structure) by enzymatic interesterification was carried out in a continuous enzyme bed pilot scale reactor. Commercial immobilized lipase (Lipozyme IM) was used and investigations of acyl migration, pressure drop, water dependence, production efficiency, and other basic features of the process were performed. The extent of acyl migration (defined as a side reaction) occurring in the present enzyme bed reactor was compared to that in a pilot batch reactor. The continuous enzyme bed reactor was better than the batch reactor in minimizing acyl migration. Generally the former produced about one-fourth the acyl migration produced by the latter at a similar extent of incorporation. Pressure drop and production efficiency were evaluated in order to obtain a suitable yield in one reaction step. High incorporation was favored by high substrate ratios between acyl donors and oils, requiring long reaction times on the enzyme bed. Under these conditions, the pressure drop of the reactor was modeled statistically and theoretically. Residence time, water content, and effects of mass transfers were also investigated. Incorporation of medium-chain fatty acids increased with increased residence time. Approximately 40% of lipase activity was lost after a 4-wk run. External mass transfer was not a major problem in the linear flow range, but internal mass transfer did impose some transfer limitations.     

Enzyme‐catalyzed synthesis of structured phospholipids with conjugated linoleic acid

Structured phospholipids were synthesized with the functional lipid conjugated linoleic acid (CLA). The lipase‐ and phospholipase A₂‐catalyzed enzymatic acidolysis reaction between phospholipids (PL) and CLA was used for fatty acid modification. Enzymatic processes were an effective way to produce structured PL. Screening of four lipases and immobilized phospholipase A₂ and a combination of lipase and phospholipase showed that only Lipozyme RM IM and Lipozyme TL IM were effective in incorporation of CLA into PL. The maximum incorporation achieved by the latter enzyme was 16% with soy PL in 72 h.     

Production of specifically structured lipids by enzymatic interesterification in a pilot enzyme bed reactor: process optimization by response surface methodology

Pilot production of specifically structured lipids by Lipozyme IM-catalyzed interesterification was carried out in a continuous enzyme bed reactor without the use of solvent. Medium-chain triacylglycerols and oleic acid were used as model substrates. Response-surface methodology was applied to optimize the reaction system with four process para-meters, these being volume flow rate, water content in the substrates, reaction temperature, and substrate ratio. The incorporation of acyl donors, product yields, and the content of diacylglycerols were measured as model responses. Enzyme activity was not identical for the sequential experiments in the same enzyme bed due to the deactivation of the Lipozyme IM. Therefore, the results were normalized based on enzyme deactivation models. Well-fitting quadratic models were obtained after normalizing the data for the incorporation of oleic acid and the production of mono-incorporated and di-incorporated structured lipids with multiple regression and backward elimination. The coefficient of determination (R²) for the incorporation was 0.93 and that for the diincorporated products was 0.94. The optimal conditions were flow rate, 2 ml/min; temperature, 65 °C; substrate ratio, 5.5; and water content, 0.1%. The production of diacylglycerols was not well correlated with any of the parameters, and the yield generally decreased with the experimental sequence. This was due to the stoichiometric water in the substrate mixture in the packed enzyme bed being complicated by the water binding and absorption of the immobilized lipase. The main effects of parameters were also examined, and conclusions in agreement with our previous results were made.     

Lipase-catalyzed incorporation of n−3 polyunsaturated fatty acids into vegetable oils

The ability of immobilized lipases IM60 fromMucor miehei and SP435 fromCandida antarctica to modify the fatty acid composition of selected vegetable oils by incorporation of n−3 polyunsaturated fatty acids into the vegetable oils was studied. The transesterification was carried out in organic solvent with free acid and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as acyl donors. With free EPA as acyl donor, IM60 gave higher incorporation of EPA than SP435. However, when ethyl esters of EPA and DHA were the acyl donors, SP435 gave higher incorporation of EPA and DHA than IM60. When IM60 and free acid were used, the addition of 5 μL water increased EPA incorporation into soybean oil by 4.9%. With ethyl ester of EPA as acyl donor, addition of 2 μL water increased EPA incorporation by 3.9%. For SP435, addition of water up to 2μL resulted in increased EPA incorporation, but the incorporation declined when the added water exceeded this amount. The addition of water increased the EPA incorporation into Trisun 90 after 24 h reaction but not the reaction rate at early stages of the reaction.     

Continuous production of structured phospholipids in a packed bed reactor with lipase from <Emphasis Type="Italic">Thermomyces lanuginosa</Emphasis>

The possibilities of producing structured phospholipids between soybean phospholipids and caprylic acid by lipase-catalyzed acidolysis were examined in continuous packedbed enzyme reactors. Acidolysis reactions were performed in both a solvent system and a solvent-free system with the commercially immobilized lipase from Thermomyces lanuginosa (Lipozyme TL IM) as catalyst. In the packed bed reactors, different parameters for the lipase-catalyzed acidolysis were elucidated, such as solvent ratio (solvent system), temperature, substrate ratio, residence time, water content, and operation stability. The water content was observed to be very crucial for the acidolysis reaction in packed bed reactors. If no water was added to the substrate during reactions under the solvent-free system, very low incorporation corporation of caprylic acid was observed. In both solvent and solvent-free systems, acyl incorporation was favored by a high substrate ratio between acyl donor and phospholipids, a longer residence time, and a higher reaction temperature. Under certain conditions, the incorporation of around 30% caprylic acid can be obtained in continuous operation with hexane as the solvent. Presented at the 95th American Oil Chemists' Society Annual Meeting and Expo in Cincinnati, Ohio, May 10, 2004.     

Lipase‐catalyzed alcoholysis of vegetable oils

Fatty acid alkyl esters were produced from various vegetable oils by transesterification with different alcohols using immobilized lipases. Using n‐hexane as organic solvent, all immobilized lipases tested were found to be active during methanolysis. Highest conversion (97%) was observed with Thermomyces lanuginosa lipase after 24 h. In contrast, this lipase was almost inactive in a solvent‐free reaction medium using methanol or 2‐propanol as alcohol substrates. This could be overcome by a three‐step addition of methanol, which works efficiently for a range of vegetable oils (e.g. cottonseed, peanut, sunflower, palm olein, coconut and palm kernel) using immobilized lipases from Pseudomonas fluorescens (AK lipase) and Rhizomucor miehei (RM lipase). Repeated batch reactions showed that Rhizomucor miehei lipase was very stable over 120 h. AK and RM lipases also showed acceptable conversion levels for cottonseed oil with ethanol, 1‐propanol, 1‐butanol and isobutanol (50‐65% conversion after 24 h) in solvent‐free conditions. Methyl and isopropyl fatty acid esters obtained by enzymatic alcoholysis of natural vegetable oils can find application in biodiesel fuels and cosmetics industry, respectively.     

Effect of phospholipids on enzyme-catalyzed transesterification of oils

The effect of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) on the activity of an immobilized lipase, Lipozyme, during transesterification of oils has been studied. PC concentrations less than 0.05% did not affect the initial rate of transesterification. Higher concentrations of PC as well as PE and PI-rich phospholipids at the 0.5% level caused a reduction in the initial reaction rate, but after ten hours transesterification had progressed to the same extent as the control sample. Reuse of Lipozyme for ten batch reactions showed that a PC content of less than 0.05% did not cause significant inactivation of the enzyme, but PC contents above this level caused progressive inactivation of the enzyme and the inactivation increased with PC content. The ability of phospholipids to inactivate the Lipozyme was in the order PE>PC>PI. Variation in the acyl groups of PC did not significantly affect inactivation of the enzyme. It is concluded that the phospholipid content of edible oils should be reduced below 200 ppm by degumming if unacceptable inactivation of Lipozyme is to be avoided.     

Lipase-catalyzed transesterification of phosphatidylcholine at controlled water activity

The incorporation of a free fatty acid into thesn-1 position of phosphatidylcholine by lipase-catalyzed transesterification was investigated. The thermodynamic water activity of both the enzyme preparation and the substrate solution was adjusted to the same value prior to the reaction. The reaction rate increased with increasing water activity but the yield of modified phosphatidylcholine decreased due to hydrolysis. By using a large excess of the free fatty acid (heptadecanoic acid), the hydrolysis reaction was slowed down, so a higher yield was obtained at a given degree of incorporation. The best results were obtained withRhizopus arrhizus lipase immobilized by adsorption on a polypropylene support. With this preparation, a yield of 60% and nearly 50% incorporation of heptadecanoic acid (100% incorporation in thesn-1 position) was obtained at a water activity of 0.064. The enzyme preparation had good operational stability and position specificity. Little incorporation (<1%) was observed in thesn-2 position, when almost all the fatty acid in thesn-1 position was exchanged.     

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.     

Application of lipase immobilized on nylon‐6 for the synthesis of butyl acetate by transesterification reaction in n‐heptane

A purified alkaline thermotolerant bacterial lipase from Bacillus coagulans BTS‐3 was immobilized on nylon‐6 matrix activated by glutaraldehyde. The matrix showed ～ 70% binding efficiency for lipase. The bound lipase was used to perform transesterification in n‐heptane. The reaction studied was conversion of vinyl acetate and butanol to butyl acetate and vinyl alcohol. Synthesis of butyl acetate was used as a parameter to study the transesterification reaction. The immobilized enzyme achieved ～ 75% conversion of vinyl acetate and butanol (100 mmol/L each) into butyl acetate in n‐heptane at 55°C in 12 h. When alkane of C‐chain lower or higher than n‐heptane was used as an organic solvent, the conversion of vinyl acetate and butanol to butyl acetate decreased. During the repetitive transesterification under optimal conditions, the nylon bound lipase produced 77.6 mmol/L of butyl acetate after third cycle of reuse. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Lipase‐catalysed biodiesel production from Jatropha curcas oil

Biodiesel as fatty acid alkylesters has become attractive because of its environmental benefits. A non‐edible oil as starting material for biodiesel production appears desirable and does not compromise the edible oils used mainly for food and feed. The present article discusses the enzymatic alcoholysis of crude Jatropha curcas oil in solvent free medium for the production of valuable fatty acid alkyl esters for use as biodiesel. Among various microbial lipases commonly tested in the literature, the highest initial rate (>18 μmol h^–1 mg^–1) with different alcohols was observed with immobilized lipase from Pseudomonas cepacia, but the activity depends on the amount of water. The best conversion (93%) to produce ethyl esters was achieved with lipase immobilized on the polypropylene carrier Accurel 1282 after 16 h at low enzyme concentration (3% w/w). Moreover, the transesterification could be conducted for at least 160 h during 10 batch runs without significant loss of activity. This reduces the costs for immobilized lipase and can thus make the enzymatic biodiesel production commercially more viable, especially starting from a non‐edible plant oil.     

Biodiesel Synthesis in a Solvent-Free System by Recombinant Rhizopus oryzae Lipase. Study of the Catalytic Reaction Progress

The recombinant 1,3-positional selective Rhizopus oryzae lipase (rROL) was used to synthesize biodiesel and monoacylglycerols simultaneously. The reaction was carried out in a solvent-free system with the enzyme immobilized on octadecyl-Sepabeads. Using response surface methodology, the methyl ester yield was optimized by means of the study of the effect of water, substrate molar ratio (methanol:olive oil) and methanol stepwise addition. It was concluded that in order to prevent enzyme inactivation by methanol, alcohol should be added slowly; otherwise a large amount of water would be present. Taking the best conditions, a 50.3 % yield was achieved in 3 h, which corresponds to 75.4 % of the acyl groups at the 1,3-position undergoing transesterification. It was also concluded that methyl esters result from the esterification of the free fatty acid hydrolyzed by the enzyme and also from a direct transesterification of oil. In addition, the fatty acid selectivity of rROL was found not to favor one fatty acid in olive oil over another.     

假丝酵母99-125脂肪酶促酯化合成生物柴油的研究

1 INTRODUCTION Biodiesel, that is long-chain fatty acid short-chain alcohol esters (methyl, ethyl, propyl and butyl ester), is produced by esterification of fatty acids or inter- esterification of oils and fats. These fatty acid alcohol esters are not only used as important industrial addi- tives and surfactants, but also used for biofuel. The biodiesel is a biodegradable, environmental friendly, renewable substitute of diesel fuel[1]. The traditional production of biodiesel is by chem- i…     

Selective,Green Synthesis of Six‐Membered Cyclic Carbonates by Lipase‐Catalyzed Chemospecific Transesterification of Diols with Dimethyl Carbonate

A facile and green synthesis of six‐membered cyclic carbonates, the potential monomers for isocyanate‐free polyurethanes and polycarbonates, was achieved by transesterification of diols with dimethyl carbonate catalyzed by immobilized Candida antarctica lipase B, Novozym®435, followed by thermal cyclization in a solvent‐free medium. The difference in the chemospecificity of the lipase for the primary, secondary and tertiary alcohols as acyl acceptors was utilized to obtain a highly chemoselective synthesis of the cyclic carbonate in high yield. In the lipase‐catalyzed reaction with diols, the product contained almost equal proportions of mono‐ and di‐carbonates with 1,3‐propanediol having two primary alcohols, a higher proportion of mono‐carbonate with 1,3‐butanediol having a primary and a secondary alcohol, and mainly mono‐carbonate with 3‐methyl‐1,3‐butanediol having a primary and a tertiary alcohol. The chemospecificity of cyclic carbonates formed by thermal treatment at 90 °C was closely related to the proportion of mono‐carbonate. The yield of cyclic carbonate was 99.3% with 3‐methyl‐1,3‐butanediol, 85.5% with 1,3‐butanediol, and 43.2% with 1,3‐propanediol.     

Enzymatic production of fatty acid alkyl esters with a lipase preparation from Candida sp. 99‐125

A lipase preparation developed from Candida sp. 99‐125 was used for fatty acid alkyl ester synthesis by both enzymatic esterification of fatty acids, and transesterification of oils and fats. The lipase preparation was chosen based on screening of lipases from commercial sources as well as those produced in the laboratory. The effects of enzyme dosage, solvent types, water absorbent additions, inhibition of short‐chain alcohols, alcohol and acid types, molar ratio of substrates, and reusability of the lipase preparation in esterification were studied. Degree of esterification between oleic acid and methanol under optimal conditions reached 92%. Purity of the methyl ester after washing with water and distillation was 98%. Half‐life of the lipase preparation was calculated to be approximately 340 h. For transesterification of rapeseed oil with the same lipase preparation, the amount of methanol and mode of methanol addition to the reaction were also conducted. Transesterification of the oil with stepwise methanol addition reached 83% after 36 h reaction time.     

Solvent‐free preparation of phytosteryl esters with fatty acids from butterfat in equimolecular conditions in the presence of a lipase from Candida rugosa

BACKGROUND: Enzymatic esterification of phytosterols with fatty acids from butterfat in equimolecular conditions to produce phytosteryl esters was performed in solvent‐free medium. Commercial and immobilized Candida rugosa lipases were used as biocatalysts for the reaction. RESULTS: By this methodology, under simple and mild reaction conditions (without solvents, 50 °C and short reaction times), 94% and 99% (w/w) of phystosteroyl esters were obtained in 48 h and 9 h with the commercial and the immobilized lipase, respectively. The effects of temperature, fatty acid specificity, enzyme amount and residual activity of each lipase were also evaluated. CONCLUSIONS: The phytosteryl esters from butterfat produced in this study are expected to have lower melting point, improved oil and fat solubility and bioavailability compared to that of their corresponding free phytosterols. Copyright © 2008 Society of Chemical Industry     

Biodiesel production using immobilized enzyme-current state and future perspective

近年来由于化石燃料的全球性短缺、原油价格的过度上涨和环境问题的加剧致使生物柴油的生产迅速增长。生物柴油生产的关键反应是化学或生物催化的转酯反应,酶催化的转酯反应与化学催化相比有相对节能、副产物甘油易回收及适合高含量游离脂肪酸油脂作为底物等明显优势。本文综述了固定化脂肪酶生产生物柴油的现状及最新进展,包括生物柴油的原料、脂肪酶的来源、酶的固定化技术、甲（乙）醇及甘油对脂肪酶的失活作用,展望了固定化脂肪酶生产生物柴油的未来前景。     

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.