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Critical role of different immobilized biocatalysts of a given lipase in the selective ethanolysis of sardine oil Sonia Moreno-Pérez, Daniela Flavia Machado Turati, Janaina Pires Borges, Pilar Luna, Francisco Javier Señoráns, Gloria Fernandez-lorente, and José Manuel Guisán J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016

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Journal of Agricultural and Food Chemistry

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Critical role of different immobilized biocatalysts of a given lipase in the selective

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ethanolysis of sardine oil

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Sonia Moreno-Perez 1, Daniela Flavia Machado Turati 2, Janaina Pires Borges 3, Pilar Luna5,

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Francisco Javier Señorans 5, Jose M. Guisan1* and Gloria Fernandez-Lorente 4 *

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1. Instituto de Catálisis. CSIC. Campus UAM-CSIC. 28049 Madrid

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2. Department of Biochemistry and Microbiology, Univ Estadual Paulista at Rio Claro – 8

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UNESP, Rio Claro, SP 13506-900, Brazil.

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3. Departamento de Química e Tecnologia. Instituto de Química – UNESP. 14800-069, 10

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Araraquara –SP, Brasil.

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4. Instituto de Investigación en Ciencias de la Alimentación (CIAL) CSIC-UAM. 28049.

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Madrid.

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5. Departamento de Química Física Aplicada. Universidad Autónoma. 28049 Madrid

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Corresponding authors: Jose M. Guisan ([email protected]) and Gloria Fernandez-

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Lorente ([email protected])

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ABSTRACT

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Different immobilized derivatives of two lipases were tested as catalyst of the synthesis of

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ethyl esters of omega-3 fatty acids during the ethanolysis of sardine oil in solvent-free

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systems at 25 ºC. Lipases from Thermomyces lanuginosus (TLL) and Lecitase Ultra (a

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phospholipase with lipolytic activity) were studied. Lipases were adsorbed on hydrophobic

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Sepabeads C18 through the open active center and on an anion exchanger Duolite with

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the active center exposed to the reaction medium. TLL-Sepabeads derivatives exhibit a

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high activity of 9 UI/mg of immobilized enzyme and they are 20 fold more active than TLL-

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Duolite derivatives and almost 1000 fold more active than Lipozyme TL IM (the

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commercial derivative from Novozymes). Lecitase-Sepabeads exhibit a high selectivity for

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the synthesis of the ethyl ester of EPA that is 43 fold faster than the synthesis of the ethyl

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ester of DHA.

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Keywords: Selectivity EPA vs DHA, Lipases in solvent-free systems, Lipases Stability ,

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Lipase Immobilization.

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1.- INTRODUCTION

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Polyunsaturated fatty acids, especially omega-3, are essential for human health at all

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stages of the life. These are principally abundant in some fish oils1-3. Docosahexaenoic acid

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or DHA is particularly important for enhancing brain capacities and it is an essential

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nutrient during the first months of life. Therefore, many organizations in the field of

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health recommend that it should be included in infant formulas. The eicosapentaenoic

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acid or EPA is very useful to prevent cardiovascular risks and consequently it is essential in

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the diet of elderly people4. The resulting ethyl esters or triglycerides highly enriched in

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omega-3 can be added to almost any type of food. Thus, a nutrient present almost

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exclusively in a little consumed food (such as fish oil) could be incorporated into a wide

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variety of food (milk, yogurt, cookies, soft drinks, bread, etc.) useful for all ages and

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gastronomic cultures5, 6.

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The use of enzymatic processes for the production of functional ingredients (e.g., omega-3

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fatty acids ethyl esters) allows us to obtain components as "natural" products without

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traces of reactive or toxic solvents, and through processes performed under very mild

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reaction conditions, contrary to chemical processes7-9. In addition to that, enzymatic

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ethanolysis may provide a possible discrimination between the synthesis of EE-EPA versus

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EE-DHA. Both omega-3 fatty acids have small structural differences but perhaps they

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could be discriminated by some lipase derivative in a given reaction medium. This ability

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of lipases to discriminate between EPA and DHA was hardly studied by other authors but

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it was already observed by our group in previous papers10,11. 3 ACS Paragon Plus Environment


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Ethyl esters of omega-3 fatty acids are usually suitable formulations as functional

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ingredients. In addition to that, ethyl esters of omega-3 fatty acids are also very good

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precursors for the production of more interesting omega-3 fatty acids formulations as

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triacylglycerol esters10 or sn-2 monoacylglycerol esters12. These two latter formulations

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could to be the ideal food ingredients based on omega-3 fatty acids13.

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In a previous paper we have reported the synthesis of ethyl esters of omega-3 fatty acids

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by ethanolysis of oils in organic solvents with 3 commercial lipases from Novozymes:

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CALB, RML and TLL10. The process was strongly modulated by the different lipases, by

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different immobilized derivatives of a given lipase (with a different orientation of the

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lipase on the support surface) and by the solvent. The best results were obtained for TLL

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Now in this paper the synthesis of ethyl esters of omega-3 fatty acids by ethanolysis of

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sardine oil in solvent-free systems catalyzed by different derivatives of TLL including the

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commercial immobilized preparation from Novozymes is reported. Now in the absence of

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solvents, the reaction may be more intensive. In addition to that, lipasecatalyzed

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ethanolysis in a solvent-free system is important in industrial applications, mainly in food

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technology14: e.g., by avoiding the problems of separation, toxicity, and flammability of

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organic solvents. The reaction was conducted at room temperature, and the combination

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of these very mild reaction conditions plus the absence of inorganic acids should prevent

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any modification to the omega-3 fatty acids. Commercial TLL and Lecitase Ultra were

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studied. Lecitase Ultra is a phospholipase that also exhibits lipase activity15.

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The results were evaluated by considering three main parameters with industrial

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relevance: activity, stability and selectivity (discrimination between synthesis of EE-EPA

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and synthesis of EE-DHA).

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The following hypothesis is proposed:

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The transition between the closed and the open structure of the active centers of a given

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lipase in anhydrous media involves dramatic conformational changes in the region where

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the active center is placed. In addition to that, relevant conformational changes in other

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regions of the enzyme surface, even far from the active center, may also occur16. The

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presence of the support surface very close to some of these regions may prevent or

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modulate some of those conformational changes. In this way the opening of the active

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center of the lipase can be slightly modified yielding different forms of the open active

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center and the catalytic properties of the lipase can be modulated.

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The different orientation of a given lipase, immobilized on different supports (e.g., ionic

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adsorption vs adsorption on silica), may promote different open forms of the lipase and

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different catalytic properties. On the other hand, when the lipase is adsorbed on

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hydrophobic supports its open active center is fixed on the support. However, the

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additional interaction with solvents or oils may also promote slight changes in the shape

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of the open form already fixed on the support. A critical role of different derivatives of a

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given lipase (with different orientation on the support) has already been observed with

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several lipases working in aqueous11,17 or in anhydrous media18,19. Some possible different

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lipase orientations are schematically represented in the Scheme 1.



Different orientations of a given immobilized lipase

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Different mechanisms of Hyperactivation

Hyperactivation Promoted by Organic solvents, oils, esters of fatty acids, etc.

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Scheme1

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From the proposed hypothesis, the main objective of this paper is the evaluation of

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different immobilized derivatives of lipases (with a different orientation of the enzyme on

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the support) as catalyst of ethanolysis of sardine oil in solvent-free systems.

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2.- MATERIALS AND METHODS.

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2.1.- Materials.

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Triton® X-100 (TX), p-nitrophenyl butyrate (p-NPB), Polyethyleneimine (PEI) (MW 25,000),

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Ethanolamine hydrochloride, 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC), were

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from Sigma Chemical Co. (St. Louis, USA). Sardine oil was donated by BTSA (Madrid,

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Spain) and it contains 18% of EPA and 12% of DHA as reported by BTSA. Octyl SepharoseTM

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CL-4B was purchased from GE Healthcare (Uppsala, Sweden). Lipozyme TLL IM (the

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enzyme adsorbed on silica) was from Novozymes. Duolite A568, a weak base ion-exchange 6 ACS Paragon Plus Environment

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resin, was provided from Rohm and Haas (USA). Sepabeads-C18 was kindly donated by

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Resindion S.R.L. Lipases form Thermomyces lanuginosus (TLL) and Lecitase Ultra were

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generously donated by Novo Nordisk (Denmark). Other reagents and solvents used were

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of analytical or HPLC grade.

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2.2 Methods.

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All experiments were made by triplicate and standard deviations were always less than 5

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%.

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2.2.1.- Hydrolytic activity of different lipases (soluble and immobilized).

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To follow the immobilization process, the activity of soluble and immobilized lipase

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preparations were analyzed by using a spectrophotometric assay (with magnetic stirring)

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as previously described10.

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2.2.2.- Purification of lipases by selective adsorption on octyl-Sepharose

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Lipases were purified by interfacial adsorption, on hydrophobic octyl–Sepharose, at 25ºC,

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in sodium phosphate buffer pH 7 and at a low ionic strength (5 mM)20.

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2.2.3.- Immobilization of Lipases on Duolite A568 or Sepabeads-C18 resins

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The purified lipases were adsorbed on Duolite A568 or on Sepabeads-C18 as previously

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described10. The different lipase derivatives were named as TLL-Sepabeads, TLL-Duolite,

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Lipozyme TL IM (adsorbed on silica and prepared by Novozymes), Lecitase-Sepabeads and 7 ACS Paragon Plus Environment


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Lecitase-Duolite. The lipase loading in all derivatives was 20 mg per mL, similar to the one

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of Lipozyme TL IM and close to the maximum loading capacity of the supports.

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2.2.4.- Drying of derivatives.

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After obtaining our derivatives, we conducted a drying process. They were washed and

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dried several times with filtration plates. 20 mL of distilled water-acetone solution were

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added per gram of derivative for this purpose. At first place, they were washed with

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water, and then, the percentage of acetone in the solution was gradually increased up to

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100% of pure acetone. Acetone was fully eliminated after overnight incubation of

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derivatives at room temperature and then, completely dried derivatives were obtained.

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2.2.5.- Enzymatic synthesis of ethyl esters of omega-3 fatty acids.

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The dried immobilized lipase (0.1 g) was added to a substrate solution with 1.9 mmols of

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sardine oil and 15.4 mmols of ethanol10. To obtain activity values in the absence of water,

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200 mg of dry molecular sieves were also added to the reaction mixture. The

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concentration of sardine oil was 700 mM (it contains 210.3 mM of glyceryl-EPA+DHA

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according the information of the company BTSA). The reactions were carried out in

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thermostatic orbital incubator (100 rpm) in order to stir the immobilized biocatalyst and in

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order to maintain the reaction temperature (25 °C). The ethanolysis was carried out for 24

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h.

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2.2.6.- HPLC analysis 8 ACS Paragon Plus Environment

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The reaction was easily followed by the synthesis of ethyl esters of PUFAs and analyzed by

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isocratic UV-HPLC. Experiments were carried out in triplicate, and the standard error was

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never over 5%. PUFAs are not very apolar compounds and they are easily eluted from

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isocratic reverse-phase HPLC. On the other hand, the number of double bonds in each

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molecule promotes an interesting absorbance at 215 nm. Furthermore, ethyl esters of

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PUFAs are the most interesting products obtained by ethanolysis of fish oils. The synthesis

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of esters of omega-3 fatty acids was analyzed by RP-HPLC (Spectra Physis SP 100 coupled

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with an UV detector Spectra Physic SP 8450) using a reversed-phase column (Ultrabase-

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C8, 150 x 4.6 mm, 5µm). The flow rate was 1.5 mL/min with acetonitrile/water/CH3COOH

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(80:20:0.1, by vol.) and pH 3. The UV detection was carried out at 215 nm. The synthetic

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yields were calculated according the area of the peaks corresponding to different

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concentrations of the pure compounds. The retention times were 9 minutes for EE-EPA

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and 12 minutes for EE-DHA 10.

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2.2.7.- Inactivation of different immobilized lipases.

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The immobilized enzyme derivatives (0.1 g) were incubated with 1.9 mmols of sardine oil

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at different temperatures (25 °C). Then, at various times, 15.5 mmols of ethanol were

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added to initiate the ethanolysis reaction at 25 °C. The synthesis of the ethyl esters was

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followed by HPLC–UV analysis.

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2.2.8.- Consecutive reaction cycles.



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First, the enzymatic synthesis of ethyl esters of omega-3 fatty acids was performed under

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the conditions described above. The course of the reaction was measured by HPLC, and

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when it reached 100%, the reaction mixture was filtered, washed and the derivative was

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dried again with acetone to remove all the substrate and product. Then the substrates

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were added again under the same conditions several times repeating the same process

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again.

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3.- RESULTS AND DISCUSSION

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3.1.- Effect of the immobilized TLL biocatalyst on synthesis of ethyl esters of PUFAs

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during the ethanolysis of sardine oil in solvent-free systems.

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Activity (synthesis of EE-EPA plus EE-DHA) and selectivity (discrimination between EPA and

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DHA) of 3 different immobilized TLL derivatives are shown in Table 1. TLL–Sepabeads was

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the most active and selective one. For example, this derivative was more than 900191 fold

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more active than the commercial immobilized TLL from Novozymes (Lipozyme® TL IM). In

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addition to that, the selectivity of the commercial derivatives was almost 5-fold lower.

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Compared to the synthesis of ethyl esters of omega-3 fatty acids during ethanolysis in the

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presence of cyclohexane, now in solvent-free systems catalytic activities were more than

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4-fold higher10.

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In figure 1 the results of the inactivation of different TLL derivatives in the presence of

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pure sardine oil are shown. Again, TLL adsorbed on hydrophobic supports was much more

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stable than commercial derivative Lipozyme TL IM. After 48 hours, the commercial


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derivative preserved only 20% of activity and under the same conditions the best

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derivative (TLL–Sepabeads) preserved 90 % of initial activity.

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The critical role of the immobilized TLL derivatives was observed for 3 parameters with

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industrial relevance: activity, selectivity EPA vs DHA and stability. In this case hydrophobic

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adsorption of TLL seems to be the best immobilization strategy to design biocatalysts for

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solvent-free ethanolysis. The critical role of the immobilized lipase derivatives has been

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very clearly observed in this paper. TLL adsorbed on a hydrophobic support through the

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open active center (TLL-Sepabeads) exhibited a very high activity for the synthesis of ethyl

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esters of PUFAs by the ethanolysis of fish oil (9 IU per mg of immobilized enzyme) under

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very mild conditions (25ºC), and this activity was more than 20 fold higher than the one

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exhibited by TLL adsorbed on one anion exchanger (Duolite) through its region with the

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high density of carboxyl groups, and almost 1000 fold higher than the one exhibited by TLL

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adsorbed on silica (the commercial derivative from Novozymes ). High loaded TLL

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derivatives may contain up to 25 mg of pure TLL per gram of biocatalyst and this means a

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catalytic activity of 225 IU per gram of biocatalyst, the highest activity reported in

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literature. In addition to that, the most active derivative was also the most stable one in

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the presence of fish oil (e.g. more than 10 fold more stable than commercial TLL

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derivative). Moreover, TLL-Sepabeads exhibited an interesting discrimination between

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EPA and DHA. In fact, EE-EPA was synthesized 14 fold faster than EE-DHA.

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3.2.- Operational stability of TLL-Sepabeads derivatives for the synthesis of esters of

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omega-3 fatty acids by solvent-free ethanolysis of sardine oil. 11 ACS Paragon Plus Environment


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4 reaction cycles were carried out with the same biocatalyst. Activity was measured as the

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synthesis of ethyl esters of PUFAS (EPA+DHA). The derivative preserves 80 % of activity

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after the forth cycle. For industrial application, TLL derivatives should undergo additional

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stabilization.

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3.3.- Effect of the immobilized Lecitase Ultra biocatalyst on the synthesis of ethyl esters

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of PUFAs by ethanolysis of sardine oil in solvent-free systems.

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Two different Lecitase derivatives were compared: Lecitase Ultra adsorbed on

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hydrophobic support (Lecitase-Sepabeads) and Lecitase Ultra adsorbed on an anionic

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exchanger (Lecitase-Duolite).

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expressed as the synthesis of ethyl esters of PUFAs but Lecitase-Sepabeads was 10-fold

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more active than Lecitase-Duolite (Table 2). On the other hand, the selectivity of Lecitase

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Sepabeads was extraordinary (the ethanolysis of EPA was 43-fold faster than ethanolysis

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of DHA). In this way, almost pure EPA-EE could be obtained in the first stages of hydrolysis

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of sardine oil in solvent free systems. In addition to that, the stability of Lecitase

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Sepabeads was excellent (Figure 2). Under optimal reaction conditions the immobilized

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biocatalyst preserved 100 % of catalytic activity after 24 hours. In addition to that, the

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derivative preserved 100% of catalytic activity when it was re-used for 5 reaction cycles.

Both derivatives exhibited a very low catalytic activity

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3.4.- Effect of the immobilized Lecitase Ultra biocatalyst on the ethanolysis of sardine

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240 oil in the presence of solvents.


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For comparative purposes, ethanolysis by immobilized derivatives of Lecitase Ultra was

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also studied in the presence of two solvents: a fairly polar one (tert-amyl alcohol) and a

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more apolar one (cyclohexane).

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Different derivatives and different solvents promoted very different activities and

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selectivities. The activity (e.g., Lecitase –Sepabeads) in the presence of tert-amyl alcohol

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was more than 10-fold higher than the activity of the same biocatalyst in solvent-free

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systems (Table 2). However, selectivities were much lower than the extraordinary one

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obtained in solvent-free systems. In addition to that, stability in tert-amyl alcohol was

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much worse than stability in solvent-free systems (figure 2). Different derivatives of

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Lecitase Ultra also exhibited a very different activity and selectivity (discrimination

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between EPA and DHA). Both Lecitase Ultra derivatives were poorly active for ethanolysis

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but Lecitase Ultra adsorbed on a hydrophobic support (Lecitase-Sepabeads) exhibits an

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extraordinary capacity of discrimination between EPA and DHA. The synthesis of EE-EPA

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that is 43 fold faster than the synthesis of EE-DHA. This extraordinary discrimination

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allows the preparation of EE-EPA almost 100 % pure and similar values have not been

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reported in literature.

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3.5.- Effect of different immobilized derivatives and different anhydrous media.

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In Table 3 the complex interrelation between different enzyme derivatives and different

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anhydrous media can be observed. The open form of immobilized lipases working in

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anhydrous media should be stabilized by the solvents or the substrates (oils, esters, etc.).

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Solvents with different polarity, tert-amyl alcohol, cyclohexane, oils, etc. should promote 13 ACS Paragon Plus Environment


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different mechanisms of opening of the lipase active center and hence different active

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centers for the same derivative of the same lipase could be obtained when the enzyme is

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working in different anhydrous media.

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The different orientation of the derivatives should also play a critical role as commented

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before (Scheme 1). Some relevant examples shown in Table 3 are: TLL-Sepabeads are

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more active but less selective in solvent-free systems than in the presence of solvents. On

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the contrary, Lecitase- Sepabeads are less active but much more selective in solvent-free

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systems. Obviously the preparation of very different lipase derivatives plus the use of a

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wide variety of anhydrous media should provide a wide range of critical parameters for

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each reaction: activity, selectivity, yield and stability. A critical role of different derivatives

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of a given lipase has already been observed with several lipases working in aqueous13, 14 or

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in anhydrous media15, 16. Now, the effect of the solvents plus the effect of the enzyme

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orientation on the support promote a great diversity of open catalytic centers of a given

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lipase and hence a great diversity in the catalytic properties of each enzyme

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Acknowledgments

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This work was sponsored by the Spanish Ministry of Science and Innovation (projects AGL-

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2009-07526 and BIO2012- 36861). We gratefully recognize the Spanish Ministry of Science

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and Innovation for the “Ramón y Cajal” contract for Dr. Fernandez-Lorente and for the FPI

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contract to Sonia Moreno-Perez. We would like to thank Novozymes and Ramiro Martinez

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for the generous gift of commercial lipases. We also thank Fundação de Amparo à


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Pesquisa do Estado de São Paulo (FAPESP) for granting the 284 scholarship to Daniela

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Flavia Machado Turati (BEPE process 2014/04925-1).

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lipase on its stability and fish oil hydrolysis. J. Mol. Catal. B: Enzym. 2012, 78, 111-118.

352

18. Moreno-Perez, S.; Filice, M.; Guisan, J. M.; Fernandez-Lorente, G., Synthesis of

353

ascorbyl oleate by transesterification of olive oil with ascorbic acid in polar organic media

354

catalyzed by immobilized lipases. Chem. Phys. Lipids 2013, 174, 48-54.



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355

19. Guisan, J.; Sabuquillo, P.; Fernandez-Lafuente, R.; Fernandez-Lorente, G.; Mateo, C.;

356

Halling, P.; Kennedy, D.; Miyata, E.; Re, D., Preparation of new lipases derivatives with high

357

activity–stability in anhydrous media: adsorption on hydrophobic supports plus

358

hydrophilization with polyethylenimine. J. Mol. Catal. B: Enzym. 2001, 11, 817-824.

359

20. Bastida, A.; Sabuquillo, P.; Armisen, P.; Fernandez-Lafuente, R.; Huguet, J.; Guisan, J.

360

M., A single step purification, immobilization, and hyperactivation of lipases via interfacial

361

adsorption on strongly hydrophobic supports. Biotechnol. Bioeng. 1998, 58, 355 486-493.

362 363 364 365

366

FIGURE LEGENDS

367

Figure 1.- Time-course of inactivation of different immobilized biocatalysts of TLL.

368

Derivatives were incubated in oil at 25 °C. At different times samples of the suspensions

369

were withdrawn, ethanol was added (the volume and ratio described in Methods) and the

370

reaction of ethanolysis of sardine oil at 25 ºC was followed (as described in Methods).

371

Rhombus: TLL-Sepabeads ; triangles: TLL-Duolite; crosses: Lipozyme® TL IM.

372

The derivative was

373

Figure 2.- Time-course of inactivation of Lecitase-Sepabeads.

374

incubated in oil (solvent-free system) or in solvent (tert-amyl alcohol) at 25 °C. At different

375

times samples of the suspensions were withdrawn and ethanol was added (in the volume 18 ACS Paragon Plus Environment

Page 19 of 27


376

and ratio described in Methods) and the reaction of ethanolysis of sardine oil at 25 ºC was

377

followed (as described in Methods). Circles: solvent-free system and squares: tert-amyl

378

alcohol.



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Page 21 of 27


100

80

60

40

20

0 0

6

12

18

24

Time (h)

Figure 2

Table 1.- Synthesis of ethyl esters of omega-3 fatty acids by ethanolysis of Sardine oil with different immobilized derivatives of TLL: Influence of solvent on reaction. Synthesis was carried out solvent and in solvent-free system at 25 ºC.



Condition

a

Catalyst

Solvent-free

Cyclohexane

Page 22 of 27

b

Reference

Initial Activity

Selectivity

TLL-Sepabeads

9.96

14

TLL-Duolite

0.45

12

Lipozyme® TL IM

0.01

3

This manuscript

TLL-Sepabeads

2.24

29

(Moreno-

TLL-Duolite

0.5

12

Perez et al., 2014) Lipozyme® TL IM

a

-

-

Initial activity is expressed as µmols of ethyl ester of PUFAS (EPA+DHA)

synthesized per minute and per mg of immobilized lipase and measured at 10% conversion to FAEE. b

Selectivity is expressed as the molar ratio between synthesized EE- EPA and

synthesized EE-DHA.

Table 2.- Ethanolysis of Sardine oil with different immobilized derivatives of Lecitase: Influence of solvent on reaction parameters. Synthesis was carried out in anhydrous systems at 25 ºC with solvents or in 413 solvent-free systems. 22 ACS Paragon Plus Environment

Page 23 of 27


Initial Activitya

Condition

Catalyst

Solvent-free

Lecitase-Sepabeads 0.010

Selectivityb 43

8 Lecitase-Duolite tert-amyl Alcohol

0.001

Lecitase-Sepabeads 0.114

Lecitase-Duolite

Lecitase-Sepabeads

20

0.029

12

0.043

18

0.011

5

Cyclohexane Lecitase-Duolite a

Initial activity is expressed as the sum of µmols of ethyl ester of PUFAS (EPA+DHA) synthesized per minute and per mg of immobilized lipase and measured at 10% conversion to ethyl esters. b

Selectivity is expressed as the molar ratio between synthesized EE- EPA and

synthesized EE-DHA.



Page 24 of 27

Table 3.- Ethanolysis of Sardine oil catalyzed by the best immobilized derivatives of Lecitase and of lipase from Thermomyces lanuginosus. Synthesis was carried out in anhydrous media at 25 ºC.

Enzyme

Condition

TLL

Lecitase

a Initial Activity

b Selectivity

Solvent-free

9.96

14

Cyclohexane

2.24

29

Solvent-free

0.010

43

tert-amyl alcohol

0.11

20

a Initial activity is expressed as µmols of ethyl ester of PUFAS (EPA+DHA) synthesized per minute and per mg of immobilized lipase and measured at 10% conversion. b Selectivity is expressed as the molar ratio between synthesized EE- EPA and synthesized EE-DHA.


Page 25 of 27




Page 26 of 27


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Different orientations of a given Immobilized lipase

TOC Graphic

Different mechanisms of hyperactivation

Hyperactivation

Promoted by Organic solvents, oils, esters of fatty acids, etc.


Critical Role of Different Immobilized Biocatalysts ... - ACS Publications

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