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Referencias bibliográficas: • Becker L. R., & Hermosura B. A. (2019) Simulation Education Theory. In S. Deering, T. Auguste, & D. Goffman (Eds.) Comprehensive Healthcare Simulation: Obstetrics and Gynecology. Comprehensive Healthcare Simulation (pp. 11-24). Cham: Springer. doi: https://doi.org/10.1007/978-3-319-98995-2_2 • Candiago, A., & Kawamoto, L. T. (2016). Development and Validation of a Serious Game for Chemistry Laboratories. Spaces, 37(11), E-1. • Carrillo, J. L., & Cortés, J. A. (2016). Secuencias didácticas con realidad virtual: En el área de geometría en educación básica. F@ro: revista teórica del Departamento de Ciencias de la Comunicación, 1(23), 2. • Cook, D. A., Hatala, R., Brydges, R., Zendejas, B., Szostek, J. H., Wang, A. T., . . . Hamstra, S. J. (2011). Technology-enhanced simulation for health professions education: a systematic review and meta-analysis. Journal of the American Medical Association, 306(9), 978-988. doi:https://doi.org/10.1001/jama.2011.1234 • Corvetto, M., Bravo, M. P., Montaña, R., Utili, F., Escudero, E., Boza, C., . . . Dagnino, J. (2013). Simulation in medical education: a synopsis. Revista médica de Chile, 141(1), 70-79. doi:https://doi.org/10.4067/S0034-98872013000100010 • Curcio, I. D. D., Dipace, A., & Norlund, A. (2016). Virtual realities and education. Research on Education and Media, 8(2), 60-68. doi:https://doi.org/10.1515/rem-2016-0019GS • Dalgarno, B., & Lee, M. J. W. (2010). What are the learning affordances of 3-D virtual environments? British Journal of Educational Technology, 41(1), 10-32. doi:https://doi.org/10.1111/j.1467-8535.2009.01038.x • De Freitas, S. (2006). Learning in immersive worlds: A review of game-based learning. Joint Information Systems Committee. Retrieved from: https://bit.ly/3aCB8GP • Facebook Technologies, LLC. (2018). RCSI Medical Training Sim. Retrieved from: https://ocul.us/2Y6byr6 • Facebook Technologies, LLC. (2019). Oculus. Retrieved from: https://www.oculus.co, El uso de realidad virtual (VR) y realidad aumentada (AR) para la mejora del aprendizaje es un tema de actualidad en la Educación Superior. El objetivo de este trabajo es evaluar el impacto de la simulación con realidad virtual (del inglés, VR-SBL) en el aprendizaje y satisfacción del estudiante. Para ello, se presentan dos casos llevados a cabo en la Universidad Europea de Madrid (UEM): el primero consiste en una experiencia de formación en primeros auxilios ante una emergencia de tráfico mediante el uso de tecnología de realidad virtual inmersiva, donde se ha comparado cómo aprenden los estudiantes con esta nueva metodología respecto a la clase magistral tradicional. El segundo caso consiste en la simulación de accidentes en un laboratorio mediante el uso de tecnología de realidad virtual de escritorio, donde se ha comparado cómo aprenden los estudiantes con esta metodología respecto a una simulación de dichos accidentes en el laboratorio real. En ambos casos se ha utilizado un diseño cuasi-experimental con un grupo experimental y un grupo de control. Además, se ha aplicado un diseño pretest-postest, que consiste en la realización de un test de conocimientos previo a la experiencia (pretest) y un cuestionario de conocimientos posterior a la misma (postest). Para medir la satisfacción con la actividad y la adecuación de las herramientas empleadas se ha utilizado un cuestionario de experiencia de usuario. Como conclusión, la satisfacción de los estudiantes en los dos casos denota una valoración alta. Además, en el primer caso, el grupo experimental que utiliza la realidad virtual inmersiva mejora significativamente el rendimiento académico. En cuanto al segundo caso, no se han encontrado diferencias significativas en cuanto al aprendizaje, por lo tanto, el nivel de aprendizaje en ambos grupos (simulación con realidad virtual y simulación en escenario real) es el mismo., Santander Universidades, Depto. de Estudios Educativos, Fac. de Educación, TRUE, pub

Immobilized enzymes have a very large surface region which is not in contact with the support surface and, thus, have potential as a target for novel stabilization strategies. In this paper, coating the surfaces of such enzymes with a highly hydrophilic and compact cross-linked poly-aminated polymer as a strategy to increase the thermal stability of the immobilized enzymes is proposed. In particular, Rhizomucor miehei lipase (RML) was immobilized by interfacial adsorption onto octyl-agarose and further coated with poly-allylamine (PAA), a polymer that is very rich in primary amino groups. Cross-linking of the PAA layer to coat the immobilized enzyme was carried out, in situ, by reaction with freshly oxidized dextran (aldehyde–dextran). The PAA layer only exerted moderate stabilizing effects (around 4-fold), but further cross-linking with aldehyde–dextran highly increased the stabilizing effects; the new derivative was 440-fold more stable than uncoated derivative at 55 °C and pH 7 and exhibited 6-fold more catalytic activity compared to the soluble enzyme used for immobilization. We hypothesize that the hydrophilicity of PAA reduces the exposure of internal hydrophobic pockets to the enzyme surface at high temperatures. Besides, the compactness of the polymer may reduce distortion of the enzyme surface during inactivation.

Lipase stability in organic solvent is crucial for its application in many biotechnological processes as biocatalyst. One way to improve lipase’s activity and stability in unusual reaction medium is its immobilization on inert supports. Here, lipases from different sources and immobilized through weak chemical interactions on hydrophobic and ionic supports had their transesterification ability dramatically dependent on the support and also on the solvent that had been used. The ethanolysis of sardine oil was carried out at the presence of cyclohexane and tert-amyl alcohol, in which Duolite A568-Thermomyces lanuginosa lipase derivative achieved 49% of ethyl esters production after 24 h in cyclohexane. The selectivity of immobilized lipases was also studied and, after 3 h of synthesis, the reaction with Duolite A568-Thermomyces lanuginosa derivative in cyclohexane produced 24% ethyl ester of eicosapentaenoic acid and 1.2% ethyl ester of docosahexaenoic acid, displaying a selectivity index of 20 times the ethyl ester of eicosapentaenoic acid. Different derivatives of Candida antarctica lipases fraction B (CALB) and phospholipase Lecitase® Ultra (Lecitase) were also investigated. Along these lines, a combination between these factors may be applied to improve the activity and selectivity of immobilized lipases, decreasing the total cost of the process.

Funding Este trabajo contó con el apoyo del Ministerio de Economía, Industria y Competitividad de España (proyectos BIO2012-36861 y CTQ2015-70348). Javier Rocha-Martin agradece la beca Juan de la Cierva (número de subvención IJCI-2014-19260 ) financiada por el Ministerio de Economía, Industria y Competitividad de España . También agradecemos a la Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) por la concesión de la beca a Erick Abreu-Silveira y por las becas 2016/10636-8 , 2015/10530-2 y 2013/20826-0 ., Different immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL) exhibited different properties for the ethanolysis of high oleic sunflower oil in solvent-free systems. TLL immobilized by interfacial adsorption on octadecyl (C-18) supports lost its 1,3-regioselectivity and produced more than 99% of ethyl esters. This reaction was influenced by mass-transfer limitations. TLL adsorbed on macroporous C-18 supports (616 Å of pore diameter) was 10-fold more active than TLL adsorbed on mesoporous supports (100–200 Å of pore diameter) in solvent-free systems. Both derivatives exhibited similar activity when working in hexane in the absence of diffusional limitations. In addition, TLL adsorbed on macroporous Purolite C-18 was 5-fold more stable than TLL adsorbed on mesoporous Sepabeads C-18. The stability of the best biocatalyst was 20-fold lower in anhydrous oil than in anhydrous hexane. Mild PEGylation of immobilized TLL greatly increased its stability in anhydrous hexane at 40 °C, fully preserving the activity after 20 days. In anhydrous oil at 40 °C, PEGylated TLL-Purolite C-18 retained 65% of its initial activity after six days compared to 10% of the activity retained by the unmodified biocatalyst. Macroporous and highly hydrophobic supports (e.g., Purolite C-18) seem to be very useful to prepare optimal immobilized biocatalysts for ethanolysis of oils by TLL in solvent-free systems., Ministerio de Economía, Industria y Competitividad (MINECO), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

The commercial enzyme Depol™ 333MDP (D333MDP) was immobilized by multipoint covalent attachment onto 10% cross-linked agarose beads support highly activated with aldehyde groups. The enzyme immobilization process was very efficient, retaining 86% of its initial catalytic activity. Thermal stability of the immobilized D333MDP biocatalysts varied according to the incubation time of the enzyme-support. The optimal immobilized biocatalyst was produced after 24 h of incubation under alkaline conditions and longer incubation times resulted in a loss of stability. The optimal immobilized biocatalyst was 60- and 50-fold more stable at pH 5.5 and pH 7 at 50 °C than the soluble enzyme, respectively. Activity and stability at pH 5.5 were enhanced when the optimal immobilized biocatalyst was modified by chemical amination of the enzyme surface. The chemical amination of the immobilized enzyme surface was 5-fold more stable at pH 5.5 and 50 °C compared with the unmodified immobilized biocatalyst. The best immobilized biocatalysts (containing 100 UI/g of support) were evaluated in the beechwood xylan hydrolysis reaction at 50 °C and pH 5.5. 80% of the reducing sugars were released after 6 h of hydrolysis with the aminated biocatalyst. Xylan hydrolysis reaction with the aminated biocatalyst was 80% faster than with the non-aminated one. The final composition of the xylooligosaccharides (XOS) obtained was identified and quantified by HPAEC-PAD which showed it was composed of 90% of xylobiose and 5% of xylotriose and xylose. The aminated immobilized-stabilized biocatalyst was used for four cycles of hydrolysis with no loss of catalytic activity, resulting in highly active and stable derivative suitable for industrial processes.

The present study focuses on the development and optimization of a packed-bed reactor (PBR) for continuous production of xylooligosaccharides (XOS) from xylan. For this purpose, three different methacrylic polymer-based supports (Relizyme R403/S, Purolite P8204F and Purolite P8215F) activated with glyoxyl groups were morphologically characterized and screened for the multipoint covalent immobilization of a xylanase. Based on its physical and mechanical properties, maximum protein loading and thermal stability, Relizyme R403/S was selected to set up a PRB for continuous production of XOS from corncob xylan. The specific productivity for XOS at 10 mL/min flow rate was 3277 gXOS genzyme−1 h−1 with a PBR. This PBR conserved >90% of its initial activity after 120 h of continuous operation.

The oleaginous yeast Moniliella spathulata R25L270 was the first yeast able to grow and produce extracellular lipase using Macaúba (Acrocomia aculeate) cake as substrate. The novel lipase was recently identified, and presented promising features for biotechnological applications. The M. spathulata R25L270 lipase efficiently hydrolyzed vegetable and animal oils, and showed selectivity for generating cis-5,8,11,15,17-eicosapentaenoic acid from sardine oil. The enzyme can act in a wide range of temperatures (25–48 °C) and pH (6.5–8.4). The present study deals with the immobilization of M. spathulata R25L270 lipase on hydrophobic, covalent and ionic supports to select the most active biocatalyst capable to obtain omega-3 fatty acids (PUFA) from sardine oil. Nine immobilized agarose derivatives were prepared and biochemically characterized for thermostability, pH stability and catalytic properties (KM and Vmax). Ionic supports improved the enzyme–substrate affinity; however, it was not an effective strategy to increase the M. spathulata R25L270 lipase stability against pH and temperature. Covalent support resulted in a biocatalyst with decreased activity, but high thermostability. The enzyme was most stabilized when immobilized on hydrophobic supports, especially Octyl-Sepharose. Compared with the free enzyme, the half-life of the Octyl-Sepharose derivative at 60 °C increased 10-fold, and lipase stability under acidic conditions was achieved. The Octyl-Sepharose derivative was selected to obtain omega-3 fatty acids from sardine oil, and the maximal enzyme selectivity was achieved at pH 5.0.

Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application

Different immobilized derivatives of two lipases were tested as catalysts of the synthesis of ethyl esters of omega-3 fatty acids during the ethanolysis of sardine oil in solvent-free systems at 25 °C. Lipases from Thermomyces lanuginosus (TLL) and Lecitase Ultra (a phospholipase with lipolytic activity) were studied. Lipases were adsorbed on hydrophobic Sepabeads C18 through the open active center and on an anion-exchanger Duolite with the active center exposed to the reaction medium. TLLSepabeads derivatives exhibit a high activity of 9 UI/mg of immobilized enzyme, and they are 20-fold more active than TLLDuolite derivatives and almost 1000-fold more active than Lipozyme TL IM (the commercial derivative from Novozymes). Lecitase-Sepabeads exhibit a high selectivity for the synthesis of the ethyl ester of EPA that is 43-fold faster than the synthesis of the ethyl ester of DHA.

Lipases are promising enzymes that catalyze the hydrolysis of triacylglycerol ester bonds at the oil/water interface. Apart from allowing biocatalyst reuse, immobilization can also affect enzyme structure consequently influencing its activity, selectivity, and stability. The lipase from Penicillium sp. section Gracilenta (CBMAI 1583) was successfully immobilized on supports bearing butyl, phenyl, octyl, octadecyl, and divinylbenzyl hydrophobic moieties wherein lipases were adsorbed through the highly hydrophobic opened active site. The highest activity in aqueous medium was observed for the enzyme adsorbed on octyl support, with a 150% hyperactivation regarding the soluble enzyme activity, and the highest adsorption strength was verified with the most hydrophobic support (octadecyl Sepabeads), requiring 5% Triton X-100 to desorb the enzyme from the support. Most of the derivatives presented improved properties such as higher stability to pH, temperature, and organic solvents than the covalently immobilized CNBr derivative (prepared under very mild experimental conditions and thus a reference mimicking free-enzyme behavior). A 30.8- and 46.3-fold thermostabilization was achieved in aqueous medium, respectively, by the octyl Sepharose and Toyopearl butyl derivatives at 60 °C, in relation to the CNBr derivative. The octyl- and phenyl-agarose derivatives retained 50% activity after four and seven cycles of p-nitrophenyl palmitate hydrolysis, respectively. Different derivatives exhibited different properties regarding their properties for fish oil hydrolysis in aqueous medium and ethanolysis in anhydrous medium. The most active derivative in ethanolysis of fish oil was the enzyme adsorbed on a surface covered by divinylbenzyl moieties and it was 50-fold more active than the enzyme adsorbed on octadecyl support. Despite having identical mechanisms of immobilization, different hydrophobic supports seem to promote different shapes of the adsorbed open active site of the lip

The enzymatic transesterification of docosahexaenoic acid (DHA) ethyl ester with glycerol was performed with several lipases in a solvent-free system and it involves the initial formation of sn-2 docosahexaenyl monoacylglyceride. This DHA derivative is highly relevant for improving the bioavailability of DHA and it has received increasing interest in the field of nutrition. Three commercial lipases, from Rhizomucor miehei (RML), Alcaligenes sp (QL), and Candida antarctica-fraction B (CALB) were tested. In certain cases (CALB), using an excess of DHA ethyl ester and high temperatures the transesterification reaction continues to the formation of triacylglycerides, but in other cases, sn-2 monoacylglyceride (2-MG) is the unique synthetic product even in the presence of high concentrations of DHA ethyl ester. At low temperatures (e.g. 37°C), RML derivatives synthesize only 2-MG in 15 min. These very mild conditions are very interesting for the thermal oxidative stability of the omega-3 fatty acid and for the thermal stability of the biocatalyst. Using Normal Phase HPLC-ELSD and accurate markers, the formation of the 2-MG was confirmed.

En la última década, ha aumentado el interés en la síntesis lípidos estructurados (SL) conteniendo ácidos grasos esenciales, ácidos grasos poliinsaturados w-3 y w-6 (PUFA), en la posición sn-2, y ácidos grasos de cadena media (MCFA) en las posiciones sn-1,3, conocidos como MLM-SL, debido a los numerosos efectos beneficiosos que se han descrito para ellos, sobre personas con necesidades dietéticas especiales. Estos SL pueden ser producidos químicamente o enzimáticamente utilizando lipasas como catalizadores (triacilglicerol acilhidrolasas, EC 3.1.1.3). Las lipasas son enzimas usadas para hidrolizar TAGs, pero también pueden catalizar la reacción inversa de hidrólisis, es decir, la esterificación bajo un ambiente hidrófobo. En este trabajo se estudia la reacción enzimática de Trioleina con diferentes derivados del ácido caprílico para obtener aceites hipocalóricos. Utilizando ligeros excesos del donador de acilo, se obtienen buenos rendimientos de síntesis de COO (monocapril-dioleina) y estos rendimientos eran similares cuando se usaba el ácido caprílico, ester metílico de ácido caprílico o tricaprilina siendo también similares para las tres lipasas comerciales (de Candida antarctica-fraccion B (CALB), de Thermomyces lanuginosa (TLL) y de Rhizomucor miehiei (RML)), adsorbidas sobre soportes hidrofóbicos ensayadas. Sin embargo, las mejores velocidades de síntesis se obtenían con TLL. Utilizando excesos significativos del donador de acilo, se obtenían buenos rendimientos de síntesis de COC y estos rendimientos eran mejores con tricaprilina y utilizando como catalizador diferentes derivados de TLL, consiguiendo buenos parámetros de actividad catalítica, rendimientos altos en COC y bajos rendimientos en COO. En principio la combinación TLL y tricaprilina era la mejor solución para la optimización en la producción de COC, como aceite hipocalórico. Sin embargo, se comprobó como la clave en la síntesis era el estudio de la ingeniería del biocatalizador, ya que diferentes der

The active site of lipase from Bacillus thermocathenolatus was selectively modified with allyl and naphthyl chains at different positions. Lipase immobilization and selective tethering of a naphthyl side chain to its position 320 improve both the hydrolysis rate of fish oils and the selectivity towards the eicosapentaenoic acid acyl chains.

We report the synthesis of polyurethane (PU) with polyethylene glycol (PEG) by miniemulsion polymerization as technique to produce a support for enzyme immobilization and stabilization. In order to study the influence of molar weight and concentration of PEG in the enzyme-support interaction, different PEG (400, 4000 and 6000 Da) was used during the synthesis of PU-PEG support to immobilize Thermomyces lanuginosus lipase (TLL). The enzymatic derivatives were effectively used in the production of polyunsaturated fatty acids (PUFAs), particularly derivative TLL-PU-PEG6000. This derivative showed the best results in terms of Km (0.183 mM) and Vmax (45.79 mmol/min/mL), calculated by Lineweaver-Burk plot (double reciprocal plot) and catalytic efficient (250.22), 100% of yield immobilization and recovered activity, reaching the highest values to the ethyl esters production (260 mM ethyl esters. U-1) making this derivative a potential low cost catalyst for ethanolysis reactions in solvent free system

The synthesis of docosahexaenoyl triacylglycerides at low temperature (e.g., 50°C) using biocatalysts of 6 commercial lipases adsorbed on hydrophobic supports was studied. In general, the triacylglyceride yields were very low with the exceptions of those produced with the enzymes from Candida antarctica fraction B, CALB (82%), and those produced with the enzyme from Pseudomonas fluorescens, PFL (57%). The reactions were performed under vacuum to remove the released ethanol. The yields varied widely when different derivatives of CALB were used, and they were higher when CALB adsorbed on hydrophobic supports was used (82%). One interesting by-product (18% of sn-2 monoacylglyceride of DHA) remained at the end of the synthetic process. CALB adsorbed on Sepabeads exhibited better activity and stability than did the commercial derivative Novozym 435. The best CALB biocatalyst preserved 90% of the activity after 30 days under the reaction conditions.

Soluble lipase from Thermomyces lanuginosa (TLL) was dramatically hyper-activated by a cationic surfactant, cetyltrimethylammonium bromide (CTAB). The greatest hyperactivation (above 340-fold) was observed with 0.005% CTAB. In addition to that, very high hyperactivation was also observed with much higher concentrations of surfactant (100-fold in the presence of 0.3% CTAB). Without surfactant or with very low surfactant concentrations, TLL was not adsorbed to the cationic exchangers. However, in the presence of high concentrations of surfactant (0.3%), the lipase was completely and strongly adsorbed on sulfopropyl-sepharose. The adsorbed enzyme remained hyperactivated (80-fold more active than the soluble enzyme) after elimination of the excess of surfactant. Complete desorption of the hyperactivated TLL from the cationic exchanger is only achieved at 2 M NaCl. The same level of hyperactivation was observed for the hydrolysis of a large substrate: fish oil. The release of EPA (eicosapentaenoic acid) was 80-fold more rapid with hyperactivated TLL derivatives than with TLL very mildly immobilized on CNBr activated agarose. Hyperactivated derivatives were very stable at 25 °C and 37 °C. Full activity was preserved after 1 week.

The binding of enzymes on carriers with a high degree of activation with glyoxyl groups is an excellent method for improving enzyme stability by multipoint covalent attachment on a pre-existing carrier. Glyoxyl groups are short aliphatic aldehyde groups (Support – O-CH2 – CHO) that can be obtained by periodate oxidation of glyceryl groups (Support –O-CH2-CHOH-CH2OH). The unique features of glyoxyl groups are as follows: a.- The immobilization of enzymes through their amino groups has to occur via multipoint attachment. b.- The glyoxyl groups are stable at pH 10, which allows for the participation of Lys in the immobilization process. c.- The glyoxyl groups are very stable at pH 10, which allows for a long-term incubation between the immobilized enzyme and the activated support to promote a very intense enzyme-support multipoint covalent attachment. Using this protocol, more than 100 industrial enzymes were highly stabilized. In many cases, stabilizations of greater than 1000-fold compared with immobilized derivatives generated by conventional methods were obtained. Although dramatic stabilization was achieved, the immobilized enzymes maintained only 50 to 90 % of the catalytic activity of the corresponding soluble enzyme. Stabilization of industrial enzymes is a key step in immobilization protocols. Enzymes are immobilized for use at industrial scales for a number of reaction cycles.

El ácido ascórbico es un agente antioxidante que previene la oxidación de los ácidos grasos poliinsaturados (AGPI). Sin embargo es hidrosoluble y necesitamos hacerlo liposoluble para incorporarlo a los derivados de AGPI para su uso como ingrediente funcional en alimentos, previniendo así su oxidación. La condensación de los aceites (de oliva) y ácido ascórbico catalizadas por lipasas inmovilizadas pueden ser un protocolo adecuado para la preparación de antioxidantes liposolubles (oleato de ascorbilo). Este proceso de síntesis es muy complejo, debido a la diferencia de polaridad de los sustratos utilizados, es necesario el uso de disolventes polares para conseguir la solubilización de ambos sustratos, siendo estas condiciones muy nocivas para la actividad y estabilidad de las enzimas. Por ello, una clave para el desarrollo de este tipo de reacciones es el diseño de estrategias de inmovilización que nos permitan aumentar la estabilidad de las enzimas en disolventes polares útiles para uso alimentario. El estudio se llevó a cabo con tres lipasas comerciales inmovilizadas por tres protocolos diferentes: adsorción hidrofóbica, intercambio aniónico y unión covalente multipuntual. En la optimización del proceso, se ha conseguido obtener un rendimiento de hasta el 80% de oleato de ascorbilo en menos de 24 horas sin la formación de productos secundarios. Los biocatalizadores más activos fueron también los más estables mostrando una vida media de tres días a 75ºC y toda su actividad después de 28 días a 45ºC en disolventes orgánicos polares.

Selective ethanolysis of fish oil was catalyzed by immobilized lipases and their derivatives in organic media. Lipases from Candida antarctica B (CALB), Thermomyces lanuginosa (TLL) and Rhizomucor miehei (RML) were studied. The three lipases were immobilized by anion exchange and hydrophobic adsorption. The discrimination between the ethyl ester of eicosapentaenoic acid (EE-EPA) and the ethyl ester of docosahexaenoic acid (EE-DHA) depends on the lipase, the immobilization support, the physico-chemical modifications of the immobilized lipase derivatives and on the solvents used. TLL and RML were much more selective than CALB. EE-EPA is released 20-fold faster than EE-DHA when ethanolysis was catalyzed, in cyclohexane, by TLL hydrophobically adsorbed on Sepabeads C18. The selectivity and stability of the different derivatives in these polar organic solvents were further improved after physico-chemical modification. The best results for activity-selectivity-stability were obtained in cyclohexane for TLL adsorbed on Sepabeads C18 and further modified via solid-phase physical modification with a polyethylenimine polymer. In this case, the initial selectivity was higher than 20, and a 80 % of EPA was released as ethyl ester after 3 h at 25 °C. At this conversion, mixtures of ethyl esters highly enriched in the ethyl ester of EPA with less than 5 % of the EE-DHA were obtained. TLL derivatives remained fully active after incubation for 24 h in anhydrous solvents. © AOCS 2013.

The reaction of transesterification between oils (e.g., olive oil) and ascorbic acid in polar anhydrous media (e.g., tert-amyl alcohol) catalyzed by immobilized lipases for the preparation of natural liposoluble antioxidants (e.g., ascorbyl oleate) was studied. Three commercial lipases were tested: Candida antarctica B lipase (CALB), Thermomyces lanuginosus lipase (TLL) and Rhizomucor miehei lipase (RML). Each lipase was immobilized by three different protocols: hydrophobic adsorption, anionic exchange and multipoint covalent attachment. The highest synthetic yields were obtained with CALB adsorbed on hydrophobic supports (e.g., the commercial derivative Novozym 435). The rates and yields of the synthesis of ascorbyl oleate were higher when using the solvent dried with molecular sieves, at high temperatures (e.g. 45 °C) and with a small excess of oil (2 mol of oil per mol of ascorbic acid). The coating of CALB derivatives with polyethyleneimine (PEI) improved its catalytic behavior and allowed the achievement of yields of up to 80% of ascorbyl oleate in less than 24 h. CALB adsorbed on a hydrophobic support and coated with PEI was 2-fold more stable than a non-coated derivative and one hundred-fold more stable than the best TLL derivative. The best CALB derivative exhibited a half-life of 3 days at 75 °C in fully anhydrous media, and this derivative maintained full activity after 28 days at 45 °C in dried tert-amyl alcohol.