Various Strategies for the Immobilization of a Phospholipase C from Bacillus cereus for the Modulation of Its Biochemical Properties.

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

Enzymatic synthesis of mono- and disubstituted phospholipids by direct condensation of oleic acid and glycerophosphocholine with immobilized lipases and phospholipase.

Lysophospholipids which contain polyunsaturated fatty acids play a key role in food and cosmetic industries because of their bioactivity. Therefore, the formation of mono- and disubstituted phospholipids is quite interesting as they could be used for the formation of different natural liposomes. Using immobilized derivatives of lipases and phospholipases, the esterification of oleic acid with glycerophosphocholine (GPC) has been studied. Thus, derivatives were quite active in completely anhydrous media and in solvent-free reaction systems where the reaction takes place. CALB biocatalyst was able to successfully form oleoyl-LPC at 60 °C in the presence of 30 % butanone, where the synthesis rate was 100 times higher than in the absence of solvents at 40 °C. On the other hand, the best synthesis rate for dioleoyl-PC was achieved with immobilized Lecitase in a solvent-free process at 60 °C, an 83 % synthesis yield was achieved with an initial synthesis rate of 4.32 mg/mL × h × g.

Immobilization of Eversa Lipases on Hydrophobic Supports for Ethanolysis of Sunflower Oil Solvent-Free.

Lipases are an important group of biocatalysts for many industrial applications. Two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 was immobilized on four different hydrophobic supports: Lewatit-DVB, Purolite-DVB, Sepabeads-C18, and Purolite-C18. The performance of immobilized lipases was investigated in the transesterification of sunflower oil solvent-free in an anhydrous medium. Interesting results were obtained for both lipases and the four supports, but with Sepabeads support the lipases Eversa showed high catalytic activity. However, the more stable and efficient derivative was Eversa® Transform immobilized on Sepabeads C-18. A 98 wt% of ethyl ester of fatty acid (FAEE) was obtained, in 3 h at 40ºC, ethanol/sunflower oil molar ratio of 3:1 and a 10 wt% of the immobilized biocatalyst. After 6 reaction cycles, the immobilized biocatalyst preserved 70 wt% of activity. Both lipases immobilized in Sepabeads C-18 were highly active and stable in the presence of ethanol. The immobilization of Eversa Transform and Eversa Transform 2.0 in hydrophobic supports described in this study appears to be a promising alternative to the immobilization and application of these news lipases still unexplored.

Capture of enzyme aggregates by covalent immobilization on solid supports. Relevant stabilization of enzymes by aggregation.

In this paper, a novel procedure for the immobilization and stabilization of enzymes is proposed: the multipoint covalent attachment of bi-molecular enzyme aggregates. This immobilization protocol allows the "capture" and fixation of the enzyme aggregate on the support surface. In addition to stabilization by multipoint attachment, enzyme aggregation promotes very interesting stabilizing effects. In the presence of low concentrations of polyethylene glycol (30 %) the dimeric amine oxidase from Pisum sativum forms soluble bi-molecular aggregates. Enzyme aggregates were analyzed by Dynamic Light Scattering and by full chemical loading of a mesoporous support (10 % agarose gels activated with glyoxyl groups). The soluble aggregate was immobilized by multipoint attachment on glyoxyl- agarose at pH 8.5 though the four amino termini of the two dimeric molecules (Lys residues are not reactive at this pH). The immobilized aggregated structure cannot undergo any movement (translational or rotational) after multipoint attachment and the aggregate is "fixed" on the support surface even after the removal of PEG. The immobilized aggregate was further incubated at pH 10 in order to allow the Lys residues to react with the glyoxyl groups on the support. Enzyme aggregation has an important effect on enzyme stabilization: the aggregated derivative was 40 fold more stable than a similar derivative of the isolated enzyme and 200 fold more than native enzymes in experiments of thermal inactivation.

Ethyl esters production catalyzed by immobilized lipases is influenced by n-hexane and ter-amyl alcohol as organic solvents.

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.

Stabilization of Glycosylated ß-Glucosidase by Intramolecular Crosslinking Between Oxidized Glycosidic Chains and Lysine Residues.

Many industrial enzymes can be highly glycosylated, including the ß-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized ß-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of ß-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of ß-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.

A mild intensity of the enzyme-support multi-point attachment promotes the optimal stabilization of mesophilic multimeric enzymes: Amine oxidase from Pisum sativum.

Stabilization of dimeric enzymes requires the stabilization of the quaternary structure as well as the 3D one. Both subunits may be easily immobilized on a highly activated support. Additional stabilization of the 3D structure may be achieved via multipoint covalent attachment (MCA) on highly activated supports. In the case of monomeric enzymes or thermophilic dimeric ones, the optimal stabilization is obtained via the most intense MCA and it is associated to a small loss of catalytic activity. However, in the case of mesophilic enzymes, a very intense MCA of both subunits may promote negative effects, e.g., associated to distortions of the assembly between subunits and a subsequent very important loss of catalytic activity. A dimeric mesophilic amine oxidase from P.sativum was stabilized by MCA on glyoxyl-agarose. Both subunits were covalently immobilized on the support through the region with the highest density in Lys residues. In addition to that, an interesting activity/stabilization binomial was obtained after only 3 h of enzyme-support multiinteraction (50 % of activity/350 fold stabilization). However, after 24 h of enzyme-support multi-interaction this binomial activity-stabilization decreased down to 30/150. A moderate multiinteraction seems to be the optimal strategy for immobilization-stabilization of mesophilic dimeric enzymes and it promotes moderate losses of activity and interesting stabilizations against the combined effect of heat, acid pH and ethanol. The control of the intensity of enzyme-support multi-interactions becomes now strictly necessary.

Fine Modulation of the Catalytic Properties of Rhizomucor miehei Lipase Driven by Different Immobilization Strategies for the Selective Hydrolysis of Fish Oil.

Functional properties of each enzyme strictly depend on immobilization protocol used for linking enzyme and carrier. Different strategies were applied to prepare the immobilized derivatives of Rhizomucor miehei lipase (RML) and chemically aminated RML (NH2-RML). Both RML and NH2-RML forms were covalently immobilized on glyoxyl sepharose (Gx-RML and Gx-NH2-RML), glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH2-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH2-RML) and heterofunctional epoxy support partially modified with iminodiacetic acid (epoxy-IDA-RML and epoxy-IDA-NH2-RML). Immobilization varied from 11% up to 88% yields producing specific activities ranging from 0.5 up to 1.9 UI/mg. Great improvement in thermal stability for Gx-DTT-NH2-RML and epoxy-IDA-NH2-RML derivatives was obtained by retaining 49% and 37% of their initial activities at 70 °C, respectively. The regioselectivity of each derivative was also examined in hydrolysis of fish oil at three different conditions. All the derivatives were selective between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. The highest selectivity (32.9 folds) was observed for epoxy-IDA-NH2-RML derivative in the hydrolysis reaction performed at pH 5 and 4 °C. Recyclability study showed good capability of the immobilized biocatalysts to be used repeatedly, retaining 50-91% of their initial activities after five cycles of the reaction.

The Science of Enzyme Immobilization.

Protocols for simple immobilization of unstable enzymes are plenty, but the vast majority of them, unfortunately, have not reached their massive implementation for the preparation of improved heterogeneous biocatalyst. In this context, the science of enzyme immobilization demands new protocols capable of fabricating heterogeneous biocatalysts with better properties than the soluble enzymes. The preparation of very stable immobilized biocatalysts enables the following: (1) higher operational times of enzyme, increasing their total turnover numbers; (2) the use of enzymes under non-conventional media (temperatures, solvents, etc.) in order to increase the concentrations of substrates for intensification of processes or in order to shift reaction equilibria; (3) the design of solvent-free reaction systems; and (4) the prevention of microbial contaminations. These benefits gained with the immobilization are critical to scale up chemical processes like the synthesis of biodiesel, synthesis of food additives or soil decontamination, where the cost of the catalysts has an enormous impact on their economic feasibility. The science of enzyme immobilization requires a multidisciplinary focus that involves several areas of knowledge such as, material science, surface chemistry, protein chemistry, biophysics, molecular biology, biocatalysis, and chemical engineering. In this chapter, we will discuss the most relevant aspects to do "the science of enzyme immobilization." We will emphasize the immobilization techniques that promote multivalent attachments between the surface of the enzymes and the porous carriers. Finally, we will discuss the effect that the structural rigidification promotes at different protein regions on the functional properties of the immobilized enzymes.

One-Point Covalent Immobilization of Enzymes on Glyoxyl Agarose with Minimal Physico-Chemical Modification: Immobilized "Native Enzymes".

The immobilization of soluble enzymes inside the porous structure of a preexisting support is one of the most interesting techniques to prepare heterogeneous biocatalysts. The main cause of inactivation of these biocatalysts is the distortion of the tridimensional structure of the immobilized enzymes. In some cases, immobilization of enzymes on preexisting supports can be used in order to improve its functional properties: stabilization by multipoint covalent immobilization, hyper-activation, and stabilization of lipases by interfacial adsorption on hydrophobic supports, etc. In other cases, the properties of the enzyme can be modified by additional interactions of the enzyme surface with the support surface: hydrophobic or electrostatic interactions.In all cases, it would be very interesting to evaluate the intrinsic tridimensional stability of native industrial enzymes. Under drastic experimental conditions, soluble enzymes may undergo undesirable aggregations, and the tridimensional stability of one enzyme is more accurately evaluated by using immobilized native enzymes. That is, immobilized derivatives associated to a minimal chemical modification of the enzyme surface placed in the proximity of a fully hydrophilic and inert support surfaces. In this chapter, the immobilization of enzymes with minimal physicochemical modification on glyoxyl agarose supports is proposed. At pH 8.5, the unique reactive amino group on the enzyme surface is the N-terminus. At the end of the immobilization, mild borohydride reduction, the primary amino terminus is simply converted into a secondary amino group, with similar physical properties, and aldehyde groups on the supports are converted into fully inert hydroxyl groups. The preparation of immobilized derivatives of penicillin G acylase (PGA) with identical properties (activity and stability) that one of the soluble enzyme is reported: preparation of immobilized native PGA.

Multi-Point Covalent Immobilization of Enzymes on Glyoxyl Agarose with Minimal Physico-Chemical Modification: Stabilization of Industrial Enzymes.

Stabilization of enzymes via immobilization techniques is a valuable approach in order to convert a necessary protocol (immobilization) into a very interesting tool to improve key enzyme properties (stabilization). Multipoint covalent attachment of each immobilized enzyme molecule may promote a very interesting stabilizing effect. The relative distances among all enzyme residues involved in immobilization have to remain unaltered during any conformational change induced by any distorting agent. Amino groups are very interesting nucleophiles placed on protein surfaces. The immobilization of enzyme through the region having the highest amount of amino groups (Lys residues) is key for a successful stabilization. Glyoxyl groups are small aliphatic aldehydes that form very unstable Schiff's bases with amino groups, and they do not seem to be useful for enzyme immobilization at neutral pH. However, under alkaline conditions, glyoxyl supports are able to immobilize enzymes via a first multipoint covalent immobilization through the region having the highest amount of lysine groups. Activation of supports with a high surface density of glyoxyl groups and the performance of very intense enzyme-support multipoint covalent attachments are here described.

Multi-Point Covalent Immobilization of Enzymes on Supports Activated with Epoxy Groups: Stabilization of Industrial Enzymes.

Commercial epoxy supports may be very useful tools to stabilize proteins via multipoint covalent attachment if the immobilization is properly designed. In this chapter, a protocol to take full advantage of the support's possibilities is described. The basics of the protocol are as follows: (1) the enzymes are hydrophobically adsorbed on the supports at high ionic strength. (2) There is an "intermolecular" covalent reaction between the adsorbed protein and the supports. (3) The immobilized protein is incubated at alkaline pH to increase the multipoint covalent attachment, thereby stabilizing the enzyme. (4) The hydrophobic surface of the support is hydrophylized by reaction of the remaining groups with amino acids in order to reduce the unfavorable enzyme-support hydrophobic interactions. This strategy has produced a significant increase in the stability of penicillin G acylase compared with the stability achieved using conventional protocols.

Immobilization of Lipases by Adsorption on Hydrophobic Supports: Modulation of Enzyme Properties in Biotransformations in Anhydrous Media.

Adsorption of lipases on hydrophobic supports is a very easy immobilization protocol and it yields very interesting immobilized lipase derivatives. The open and active form of lipase molecules becomes stabilized by strong adsorption on the support surface. By using very rigid hydrophobic supports (e.g., methacrylate), lipase derivatives are very useful to catalyze biotransformations in fully anhydrous organic media (solvents, solvent-free systems, etc.) and design of continuous flow reactors. In addition to that, the design of different lipase derivatives allows the modulation of functional properties of the derivatives. In this chapter, methodology of immobilization into hydrophobic carriers is described using as case study the preparation of immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL), and the following particular features will be discussed: 1. Adsorption on hydrophobic supports yields lipase derivatives that are much more active and stable than other immobilized lipase derivatives. 2. Regioselectivity can be modulated, for example, TLL adsorbed on divinyl benzene hydrophobic supports retains a 1,3 regioselectivity during ethanolysis of oils. On the contrary, the enzyme adsorbed on octadecyl supports loses the regioselectivity and allows the complete ethanolysis of oils (e.g., biodiesel synthesis). 3. TLL adsorbed on octadecyl supports with large pore size (60 nm) is tenfold more active for ethanolysis in solvent-free systems than TLL derivatives adsorbed on supports with small pore size (10 nm).

Stabilization of Multimeric Enzymes via Immobilization and Further Cross-Linking with Aldehyde-Dextran.

Subunit dissociation of multimeric proteins is one of the most important causes of inactivation of proteins having quaternary structure, making these proteins very unstable under diluted conditions. A sequential two-step protocol for the stabilization of this protein is proposed. A multisubunit covalent immobilization may be achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed of large internal surfaces and covered by a very dense layer of reactive groups. Additional cross-linking with polyfunctional macromolecules promotes the complete cross-linking of the subunits to fully prevent enzyme dissociation. Full stabilization of multimeric structures has been physically shown because no subunits were desorbed from derivatives after boiling them in SDS. As a functional improvement, these immobilized preparations no longer depend on the enzyme.

Co-Immobilization and Co-Localization of Multi-Enzyme Systems on Porous Materials.

The immobilization of multi-enzyme systems on solid materials is rapidly gaining interest for the construction of biocatalytic cascades with biotechnological applications in industry. The heterogenization and control of the spatial organization across porous materials of the system components are essentials to improve the performance of the process providing higher robustness, yield, and productivity. In this chapter, the co-immobilization and co-localization of a bi-enzymatic bio-redox orthogonal cascade with in situ cofactor regeneration are described. An NADH-dependent alcohol dehydrogenase catalyzes the asymmetric reduction of 2,2,2 trifluoroacetophenone using an NADH regeneration system consisting of a glutamate dehydrogenase and glutamic acid. Three different spatial organizations of the enzymes were compared in terms of cofactor-recycling efficiency. Furthermore, we demonstrated how the co-localization and uniform distribution (by controlling the enzyme immobilization rate) of the main and recycling dehydrogenases inside the same porous particle lead to enhance the cofactor-recycling efficiency of the bi-enzymatic bio-redox systems.

Synthesis of omega-3 ethyl esters from chia oil catalyzed by polyethylene glycol-modified lipases with improved stability.

Enzymatic synthesis of fatty acid ethyl esters (FAEE) from chia (Salvia hispanica L.) oil has been performed with different modified derivatives and compared with commercial immobilized lipases to produce omega-3 rich FAEE. Therefore, the main objective was to synthesize omega-3 esters from chia oil catalysed by polyethylene glycol-modified lipases using a biocatalyst with higher stability than commercial derivatives. Lipase from Thermomyces lanuginosus (TLL) was immobilized by hydrophobic adsorption on Sepabeads C-18 followed by a physicochemical coating of lipase surface with a dense layer of PEG. Ethanolysis reactions were carried out using pressurized liquid extracted chia seed oil and with different lipase derivatives to compare the omega-3 FAEE yield and ratio of reaction products, which were analysed by HPLC-ELSD. Furthermore, reutilization of lipase derivatives was studied to evaluate the stability after several reaction cycles to minimize decreasing of catalytic activity and develop a feasible enzymatic process for food industrial applications.

Biocatalyst engineering of Thermomyces Lanuginosus lipase adsorbed on hydrophobic supports: Modulation of enzyme properties for ethanolysis of oil in solvent-free systems.

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.

Co-localization of oxidase and catalase inside a porous support to improve the elimination of hydrogen peroxide: Oxidation of biogenic amines by amino oxidase from Pisum sativum.

Diamine oxidase (DAO) from Pisum sativum is an enzyme that catalyzes the degradation of biogenic amines (BA) present in wine, producing harmless aldehydes and hydrogen peroxide (H2O2). H2O2 promotes a rapid inactivation of the immobilized enzyme. At first glance, co-immobilization of DAO and catalase (CAT) could improve the elimination of the released hydrogen peroxide. Two different co-immobilized derivatives were prepared: (a) both enzymes co-localized and homogeneously distributed across the whole structure of a porous support, and (b) both enzymes we de-localized inside the porous support: DAO immobilized on the outer part of the porous support and catalase immobilized in the inner part. Co-localized derivatives were seven-fold more effective than de-localized ones for the elimination of hydrogen peroxide inside the porous support. In addition to that, the degradation of putrescine by DAO was three-fold more rapid when using both co-localized enzymes. The optimal co-localized derivative (containing 1.25 mg of DAO plus 25 mg of CAT per g of support) promoted the instantaneous elimination of 91% H2O2 released inside the porous support during putrescine oxidation. This optimal derivative preserves 92% of activity after three reaction cycles and DAO immobilized without catalase only preserves 41% of activity. Co-localization seems to be the key strategy to immobilize two sequential enzymes. When enzymes are immobilized in close proximity to each other in a co-localized pattern, the generation of byproducts as H2O2 is strongly reduced.

Stabilization of Immobilized Lipases by Intense Intramolecular Cross-Linking of Their Surfaces by Using Aldehyde-Dextran Polymers.

Immobilized enzymes have a very large region that is not in contact with the support surface and this region could be the target of new stabilization strategies. The chemical amination of these regions plus further cross-linking with aldehyde-dextran polymers is proposed here as a strategy to increase the stability of immobilized enzymes. Aldehyde-dextran is not able to react with single amino groups but it reacts very rapidly with polyaminated surfaces. Three lipases-from Thermomyces lanuginosus (TLL), Rhizomucor miehiei (RML), and Candida antarctica B (CALB)-were immobilized using interfacial adsorption on the hydrophobic octyl-Sepharose support, chemically aminated, and cross-linked. Catalytic activities remained higher than 70% with regard to unmodified conjugates. The increase in the amination degree of the lipases together with the increase in the density of aldehyde groups in the dextran-aldehyde polymer promoted a higher number of cross-links. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of those conjugates demonstrates the major role of the intramolecular cross-linking on the stabilization of the enzymes. The highest stabilization was achieved by the modified RML immobilized on octyl-Sepharose, which was 250-fold more stable than the unmodified conjugate. The TLL and the CALB were 40-fold and 4-fold more stable than the unmodified conjugate.

Production of FAME and FAEE via Alcoholysis of Sunflower Oil by Eversa Lipases Immobilized on Hydrophobic Supports.

The performance of two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 immobilized in different supports was investigated. The two lipases were adsorbed on four different hydrophobic supports. Interesting results were obtained for both lipases and for the four supports. However, the most active derivative was prepared by immobilization of Eversa® Transform 2.0 on Sepabeads C-18. Ninety-nine percent of fatty acid ethyl ester was obtained, in 3 h at 40 °C, by using hexane as solvent, a molar ratio of 4:1 (ethanol/oil), and 10 wt% of immobilized biocatalyst. The final reaction mixture contained traces of monoacylglycerols but was completely free of diacylglycerols. After four reaction cycles, the immobilized biocatalyst preserved 75% of activity. Both lipases immobilized in Sepabeads C-18 were very active with ethanol and methanol as acceptors, but they were much more stable in the presence of ethanol.