Eitel Pastor

Enzymatic acylation of di- and trisaccharides with fatty acids: choosing the appropriate enzyme, support and solvent

Enzymatic synthesis of fatty acid esters of di- and trisaccharides is limited by the fact that most biological catalysts are inactivated by the polar solvents (e.g. dimethylsulfoxide, dimethylformamide) where these carbohydrates are soluble. This article reviews the methodologies developed to overcome this limitation, namely those involving control over the reaction medium, the enzyme and the support. We have proposed the use of mixtures of miscible solvents (e.g. dimethylsulfoxide and 2-methyl-2-butanol) as a general strategy to acylate enzymatically hydrophilic substrates. We observed that decreasing the hydrophobicity of the medium (i.e. lowering the percentage of DMSO) the molar ratio sucrose diesters versus sucrose monoesters can be substantially enhanced. The different regioselectivity exhibited by several lipases and proteases makes feasible to synthesise different positional isomers, whose properties may vary considerably. In particular, the lipase from Thermomyces lanuginosus displays a notable selectivity for only one hydroxyl group in the acylation of sucrose, maltose, leucrose and maltotriose, compared with lipase from Candida antarctica. We have examined three immobilisation methods (adsorption on polypropylene, covalent coupling to Eupergit C, and silica-granulation) for sucrose acylation catalysed by T. lanuginosus lipase. The morphology of the support affected significantly the reaction rate and/or the selectivity of the process.

High-yield production of mono- and di-oleylglycerol by lipase-catalyzed hydrolysis of triolein

Abstract Lipases from several origins—in soluble form or immobilized on solid supports of different aquaphilicity—have been used for the production of mono- and di-oleylglycerol by hydrolysis of triolein. The porcine pancreatic lipase adsorbed on Celite was found to be the most effective biocatalyst tested. High activity and excellent selectivity toward the formation of intermediate acylglycerols were achieved (the conversion to diolein plus monoolein was 79% after 5 h of reaction). The time course of triolein hydrolysis with free and immobilized lipases was analyzed using a sequential kinetic model, and the apparent first-order rate constants were calculated. The differences found in the rate constants were related to differences in the regioselectivity of the enzymes and/or the aquaphilicity of the supports.

Influence of the support on the reaction course of tributyrin hydrolysis catalyzed by soluble and immobilized lipases

Lipases from different origins have been immobilized in supports chosen by its different aquaphilicity and used as biocatalysts for the hydrolysis of tributyrin. The changes of the concentration of tri-, di-, monobutyrin, glycerol, and butyric acid during the reactions catalyzed by soluble, as well as immobilized, lipases were evaluated by gas chromatography. The experimental data were fitted to a simple kinetic model for the sequential reaction of tributyrin hydrolysis. The calculated apparent rate constants were different for the biocatalysts used and were apparently related to diffusional effects and aquaphilicity of the supports. Maximal yields of dibutyrin were found with the solubleCandida lipase, whereas the highest yield of monobutyrin (90%) was obtained with the least aquaphylic derivative (Candida-Celite).

Synthesis of monobutyrylglycerol by transesterification with soluble and immobilized lipases

Transesterification between ethyl butyrate and glycerol using very different lipase preparations (native, modified, and immobilized) in a two-phase system—no extra solvent added—has been investigated. Optimal conversion was obtained with the presence of 5% water in the reaction mixture. Only monobutyrin was produced in all conditions tested. The best enzyme preparations were nativeCandida cylindracea lipase,Mucor miehei lipase immobilized on a phenol-formaldehyde exchange resin (Lipozyme™), andC. cylindracea lipase immobilized on Celite.

Enzymatic Preparation of Mono- and Di-Stearin by Glycerolysis of Ethyl Stearate and Direct Esterification of Glycerol in the Presence of a Lipase from Candida Antarctica (Novozym 435)

Mixtures of 1(3)-monostearin and distearin were prepared by direct esterification of glycerol with stearic acid or transesterification using ethyl stearate as acyl donor in the presence of Candida antarctica lipase (Novozym 435) using a variety of solvents of differing polarity. In all cases, the transesterification resulted in higher product yields. In n-heptane as reaction medium the addition of water (3%) was essential for high product yields, with mono- and distearin being produced in almost equal amounts. Using more polar solvents as reaction media, such as acetonitrile or acetone, again the highest yields were obtained in the transesterification mode; employing these solvents the reactions were much more selective towards the formation of monostearin.

Effect of chemical modification of isoenzymes A and B from C. rugosa on their activity and stability

SummaryLipases A and B from C.rugosa have been chemically modified with octanoyl chloride or polyethylene glycol (molecular weight: 750 or 5000). Although the hydrolytic activity was diminished, modification gave rise in some cases to preparations with increased synthetic activity in hexane. Furthermore, PEG5000-lipases A and B were significantly more stable (up to 20-fold) at alkaline pH values than the native enzymes.

Synthesis of mono- and dioleylglycerols using an immobilized lipase

Transesterification between ethyl oleate and glycerol, or esterification between oleic acid and glycerine by the immobilized lipase preparation SP 435 fromCandida antarctica, was investigated. Different temperatures and reactants ratios were tried. In all cases, transesterification yielded better results. When the reaction was carried out in n-heptane the addition of 3% water gave highest yields to mono and diolein; the formation of diolein can reach levels comparable to those of monoolein. However, when the reaction was carried out in oneliquid-phase only (in the presence of acetonitrile or acetone), the reaction was much more selective to monoolein.

Hept-1-ene hydroformylation on phosphinated polystyrene-anchored rhodium complexes

Abstract The catalytic performance for the hydroformylation reaction of hept-1-ene at 343 K and 30 atm total pressure has been studied over some rhodium-(I) complexes anchored to 2% DVB crosslinked phosphinated polystyrene. The catalysts were prepared from the common [RhCl(COD)] 2 precursor and characterized by FTIR and photoelectron (XPS) spectroscopy techniques. The activity profiles and the number of cycles operative in the reactor were found to depend markedly on the Rh:P ratio. A joint discussion involving the data obtained from the homogeneous process is also presented.

Chemical versus enzymatic catalysis for the regioselective synthesis of sucrose esters of fatty acids

In this work we present two processes for the regioselective synthesis of sucrose monoesters. The first is enzymatic, and is carried out in a medium constituted by two miscible solvents, namely tert-amyl alcohol and dimethylsulfoxide. The second is chemical, and is based on the use of simple basic catalyts in dimethylsulfoxide. The regioselectivity of both methodologies will be compared.

Hydrolysis and Synthesis of Butyrylglycerols by Lipasesa

Lipases (EC 3.1.3) comprise a group of enzymes whose biological function is to catalyze the hydrolysis and synthesis of triacylglycerols. For many years, Desnuelle and collaborators’ carried out detailed studies on the mode of action of lipases, mainly animal pancreatic lipase. Later, many lipases from microbial sources were investigated and were found to be promising catalysts for hydrolysis and synthesis of fats and oils.* Of these, the enzyme from Candida cylindracea (now named C. rugosa) is an interesting lipase because of its high activity in hydrolytic as well as synthetic reactions. Lipases, which act on water-insoluble substrates, can be adsorbed on hydrophobic surfaces. This stresses the importance of the hydrophobic/hydrophilic character of the matrix used in the immobilization. Besides, immobilization of enzymes that act on substrates having several bonds susceptible to reaction (lipases, nucleases, carbohydratases, etc.) may create diffusional resistances or local gradients that may change the progress of the hydrolysis reaction as compared to the native enzyme. As regards their ability to hydrolyze triacylglycerols, lipases have been classified as unspecific, 1,3-specific, and fatty acid-specific and these various specificities have been exploited in industry.’ Therefore, it may be interesting to investigate the synthesis of acylglycerols with lipases in order to see if the specificity of action is the same in hydrolysis and synthesis (i.e., if a 1,3-lipase yields I ,3-diglycerides. but no triglycerides in the synthetic reaction). On the other hand, Cesti et u I . ~ used pancreatic lipase for the transesterification of diols with ethyl carboxylates; as a result, only primary monoesters were produced. In the present work, we have investigated the time course of the hydrolysis of tributyrin by several immobilized lipases as a function of the aquaphilicity’ of the support. In addition, the transesterification between ethyl butyrate and glycerol, using different lipase preparations (soluble, immobilized, modified), has been studied. The acylation has proven to be very selective; only monobutyrin has been produced.