Continuous hydrolysis of olive oil by lipase in microporous hydrophobic membrane bioreactor

Continuous hydrolysis of olive oil byCandida cylindracea’s lipase was studied in a microporous hydrophobic membrane bioreactor. Olive oil and buffer solution, fed continuously through two compartments partitioned by membrane, caused reaction at the interface of lipase-adsorbed membrane and buffer solution. Fatty acid was obtained in a single phase without being mixed with components of other phases. At all mean residence times, countercurrent flow mode was superior to cocurrent one. The lipase was adsorbed onto the membrane, and its adsorption was suggested to be partially specific from the experiments with enzymes having various levels of purity. The percent hydrolysis depended hyperbolically on the interfacial enzyme concentration. The hydrolysis seemed to be limited by diffusion of fat or fatty acid through the micropores of the membrane at higher interfacial enzyme concentrations. The lipase was stabilized significantly by glycerol added to the buffer solution. Satisfactory performance of the membrane bioreactor was obtained in a longterm continuous operation which lasted for 24 days by feeding buffer-glycerol (18.0%) solution over the adsorbed lipase. The operational half-life of the adsorbed enzyme was 15 days at 40 C.     

Characterisation of acylglycerol synthesis in aqueous foams by lipases

Low water systems have become important for organic synthetic reactions catalysed by enzymes. Esterification reactions could be catalysed by lipase in multiphasic reaction media, such as foams, containing excess water. The esterification of glycerol with oleic acid catalysed by lipase in aqueous foams has been characterised. More than 80% of the oleic acid was converted to glycerides within 10 h. Preliminary characterisation of the reaction with respect to time, pH and temperature indicate that acylglycerol synthesis in foam media as similar to hydrolysis by the same lipase. The observed high K_m of glycerol for the esterification reaction may be due to poor surface active properties of glycerol. The unique advantages of foam as a medium to conduct lipase reactions are discussed. © 1999 Society of Chemical Industry     

玉米油酶法合成甘油二酯工艺优化研究

以玉米油、甘油为原料,在无溶剂体系中用固定化脂肪酶Lipozyme TL IM催化合成甘油二酯,通过单因素试验和响应面试验研究底物摩尔比(亚油酸/甘油)、反应温度、反应时间、酶质量分数和初始水质量分数等因素对亚油酸转化率的影响。得出:反应温度、底物摩尔比、反应时间、酶质量分数对亚油酸转化率影响较大;影响酯化反应中亚油酸转化率的主次因素依次为反应温度、底物摩尔比、反应时间、酶质量分数;最佳工艺条件为亚油酸和甘油的摩尔比为2.10∶1,反应温度为61.16℃,反应时间为12.17 h,酶质量分数为20.09%,亚油酸转化率达到75.69%。Lipozyme TL IM连续反应3批次,其相对酶活仍有65.7%。     

固定化假丝酵母脂肪酶催化合成辛酸甘油酯

以自制固定化假丝酵母脂肪酶作为催化剂,研究了无溶剂体系中辛酸和甘油直接酯化合成辛酸甘油酯的反应条件。考察了酶的种类、底物的物质的量之比、温度、酶量、甘油的初始含水量和反应时间等因素对辛酸转化率和产物组成的影响。结果证明,以纺织物作为载体制备的固定化假丝酵母脂肪酶适宜催化辛酸甘油酯的合成。最优反应条件为：辛酸与甘油的物质的量之比为2∶1,固定化假丝酵母脂肪酶加量为0.5g/0.69g甘油,温度为40 ℃,振荡培养箱转速为190 r/min。最优反应条件下辛酸转化率可以达到94%以上,经过简单处理的固定化酶可以重复使用4批。     

Enzymatische Herstellung von festen Fettsäuremonoglyceriden

Enzymatic Preparation of Solid Fatty Acid Monoglycerides Lipases can be used to synthesize monoglycerides from solid fatty acids and glycerol. We have examined the conditions of such reaction systems with a view to developing a simple technical process. The selectivity of the Penicillium cyclopium and Rhizopus sp. lipases were studied at high rates of fatty acid turnover. By the correct choice of lipase, temperature and water content, the reaction may be steered in the direction of either monoglycerides of diglycerides. Using the Penicillium lipase under narrowly defined reaction conditions a highly selective monoglyceride synthesis is possible. Di glycerides are almost the sole products when using the Rhizopus lipase at 40°C. At 20°C, but under otherwise identical conditions, the main products are monoglycerides. The Penicillium lipase catalyzes the synthesis of monoglycerides at both 20°C and 40°C, provided that the water content of the glycerine is less than 10%. At a glycerol concentration of 80% the selectivity changes such that more diglycerides are formed. The enzymatic synthesis of glycerides can be so regulated that more than 95% of the available fatty acid is incorporated into monoglyceride. After melting the reaction mixture and allowing it to stand for less than an hour, the phases separate and excess glycerol can be separated very simply. A product conforming to industry specifications can then be produced by distilling off the trace amounts of remaining glycerol from the lipid phase.     

Synthesis of medium-chain glycerides using lipase from Candida rugosa

Enzymatic synthesis of medium-chain glycerides (MCG) from capric acid and glycerol was studied using lipase from Candida rugosa. The effects of various reaction parameters such as time, molar ratio of substrates (mmol capric acid/mmol glycerol), amount of lipase, type of organic solvents, and initial water activity (a _w ) were studied. The best conditions tested for MCG synthesis at 37°C were, respectively, time, 24 h; molar ratio of substrates, 2.5; and amount of lipase, 100.0 mg. The use of organic solvents greatly influenced the activity of lipase in the synthesis of MCG. Generally, activity of lipase was high in nonpolar solvents with log P values from 3.50 to 4.50, where P is the partition coefficient between water and 1-octanol. The enzymatic synthesis of MCG was preferably carried out at an initial a _w of 0.328, which resulted in maximal yield. Analysis of the products of reaction using gas chromatography showed that lipase from Candida rugosa seemed to produce more dicaprin and tricaprin than monocaprin.     

Lipase-catalyzed esterification of glycerol and oleic acid

The enzymatic synthesis of glycerides from glycerol and oleic acid in organic solvent was studied, and the optimal conditions for glyceride synthesis by lipases were established. Of the commercially available lipases that were investigated, Candida rugosa lipase and porcine pancreas lipase resulted in the highest extent of esterification. Iso-octane and hexane were particularly useful organic solvents in glyceride synthesis. The water content in the reaction mixture was of primary importance. For C. rugosa lipase and porcine pancreas lipase, the optimal water contents were 5 and 1%, respectively. Candida rugosa lipase and porcine pancreas lipase manifested contrasting positional specificities in glyceride synthesis.     

Esterification of lauric acid using lipase immobilized in the micropores of a hollow-fiber membrane

A porous anion-exchange hollow-fiber membrane was prepared by radiation-induced graft polymerization and chemical modification to immobilize lipase for enzymatic reaction in an organic solvent. The amount of anion-exchange group introduced to the porous hollow-fiber membrane was 2.5 mol/kg_fiber. A lipase solution was allowed to permeate through the porous anion-exchange hollow-fiber membrane, and lipase molecules that adsorbed onto the grafted polymer brush were cross-linked with glutaraldehyde. The lipase was immobilized at a density of 0.14 kg_lipase/kg_fiber, which was equivalent to a degree of multilayer binding of 20. Esterification was carried out by passing a solution of lauric acid and benzyl alcohol in anhydrous issoctane through the lipase-immobilized membrane, and lipase activity was determined. A reaction percentage of 50% was achieved at space velocity 68 h⁻¹. The maximum immobilized lipase and native lipase activities were 8.9 and 0.38 mol/(h·kg_lipase), respectively. Thus, the activity of the immobilized lipase was 23.4 times higher than that of the native lipase.     

Continuous production of structured lipid containing γ-linolenic and caprylic acids by immobilized Rhizopus delemar lipase

Production of a structured lipid containing γ-linolenic acid (GLA) achieved by the continuous acidolysis of borage oil with caprylic acid (CA) using 1,3-specific Rhizopus delemar lipase as a catalyst. The lipase immobilized on a ceramic carrier was activated by feeding the borage oil/CA (1:2, w/w) mixture saturated with water into a column packed with the enzyme. However, the generation of partial glycerides (20%) in the reaction mixture showed that hydrolysis occurred concomitantly with acidolysis. The concomitant hydrolysis was completely repressed by feeding the oil/CA substrate mixture without adding additional water. When the substrate mixture was fed at 30°C and a flow rate of 4.5 mL/h into a column packed with 8 g of the carrier with immobilized lipase, the content of CA incorporated in glycerides was 50 to 55 mol%. The acidolysis activity scarcely changed even though the substrate mixture was continuously fed for 60 d; then it gradually decreased. The CA content in glycerides was decreased to 73% of the initial value after 100 d, but returned to the initial level when the flow rate was reduced to 3.1 mL/h. Molecular distillation was employed to separate the transesterified oil from the reaction mixture. No glycerides were detected in the distillate, and the transesterified oil was recovered as the residue (acid value, 2.6). Regiospecific analysis of the transesterified oil showed that only fatty acids at the 1- and 3-positions of borage oil were exchanged for CA. It was additionally found by high-performance liquid chromatography analysis that all the triglycerides contained one or two CA, and that the triglyceride with two GLA and one CA was also present, because the lipase acted on GLA very weakly.     

Designs of Bioreactor Systems for Solvent-Free Lipase-Catalyzed Synthesis of Fructose–Oleic Acid Esters

Fructose–oleic acid esters, biodegradable, biocompatible and biobased surfactants and value-added products were synthesized under solvent-free conditions at 65 °C in stirred-batch mode and using several different bioreactor systems. For a stirred-tank bioreactor (STBR) using fed-batch fructose addition and 5.0 wt.% immobilized Rhizomucor miehei lipase (Lipozyme^® IM, Novozymes, Franklinton, NC), the conversion yield was over 80%, and the initial rate of the reaction was comparable to previously obtained results using tert-butanol during the initial phase. The bioreactor systems contained a packed “desorption” column (DC) containing fructose crystals and silica gel for delivery of saccharide, and either a STBR or packed-bed bioreactor (PBBR). The liquid stream, initially containing oleic acid and a mixture of fructose–oleic acid esters at a ratio of 75/25 w/w, was continuously recirculated throughout the system. The PBBR system yielded the highest conversion (84.4%) and rate of reaction subsequent to the addition of 10 wt.% molecular sieves during the latter stage of reaction; however, the reaction rate was several-fold lower than the batch mode reactions due to the lower fructose concentrations provided by the DC.     

Lipase-catalyzed esterification of glycerol and n-3 polyunsaturated fatty acid concentrate in organic solvent

Enzymatic synthesis of glycerides from glycerol and n-3 polyunsaturated fatty acid in organic solvent was studied. Optimal conditions for glyceride synthesis by lipases were established. Of the commercially available lipases that were investigated, lipase PS-30 fromPseudomonas sp. and lipase IM-60 fromMucor miehei resulted in the highest extent of esterification. Isooctane and hexane were particularly useful organic solvents in glyceride synthesis. The water content in the reaction mixture was of primary importance. For lipase PS-30 and lipase IM-60, optimal water contents were 5 and 1%, respectively. Lipases PS-30 and IM-60 manifested contrasting positional specificities in glyceride synthesis. Glycerides containing predominantly eicosapentaenoic acid and docosahexaenoic acid can be easily synthesized.     

Selective hydrolysis of borage oil with Candida rugosa lipase: Two factors affecting the reaction

A 46% γ-linolenic acid (GLA)-containing oil was produced by selective hydrolysis of borage oil (GLA content, 22%) at 35°C for 15 h in a mixture containing 50% water and 20 units (U)/g reaction mixture of Candida rugosa lipase. The GLA content was not raised over 46%, even though the hydrolysis extent was increased by extending the reaction time and by using a larger amount of the lipase. However, 49% GLA-containing oil was produced by hydrolysis in a reaction mixture with 90% water. This result suggested that free fatty acids (FFA) that accumulated in the mixture affected the apparent fatty acid specificity of the lipase in the selective hydrolysis and interfered with the increase of the GLA content. To investigate the kinetics of the selective hydrolysis in a mixture without FFA, glycerides containing 22, 35, and 46% GLA were hydrolyzed with Candida lipase. The result showed that the hydrolysis rate decreased with increasing GLA content of glycerides, but that the release rate of GLA did not change. Thus, it was found that the apparent fatty acid specificity of the lipase in the selective hydrolysis was also affected by glyceride structure. When 46% GLA-containing oil was hydrolyzed at 35°C for 15 h in a mixture containing 50% water and 20 U/g of the lipase, GLA content in glycerides was raised to 54% at 20% hydrolysis. Furthermore, GLA content in glycerides was raised to 59% when the hydrolysis extent reached 60% using 200 U/g of the lipase. These results showed that repeated hydrolysis was effective to produce the higher concentration of GLA oil. Because film distillation was found to be extremely effective for separating FFA and glycerides, large-scale hydrolysis of borage oil was attempted. As a result, 1.5 kg of 56% GLA-containing oil was obtained from 7 kg borage oil by repeated reaction.     

Phospholipase A2-catalyzed hydrolysis of lecithin in a continuous reversed-micellar membrane bioreactor

Lysophospholipids and free fatty acids produced by lecithin hydrolysis are important natural compounds with high potential for application in the food, chemical, and pharmaceutical industries. In this work, the enzymatic hydrolysis of lecithin (essentially phosphatidylcholine) catalyzed by porcine pancreatic phospholipase A₂ (phosphatide 2-acyl-hydrolase, EC 3.1.1.4), encapsulated in mixed reversed micelles of lecithin andbis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, was carried out in a continuous reversed-micellar membrane bioreactor. A tubular ceramic membrane with a 10,000 molecular weight (MW) cutoff was installed in an ultrafiltration module to retain the phospholipase A₂ (MW 14,000) and to continuously separate the products from the reaction media. Water and co-factor (Ca⁺⁺)-containing reversed micelles of lecithin/AOT in isooctane were supplemented to the reactor to compensate for the permeation of reversed micelles and to continuously supply the substrate. The influence of relevant parameters, such as substrate, AOT and enzyme concentrations, water content and fluid hydrodynamics, on the performance of the ultrafiltration membrane bioreactor was investigated. Fluid axial velocity and substrate concentration were the major factors that affected the transport processes through the membrane. Permeate flow rate increased significantly with fluid axial velocity and decreased with substrate concentration; on the other hand, water and enzyme concentrations were identified as critical parameters for the final conversion of lecithin. The relationship between productivity and normalized residence time was analyzed for each set of experimental parameters tested. Operational stability of the bioreactor was tested in a long-term operation to confirm the high stability of this catalytic system.     

Glyceride synthesis in a solvent-free system

Synthesis of partial glycerides in a solvent-free system has been investigated with various acyl donors and glycerol as substrates and a 1,3-specific immobilized lipase to catalyze the reaction. Capric acid was the most efficient acyl donor, compared with ethyl caprate and tricaprin. However, to obtain a high yield of dicaprin and a low amount of tricaprin, ethyl caprate was the acyl donor of choice. The composition of the product mixture was determined by the ratio of ethyl caprate to glycerol; a molar ratio of 3∶1 was optimum for dicaprin synthesis. The water content in glycerol did not influence the final yield of dicaprin, but initial production of capric acid increased with increasing water content. The reaction was found to be controlled entirely by external mass transfer. The yield of diglyceride could be increased from 70 to 90% by lowering the reaction temperature, so that the diglyceride precipitated during the reaction.     

Production of medium-chain glycerides by immobilized lipase in a solvent-free system

Enzymatic synthesis of medium- chain glycerides (MCGs) was studied by using capric acid (decanoic acid) and glycerol as substrates for immobilized lipase (Lipozyme^TM) without any solvents or surfactants. Quantitative analysis of the reaction mixture was conducted by using highperformance liquid chromatography (HPLC), which enabled the exact tracing of the capric glyceride synthesis. Oleic acid was also used for comparison. The esterification activity of Lipozyme was determined at 40°C in an open batch reactor; the activities were 400 and 200 units/g for the capric glyceride and oleic glyceride synthesis, respectively. Maximum initial reaction rate was obtained at 50°C for capric and 60°C for oleic glyceride synthesis. The time course of the capric glyceride synthesis was compared in terms of different molar ratios, from which we infer that this enzyme is 1,3- specific, but not absolute, in this esterification reaction. The final conversion was greatly influenced by the methods used to remove water, among which the cold trap method resulted in a noticeable improvement. *To whom correspondence should be addressed at Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, P.O. Box 150, Chongyang, Seoul 130-650, Korea.     

Utilization of biodiesel and glycerol for the synthesis of epoxidized acyl glycerides and its application as a plasticizer

In many regions, even if waste vegetable oil is used as raw material, the profits of biodiesel (fatty acid methyl ester, FAME) enterprises still remain restricted, and they are facing environmental problems caused by the by-product glycerol. Hence, value-added utilization of FAME and glycerol is a promising alternative to improve the competitiveness of biodiesel enterprises in the economy and environmental protection. In this paper, FAME and glycerol were utilized to prepare epoxidized acyl glycerides by transesterification and epoxidation. In the transesterification, acyl glycerides were prepared by reacting FAME and glycerol catalyzed by K₂CO₃. The contents of monoacyl glyceride, diacyl glyceride, and triacyl glyceride in the acyl glycerides were about 50%, 40%, and 10%, and the optimal conditions were a temperature of 200°C and the molar ratio of FAME to glycerol of 1:1. In the epoxidation, selectivity of epoxides was significantly affected by monoacyl glyceride content, and a treatment method was developed to control the monoacyl glyceride content <5%. In addition, the plasticity of epoxidized acyl glycerides to partially substitute dioctyl phthalate in polyvinyl chloride was studied, the overall mechanical properties, thermal stability, and extraction resistance were improved. In this work, FAME and glycerol were utilized to prepare epoxidized acyl glycerides with higher value-added, which provide a potential solution for improving the market competitiveness of biodiesel enterprises.     

Kinetic resolution of 1‐phenylethanol integrated with separation of substrates and products by a supported ionic liquid membrane

BACKGROUND: In the present study, the kinetic resolution of rac‐1‐phenylethanol by transesterification with several vinyl esters catalysed by a commercial immobilized Candida antarctica lipase B (Novozym 435) was carried out in n‐hexane at different water contents. The subtrates and products involved in the kinetic resolution were separated using a membrane bioreactor containing a supported liquid membrane based on the ionic liquid 1‐butyl‐3‐methylimidazolium tetrafluoroborate [bmim⁺][BF₄^?]. RESULTS: Variables affecting the kinetic resolution performance of the enzyme were studied. First, the influence of water content of the medium on the synthetic activity, selectivity and enantioselectivity of the enzyme was analysed in order to establish the optimal amount of water. The use of vinyl esters of different alkyl chain length (vinyl propionate, vinyl butyrate and vinyl laurate) as acyl donors to kinetic resolution was studied. Finally, the integrated reaction/separation process for the resolution of rac‐1‐phenylethanol was carried out in the optimal conditions found. CONCLUSION: These investigations demonstrate that the coupling of lipase enantioselectivity with the selective separation of supported liquid membranes based on ionic liquids provides a promising basis for practical production of enantiomerically pure or enriched compounds. Copyright © 2008 Society of Chemical Industry     

Hydrogen production by aqueous-phase reforming of glycerol over nickel catalysts supported on CeO2

Nickel catalysts supported on CeO₂ were prepared and evaluated in aqueous-phase reforming of glycerol. Three different methodologies of synthesis were used: wet impregnation, co-precipitation and combustion, and the catalysts were characterized by chemical composition, textural analysis, crystalline structure and reducibility. The reaction was carried out in a batch reactor with solution of 1 and 10 wt.% glycerol, at 523 and 543 K. A maximum glycerol conversion of 30% was achieved by the catalyst prepared by combustion at 543 K using solution 1% glycerol. In the gas phase, the molar fraction of H₂ was always higher than 70% and formation of CH₄ was very low (< 1%). The increase in glycerol concentration decreases the conversion and H₂ formation.     

Oat (Avena sativa) caryopses as a natural lipase bioreactor

The caryopses of oats, when moistened and immersed in oils, constitute a natural lipase bioreactor. Hydrolysis was monitored by titration of the free fatty acids in the oil phase and by thin-layer chromatography. The optimum amount of additional water was about 20% of the weight of the caryopses, and the optimum temperature was about 40°C. The reaction was accelerated by gentle agitation, by reducing the viscosity of the oil phase by the addition of nonpolar solvents, and by increasing the amount of lipase on the caryopses. The reaction was inhibited by the accumulation of glycerol in the interior of the caryopses and free fatty acids in the oil phase. The lipase hydrolzyed all three positions of glycerol and there was little accumulation of mono- or diglyceride in the lipid phase. The time necessary to obtain 90% hydrolysis varied for a few days to several weeks. Greater degrees of hydrolysis could be obtained by replacing the caryopses when they became inhibited or by diluting the oil phase with hexane. The glycerol that was released could be recovered by extracting the caryopses with water. The moist oat bioreactor also was capable of catalyzing transesterification and interesterification reactions. Journal Paper No. 13447 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Project No. 2799.     

Enrichment of polyunsaturated fatty acids withGeotrichum candidum lipase

Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.