Response surface modelling of the production of ω-3 polyunsaturated fatty acids-enriched fats by a commercial immobilized lipase

The aim of this study was to model the production of fats, enriched with ω-3 polyunsaturated fatty acids (ω-3 PUFA) for nutraceutical purposes, via the response surface methodology. These fats were obtained by transesterification of palm oil stearin (POS) with a concentrate (EPAX 2050TG) of triglycerides enriched with ω-3 PUFA and soybean oil, catalysed by a commercial immobilized Candida antarctica lipase (“Novozym 435”).

The initial water activity (a_w) of the biocatalyst, POS and EPAX 2050TG concentrations, time and temperature showed a significant effect on the transesterification reaction, as well as on the competing reactions of hydrolysis and lipid oxidation.

Depending on the factors included, the transesterification reaction was described either by first- or second-order models.

The production of free fatty acids, which is ascribed both to the hydrolytic reaction and the mechanism of lipase-catalysed transesterification, showed a second-order dependence on the initial a_w of the biocatalyst.     

Different enzyme requirements for the synthesis of biodiesel: Novozym 435 and Lipozyme TL IM

Enzymatic syntheses of biodiesel via alcoholysis of different vegetable oils (sunflower, borage, olive and soybean) have been studied. Loss of lipase activity induced by the nucleophile is greater with methanol than with ethanol, and is greater for Lipozyme TL IM than for Novozym 435. The optimum volume of ethanol depends on the loading of solid biocatalyst and is higher for preparations of Novozym 435 than for Lipozyme TL IM. Maximum rates were obtained with Lipozyme TL IM, for a molar ratio of alcohol to FA residues of 0.33. By contrast, Novozym 435 requires at least a 2:1 ratio. Alcoholysis of the vegetable oils is faster with Lipozyme TL IM than with Novozym 435. Use of a high loading of Novozym 435 (50% w/w) and a large molar excess of ethanol are required to obtain an initial rate similar to that obtained with Lipozyme TL IM at a lower enzyme loading (10% w/w) and an equimolar ratio of ethanol and FA residues. Novozym 435 produces quantitative conversions in only 7h at 25 degrees C, but complete conversions are not obtained with Lipozyme TL IM. Three stage stepwise addition of ethanol yields 84% conversion to ethyl esters for Lipozyme TL IM. Hence use of Novozym 435 is preferred. After nine cycles in a batch reactor Novozym 435 retained 85% of its initial activity.     

Production of extremely pure diacylglycerol from soybean oil by lipase-catalyzed glycerolysis

Research work was objectively targeted to synthesize highly pure diacylglycerol (DAG) with glycerolysis of soybean oil in a solvent medium of t-butanol. Three commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM and Novozym 435) were screened, and Novozym 435 was the best out of three candidates. Batch reaction conditions of the enzymatic glycerolysis, the substrate mass ratio, the reaction temperature and the substrate concentration, were studied. The optimal reaction conditions were achieved as 6.23:1 mass ratio of soybean oil to glycerol, 40% (w/v) of substrate concentration in t-butanol and reaction temperature of 50 °C. A two-stage molecular distillation was employed for purification of DAG from reaction products. Scale-up was attempted based on the optimized reaction conditions, 98.7% (24 h) for the conversion rate of soybean oil, 48.5% of DAG in the glycerolysis products and 96.1% for the content of DAG in the final products were taken in account as the results.     

Response surface modeling of glycerolysis catalyzed by Candida rugosa lipase immobilized in different polyurethane foams for the production of partial glycerides

Monoglycerides (MG) and diglycerides (DG) are the most widely used emulsifiers in food and pharmaceutical industries. In this study, MG and DG were produced by inter-esterification of refined olive residue oil with glycerol (glycerolysis), in n-hexane, catalyzed by Candida rugosa lipase immobilized in different biocompatible hydrophilic polyurethane foams, A and B. These foams, with aquaphilicities of 3.7 and 2.8, were prepared with a toluene diisocyanate (“Hypol FHP 2002™”) and a diphenylmethane diisocyanate (“Hypol FHP X4300^TM”) pre-polymer, respectively.Response surface methodology was used for modeling the reaction, as a function of the molar ratio glycerol/triglycerides (Gly/TG, 0.5–2.0) and the initial water activity (a_w) of the biocatalyst (A, 0.24–0.91; B, 0.37–0.91). Experiments were carried out following a central composite rotatable design. With lipase in foam A, production of MG and DG could be described by first order polynomials. With foam B, MG and DG production could be fitted to concave and flat surfaces, described by a second and a first orders polynomials, respectively.The best productions of MG and DG were achieved with the lipase in the less hydrophilic foam, B: at 24 h reaction time, 32% (w/w) MG and 18% (w/w) DG were obtained, when the initial a_w of the biocatalyst was 0.83, with a Gly/TG of 1.     

Kinetics of enzymatic synthesis of L-ascorbyl acetate by Lipozyme TLIM and Novozym 435

L-ascorbyl acetate was synthesized through lipase-catalyzed esterification using Lipozyme TLIM and Novozym 435. Four solvents, including methanol, ethanol, acetonitrile, and acetone were investigated for the reaction, and acetone and acetonitrile were found to be suitable reaction media. The influences of several parameters such as water activity (a _w), substrate molar ratio, enzyme loading, and reaction temperature on esterification of L-ascorbic acid were systematically and quantitatively analyzed. Through optimizing the reaction, lipase-catalyzed esterification of L-ascorbic acid gave a maximum conversion of 99%. The results from using Lipozyme TLIM and Novozym 435 as biocatalysts both showed that a _w was an important factor for the conversion of L-ascorbic acid. The effect of pH value on lipase-catalyzed L-ascorbic acid esterification in acetone was also investigated. Furthermore, results from a kinetic characterization of Lipozyme TLIM were compared with those for Novozym 435, and suggested that the maximum reaction rate for Lipozyme TLIM was greater than that for Novozym 435, while the enzyme affinity for substrate was greater for Novozym 436.     

Wax esters synthesis from heavy fraction of sheep milk fat and cetyl alcohol by immobilised lipases

Wax esters were obtained from lipase-catalysed alcoholysis of triglycerides with cetyl alcohol, using n-hexane as solvent. The heavy triglyceride fraction (HTF), obtained by fractionation of sheep milk fat, was used as raw material. In the natural fat mixture GC analysis showed that palmitic, myristic, stearic and oleic acids are the most abundant fatty acids which are useful to produce wax esters. Reactions were tested for different amounts of Lipozyme RMIM catalyst, and the optimum concentration of 10 mg catalyst/ml solution has been determined. The formation of the four main products, i.e. cetyl myristate, cetyl palmitate, cetyl oleate and cetyl stearate, was determined by HPLC/ELSD quantitative analysis. The optimum water activity in the reaction medium a_w=0.35 in the case of Lipozyme RMIM, and a_w=0.53 for Novozym 435 was found. Lipozyme RMIM (immobilised sn-1,3-specific lipase from Rhizomucor miehei) was more active than Novozym 435 (immobilised nonspecific lipase-B from Candida antarctica) towards wax esters production. The acyl migration of 2-monoglycerides was suggested as a crucial step to explain the higher yields produced by the 1,3-specific lipase.     

Influence of compressed fluids treatment on the activity of Yarrowia lipolytica lipase

This work investigated the influence of temperature, pressure, exposure times and depressurization rate on the activity of a non-commercial immobilized lipase from Yarrowia lipolytica (YLL) submitted to compressed carbon dioxide, propane and n-butane. A high-pressure cell was employed in the experiments, in the pressure range of 10–280 bar, varying the temperature from 35 to 75 °C, exposure times from 1 to 6 h, and adopting distinct decompression rates. Results showed that significant activity losses were obtained when the treatment was conducted in carbon dioxide, while negligible losses were observed in both propane and n-butane. For the treatment with carbon dioxide, within the range studied, the decompression rate affected positively enzyme activity, while the exposure time and temperature presented an opposite effect on the non-commercial immobilized lipase from Y. lipolytica (YLL). Additionally, the performance of two commercial immobilized lipases (Lipozyme IM and Novozym 435) and the immobilized YLL in the three solvents was compared. Immobilized YLL has shown to be more suitable than Lipozyme IM for enzyme-catalyzed reactions using compressed propane and n-butane as solvents, but with inferior performance compared to Novozym 435 treated in these solvents.     

Kinetic modeling of immobilized-lipase catalyzed transesterification of n-octanol with vinyl acetate in non-aqueous media

Organic esters are employed as solvents, fragrance, flavors, and precursors in a variety of industries. Particularly, aliphatic esters are greatly used in flavor industry, mainly as fixatives and modifiers, and aromatic esters in fragrance compositions. Esters are produced by a variety of methods among which esterification and transesterification with acid catalysts under reflux conditions are prominent. The use of biocatalysts provides an opportunity for carrying out reactions under milder conditions leading to better quality products suitable in fragrance and flavor industry. Transesterification of n-octanol with vinyl acetate was studied at 30 °C as a model reaction by employing different lipases as catalysts such as Psedomonas species lipase immobilized on diatomite, free Candida rugosa lipase. Novozym 435 (lipase B from Candida antarctica; immobilized on macro-porous polyacrylic resin beads) and Lipozyme IM 20 (Mucor miehei lipase immobilized on anionic resin). Novozym 435 was found to be the most active catalyst in heptane as a solvent. A conversion of 82% with 100% selectivity of n-octyl acetate was obtained at 30 °C in 90 min using equimolar quantities of the reactants with 0.833 g l⁻¹ of Novozym 435. Transesterification of other alcohols such as n-decanol, benzyl alcohol, cinnamyl alcohol, 2-ethyl-1-hexanol, 1-phenyl ethyl alcohol, and 2-phenyl ethyl alcohol was also studied with vinyl acetate. The analysis of the initial rate data and progress curve data showed that the reaction obeys the ternary complex bi–bi mechanism with inhibition by n-octanol. The experimental and theoretical values matched very well.

The order of transesterification reactivity of vinyl acetate with various alcohols in presence of Novozym 435 under otherwise identical conditions at 30 °C was found to be as follows:

n-octanol>n-decanol>benzylalcohol>cinnamylalcohol>2-ethyl-1-hexanol>2-phenylethylalcohol>1-phenylethylalcohol.

     

Enzymatic Synthesis of Lipophilic Caffeoyl Lipids Using Soybean Oil as the Novel Acceptor

Soybean oil-based caffeoyl lipids are the novel lipophilic derivatives of caffeic acid, which can be used as UV absorbers and antioxidants in the food and cosmetic industries. In the work, the novel lipophilic structured lipids were prepared using soybean oil as the novel caffeoyl acceptor by enzymatic transesterification. The effects of the reaction variables on the transesterification were investigated, and response surface methodology was used to optimize the reaction variables. Reactions were monitored by HPLC-UV. Different enzymes (Novozym 435, Lipozyme RMIM, and Lipozyme TLIM) were used as biocatalysts, and Novozym 435 showed the best performance for the reaction. The results showed that a high lipophilic soybean oil-based caffeoyl lipids yield (73.5 ± 1.2%) was achieved under the optimal conditions (reaction temperature 85°C, substrate molar ratio 1:6 (ethyl caffeate (EC)/soybean oil), enzyme load 25% (w/w), and 60 h at atmosphere pressure). The activation energies of EC conversion, hydrophilic glyceryl caffeates (GC) and lipophilic caffeoylated acylglycerol (CAG) formations were 32.92 kJ/mol, 17.21 kJ/mol and 57.36 kJ/mol, respectively. Km and Vm were 0.022 mol/L and 0.033 × 10^-3 mol/(Lmin), respectively.     

复合脂肪酶催化生物柴油的初步研究

初步探讨了复合脂肪酶催化生物柴油的工艺。优化了复合酶配比条件和叔丁醇反应体系。在无溶剂体系中,Novozym435分别与Lipozyme TLIM和Lipozyme RMIM均以70／30质量比混合时,甲酯得率分别达到94．52％和96．25％,比Novozym435单独催化时的甲酯得率分别提高了9．52％和9．99％。在叔丁醇体系中,当Novozym435与Li-pozyme TLIM和Lipozyme RMIM分别以60／40和80／20的质量比混合时,其甲酯得率分别为85．06％和81．5％,比Novozym435单独催化的效率分别提高了9．89％和7．48％。优化叔丁醇体系中复合酶催化条件后,甲酯得率达92％。     

Immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads improves butyl acetate synthesis

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.     

Lipase-catalyzed ammonolysis of trimethylsilylmethyl acetate in organic solvent

Lipase could catalyze the ammonolysis of trimethylsilylmethyl acetate in organic solvents and Novozym 435 was the best biocatalyst for the reaction. The influences of some factors on the reaction were investigated. Cyclohexane, n-hexane and heptane were found to be suitable reaction media and ammonium carbamate was the best ammonium source. The optimal initial water activity, temperature and pH value were 0.55-0.75, 35°C and 6.5 respectively, under which a substrate conversion of 97.6% could be achieved after reaction for 140 h.     

Immobilized lipase-catalysed synthesis of cinnamyl laurate in non-aqueous media

Esters of cinnamyl alcohol find many applications in food, cosmetic and pharmaceutical industries as flavor and fragrance compounds. The current work focuses on the synthesis of cinnamyl laurate from cinnamyl alcohol and lauric acid, including screening of various immobilized lipases and optimization of reaction conditions such as catalyst loading, speed of agitation, mole ratio and temperature. Among different lipases screened such as Novozym 435, Lipozyme RM IM and Lipozyme TL IM, Novozym 435 was found to be the best catalyst with 60% conversion in 2 h at 30 °C for equimolar quantities of the reactants using 0.33% (w/v) of catalyst and toluene as solvent. An ordered bi–bi mechanism with dead-end complex of lauric acid was found to represent the kinetic data.     

Lipase-catalyzed transesterification of rapeseed oils for biodiesel production with a novel organic solvent as the reaction medium

tert-Butanol, as a novel reaction medium, has been adopted for lipase-catalyzed transesterification of rapeseed oil for biodiesel production, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. Combined use of Lipozyme TL IM and Novozym 435 was proposed further to catalyze the methanolysis and the highest biodiesel yield of 95% could be achieved under the optimum conditions (tert-butanol/oil volume ratio 1:1; methanol/oil molar ratio 4:1; 3% Lipozyme TL IM and 1% Novozym 435 based on the oil weight; temperature 35 °C; 130 rpm, 12 h). There was no obvious loss in lipase activity even after being repeatedly used for 200 cycles with tert-butanol as the reaction medium. Furthermore, waste oil was also explored for biodiesel production and it has been found that lipase also showed good stability in this novel system.     

Production of monoglycerides by glycerolysis of olive oil with immobilized lipases: effect of the water activity

The production of monoglycerides by glycerolysis of olive oil catalyzed by lipases from Candida rugosa, Chromobacterium viscosum and Rhizopus sp. immobilized in a hydrophylic polyurethane foam was investigated. The effect of the amount of aqueous phase used for foam polymerization on the competing reactions of glycerolysis and hydrolysis was studied. The highest monoglyceride production was achieved with the C. rugosa lipase which was thus selected for subsequent studies.The extent to which hydrolysis and glycerolysis occur and the influence of the initial a _w of the system on both reactions were also investigated. In glycerolytic reaction systems, initial rates of fatty acid release were always higher than in hydrolytic systems. At a _w values lower than 0.43, hydrolysis was completely repressed, although glycerolysis still occurred. This suggests that hydrolysis of the ester bond in the glyceride, promoted by glycerol, is the first reaction step.In glycerolysis, initial rates of FFA and DG production increased exponentially with the initial a _w of the system.The lowest total conversion (in terms of % TG consumed) at 48 hours was obtained at intermediate a _w values; higher conversions at extreme a _w indicated an increase in hydrolytic and glycerolytic rates, at high and low a _w respectively.The yield of MG increased with decreasing a _w . The highest yield of MG (70%, w/w) was obtained at the lowest a _w used (0.23). The initial a _w of the reaction system is an important parameter in glycerolysis.List of Symbols a _w thermodynamic activity of water - C ₁₂₀ lauric acid - C ₁₄₀ myristic acid - C ₁₈₁ oleic acid - DG dediglyceride (s) - FAME fatty acid methyl ester (s) - FFA free fatty acid (s) - FID fire ionization detector - Gly glycerol - n number of replicates - MG monoglyceride (s) - PCA principal component analysis - PU polyurethane - r correlation coefficient - TG triglyceride (s) - TO triolein - significance level The authors are grateful to Prof. L. Beirão da Costa and to Prof. L. Campos, Inst. Sup. de Agronomia (ISA), and to Prof. J.M.S. Cabral, Inst. Sup. Técnico, Lisbon, Portugal, for inspiring discussions and advice, and to Mrs. Marlene Dionísio, ISA, for invaluable help with some of the experimental work.     

Enhancement of Novozym-435 catalytic properties by physical or chemical modification

Physical (ionic exchange of ionic polymers) or chemical (aminoethylamidation, succinylation, hydroxyethylamidation) modifications of Novozym 435 have been performed and the resulting biocatalysts have been assayed in diverse reactions. The coating of the immobilized enzyme with dextran-sulphate via ionic exchange permitted to increase the asymmetric factor of the biocatalyst from A = 13 (ee = 83%) to 24 (ee > 90%) in the hydrolysis of 3-phenylglutaric acid dimethyl diester, producing the (R)-monomethyl ester. The chemical succinylation of Novozym 435 permitted to enhance the biocatalyst enantiospecificity from E = 1 to 13 in the hydrolysis of (±)-mandelic acid methyl ester. In the hydrolysis of (±)-2-O-butyryl-2-phenylacetic acid, the enantiospecificity of Novozym 435 was very high towards the S-enantiomer (E > 100) but it was inverted after the chemical hydroxyethylamidation of the immobilized enzyme (E = 6.6 towards R-enantiomer).Thus, these simple protocols seem to be a very powerful tool to generate a library of biocatalysts from Novozym 435 with very different catalytic properties.     

Glycerolysis of olive oil: batch operational stability of Candida rugosa lipase immobilized in hydrophilic polyurethane foams

The operational stability of the Candida rugosa lipase immobilized in a hydrophilic polyurethane foam was evaluated in consecutive batches for the glycerolysis of olive oil in n-hexane, aimed at the production of monoglycerides.Glycerol controlled the glycerolysis in the system under study, since it is both a substrate and a powerful water binder that reduces the water activity of the reaction medium and of the microenvironment. Two sets of experiments were carried out under different glycerol/triglyceride ratios. After 345 hours of consecutive 23 hours batches no lipase inactivation was observed.List of Symbols a_w thermodynamic activity of water - DG diglyceride (s) - FAME fatty acid methyl ester (s) - FFA free fatty acid (s) - FID flame ionization detector Gly glycerol - MG monoglyceride (s) - TG triglyceride (s) - TLC thin layer chromatography The authors are grateful to Profs. P. Adlercreutz, Technical University of Lund, Sweden, and J.M.S. Cabral, Instituto Superior Técnico, Lisbon, Portugal, for inspiring discussions and advice, to Prof. Helena Pereira, Instituto Superior de Agronomia (ISA), Lisbon, Portugal, for the use of GC equipment and to Mrs. Marlene Dionísio, ISA, for invaluable help with some of the experimental work.     

Production of human milk fat substitutes enriched in omega-3 polyunsaturated fatty acids using immobilized commercial lipases and Candida parapsilosis lipase/acyltransferase

In human milk fat (HMF), palmitic acid (20–30%), the major saturated fatty acid, is mostly esterified at the sn-2 position of triacylglycerols, while unsaturated fatty acids are at the sn-1,3 positions, conversely to that occurring in vegetable oils.This study aims at the production of HMF substitutes by enzyme-catalyzed interesterification of tripalmitin with (i) oleic acid (system I) or (ii) omega-3 polyunsaturated fatty acids (omega-3 PUFA) (system II) in solvent-free media. Interesterification activity and batch operational stability of commercial immobilized lipases from Rhizomucor miehei (Lipozyme RM IM), Thermomyces lanuginosa (Lipozyme TL IM) and Candida antarctica (Novozym 435) from Novozymes, DK, and Candida parapsilosis lipase/acyltransferase immobilized on Accurel MP 1000 were evaluated. After 24-h reaction at 60 °C, molar incorporation of oleic acid was about 27% for all the commercial lipases tested and 9% with C. parapsilosis enzyme. Concerning omega-3 PUFA, the highest incorporations were observed with Novozym 435 (21.6%) and Lipozyme RM IM (20%), in contrast with C. parapsilosis enzyme (8.5%) and Lipozyme TL IM (8.2%). In system I, Lipozyme RM IM maintained its activity for 10 repeated 23-h batches while for Lipozyme TL IM, Novozym 435 and C. parapsilosis enzyme, linear (half-life time, t_1/2 = 154 h), series-type (t_1/2 = 253 h) and first-order (t_1/2 = 34.5 h) deactivations were respectively observed. In system II, Lipozyme RM IM showed linear deactivation (t_1/2 = 276 h), while Novozym 435 (t_1/2 = 322 h) and C. parapsilosis enzyme (t_1/2 = 127 h), presented series-type deactivation. Both activity and stability of the biocatalysts depended on the acyl donor used.     

Lipase-catalyzed glycerolysis in ionic liquids directed towards diglyceride synthesis

This work examined the lipase-catalyzed glycerolysis of triglycerides (TG) in a list of commercially available ionic liquids (ILs) with varied cations and anions for the purpose of developing an efficient reaction protocol for diglyceride (DG) production and to understand whether ILs could assist the reaction systems. The reaction performances (reaction rate, TG conversion and DG yield) were found to be greatly dependent on the structure and property of ILs. The reactions in [TOMA·Tf₂N] and Ammoeng 120 produced comparable yield of DG to those most efficient conventional systems but with less by-products. Temperature enhancement generally yields positive effect on the conversion of TG, which was much more significant for the ILs with high viscosity. Unusually, increasing substrate concentration in many types of ILs led to enhanced conversion and yield; whereas the increase of glycerol/TG ratio resulted in a dramatic improvement of the reactions in the ILs with strong acidic anions. This work also sorted out some promising IL candidates, namely the ILs with good DG formation selectivity and the ones being able to achieve high TG conversion, which offered possibility to design binary IL systems. Overall, this study presented the first attempt concerning evaluation and characterization of lipase-catalyzed glycerolysis of TG for DG production in IL-based systems.     

Enzymatic synthesis of trieicosapentaenoylglycerol in a solvent-free medium

The enzymatic synthesis of trieicosapentaenoylglycerol from glycerol and eicosapentaenoic ethyl ester in a solvent-free medium is studied here. Novozym SP 435 (immobilized lipase from Candida antarctica) has appeared as a very efficient biocatalyst for this transesterification. A nitrogen bubbling has allowed a good mixing and also the shifting of the reaction toward synthesis by eliminating the ethanol formed. The effect of temperature and of the quantity of lipase has been studied. In the optimal conditions (T=80°C, 5% (w/w) of lipase, 1 mol glycerol for 3 mol ethyl ester), pure triglyceride has been obtained after 10 h.