Cristina Otero

Purification and characterization of two distinct lipases from Candida cylindracea

We have purified and characterized two isoenzymes from a commercial lipase preparation of Candida cylindracea. The purification procedure includes ethanol precipitation and DEAE-Sephacel and Sephacryl HR 100 chromatographies. Lipase A and lipase B were purified 11-fold with a 5% and 21% recovery in activity, respectively. The enzymes have similar amino acid content, N-terminal sequence and molecular weight, but differ on neutral sugar content, hydrophobicity, presence of isoforms and stability to pH and temperature. They also show some differences in the substrate specificity.

Quantitative enzymatic production of 6-O -acylglucose esters

Selective production of emulsifiers from glucose and fatty acids has been achieved using an immobilized Candida antarctica lipase. Optimization of process selectivity considers the solubilities of the sugar and its monoesters in acetone at different temperatures, the percentage of this organic solvent in the reaction mixture, and the reaction temperature. The solvent (acetone) is both easily eliminated and accepted by the European Community for use in the manufacture of foods and/or food additives. Different fatty acids with a longer length chain than that of caprylic acid may be employed. For saturated fatty acids longer than lauric acid, continuous precipitation of the monoester as it is formed at 40 degrees C permits nearly complete conversion (98%) of glucose to the monoester within 2-3 days. The procedure does not require total dissolution of the sugar, and precipitation of the monoester permits selective conversion of charges of glucose higher than 100 mg/mL solvent. A scaleup of the process under the optimum conditions gives high yields of 6-O-lauroyl glucose, which may be readily prepared on a gram scale. Copyright 1998 John Wiley & Sons, Inc.

Enzymatic synthesis of amide surfactants from ethanolamine.

The condensation of a primary amine with fatty acids has been studied to determine optimum conditions for selective formation of amide surfactants via enzymatic amidification. Monoacylated ethanolamide and the diacylated amide-ester can be isolated from the reaction mixture, but the monoacylated ester cannot be isolated. The selectivity of the reaction depends on the solubility of the intermediate amide. Continuous precipitation of this product decreases the amount of amide-ester produced. Solubility values of the desired product (amide) are reported for different conditions.In acetonitrile, the ethyl ester of the corresponding fatty acid has been used successfully to avoid formation/precipitation of the ion-pair of the precursor reagents. In this medium, use of the transacylation reaction permits one to accelerate the reaction without producing a significant change in the selectivity toward the intermediate amide. This strategy is not successful in n-hexane where the solubilities of both ethanolamine and its ion-pair with lauric acid are similar.Results obtained for high loadings of substrates have been analyzed. In n-hexane and acetonitrile, the kinetics of the direct acylation reactions are controlled by the limited solubility of the ion pair formed by the two precursor reagents For the transacylation reaction in acetonitrile, at a sustrate loading of 2 mol l(-1,) selective production of as much as 92 mole percent N-acyl ethanolamine was observed in only 1.5 h.

Biphasic aqueous media containing polyethylene glycol for the enzymatic synthesis of oligosaccharides from lactose

Abstract The behavior of aqueous two-phase systems (ATPS) as media for the synthesis of galacto-oligosaccharides (GalOS) from lactose has been studied to determine the effects of changes in the phase compositions on the yield and selectivity of this reaction. Transglycosylation was accomplished using a commercial enzyme preparation (Pectinex Ultra-SP) in biphasic media containing polyethylene glycol (PEG). Factors such as the molecular weight of PEG, the concentration of NaCl, and the pH affected the yield of trisaccharides. The results obtained in the ATPS were better (33% increase in the yield of 6′-galactosyl lactose and 21% higher selectivity to this product) than those obtained in conventional aqueous solutions with the same enzymatic preparation. These results are attributed to the partitioning of the sugars (especially the desired product and inhibitors) and the biocatalyst between the two phases of the system. The ratio of the volumes of the two phases, partition coefficients, and the concentrations of enzyme and lactose in each phase were also determined.

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).

Enzyme, medium, and reaction engineering to design a low-cost, selective production method for mono- and dioleoylglycerols

The selective enzymic production of mono- and diolein (MO, DO) was optimized at high yields. A comparative study of the following distinct enzymic reactions was conducted: ethyl oleate glycerolysis, triolein (TO) glycerolysis, and direct esterification Solvent-free systems were compared with media that contained different solvents. Native, modified (with polyethylene glycol), and immobilized lipases were used. Mechanical resistance, the support effect on enzyme and glycerol dispersion and on process reproducibility, and hydrophilicity of the support were considered in the process optimization. We report the use of an immobilized lipase on an inorganic support (Celite), which has high activities in both solid-phase glycerolysis (99% reaction conversion) and esterification (100% conversion). The optimum conditions for the distinct reactions were compared by considering their selectivities, conversions, yields, and cost of the substrates. We found less costly and more selective processes in the absence of solvents for glycerolysis of triolein and direct esterification. Although glycerolysis was the most interesting process to produce diolein, esterification was better for monoolein preparation with this biocatalyst. The esterification reaction yielded 93 wt% of MO, in the absence of either TO or oleic acid (OA), at low cost because of the 100% reaction conversion. Similar costs of the substrates (10.6 and 10.1

Part III. Direct enzymatic esterification of lactic acid with fatty acids

/g) were necessary to obtain 67 and 80 wt% of DO in esterification and glycerolysis, respectively. The glycerolysis conversion was 96%. In esterification, the product mixture was impure, with a high amount of residual OA due to the low conversion (59%). The high activity of PSL-Celite in these solid-phase reactions has an advantage over the reactions with nonimmobilized lipases due to the ease of enzyme recovery. The absence of organic solvents reduces the need for solvent removal from the reaction mixtures.

Part I. Enzymatic synthesis of lactate and glycolate esters of fatty alcohols

Lipase catalyzed esterification reactions between lactic acid and several fatty acids have been studied. Difficulties arise in esterifying lactic acid because of the potential for this substance to act both as an acyl donor and as a nucleophile. These difficulties were minimized via strategies which greatly increased the yield of the desired ester. Use of the companion fatty acid in excess with respect to lactic acid in an apolar solvent (n-hexane) in which the lactic is not completely dissolved has been employed to minimize the potential for lactic acid to act as an acyl donor in a self-polymerization reaction.Beneficial and sinergistic effects of both silica gel and molecular sieves on conversion to the desired product are described. However, careful control of the amount of molecular sieves used is required. This fact is a consequence of two opposing effects of this material: i.e. adsorption of both lactic acid and water from the reaction mixture. For reaction between caprylic and lactic acids, use of an excessive amount of enzyme reduces the extent of conversion to 2-O-caproyl-lactic acid.A very pure ester of the L-enantiomer (optical rotation of [alpha]D(25) = -23.5) can be prepared in n-hexane using a four fold excess of caprylic acid and Candida antarctica lipase. Optimum reaction conditions lead to 35% yield of 2-O-caproyl-lactic acid, a result which is close to the maximum yield that can be enantioselectively obtained from commercial grade lactic acid (68 mole per cent monomer).

Quantitative enzymatic production of 1,6-diacyl fructofuranoses

Abstract Optimum conditions were determined for the esterification reactions of lactic and glycolic acids with fatty alcohols (C8–C16) in the presence of a lipase from Candida antarctica . This synthetic method gives nearly complete conversion to the desired ester in a relatively short time with high volumetric productivity. In acetonitrile the maximum yields of dodecyl lactate (95% in 48 h) and glycolate (87% in 24 h) were obtained in the presence of a desiccant and 0.28% (w/w) added water, respectively. The procedure permits one to increase substrate concentrations without significant adverse effects on the yield of lactate ester for lactic acid concentrations of 0.25 to 1 M. Similar yields of lactate ester (94–96%) were obtained for alcohol chain lengths from C8 to C16. Although esterification of lactic acid with fatty alcohols is not favored in apolar solvents (e.g. n-hexane), esterification of glycolic acid in n-hexane produces high yields of the glycolate ester (96% in 4 h). In the solvent-free system, esterification of lactic acid with a fatty alcohol requires the presence of desiccant from the beginning of the process (yield of 70% in 48 h). For the reactions of glycolic acid, a strategy in which a desiccant is added after 24 h of reaction gives the maximum yield of the glycolate ester (91%) in a shorter time (48 h of total reaction time). Alternatively, transesterification between the alcohol of interest and ethyl lactate increases the maximum yield of dodecyl lactate (87% in 24 h).

Regioselective deacylation of 1,6-anhydro-β-D-galactopyranose derivatives catalyzed by soluble and immobilized lipases

Three different 1,6-diacyl fructofuranoses have been prepared enzymatically. At low temperature (5°C), the synthesis produces quantitative yields of the diester by simple addition of the original sugar to a solution of the fatty acid in a solvent (acetone) which is accepted by the EEC for use in the manufacture of food additives. A strategy to reduce the reaction times is also reported. The method is not limited by the low solubility of the sugar in the medium. In contrast with alternative enzymatic methods, the indicated method minimizes the solvent/sugar ratio. The stability of the biocatalyst (Novozym 435) is high relative to the required reaction time.