J.L. Iborra

Over-stabilization of Candida antarctica lipase B by ionic liquids in ester synthesis

Four different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for butyl butyrate synthesis catalyzed by free Candida antarctica lipase B at 2% (v/v) water content and 50 °C. Lipase had enhanced synthetic activity in all ionic liquids in comparison with two organic solvents (hexane, and 1-butanol), the enhanced activity being related to the increase in polarity of ionic liquids. The continuous operation of lipase with all the assayed ionic liquids showed over-stabilization of the enzyme. The reuse of free lipase in 1-butyl-3-methylimidazolium hexafluorophosphate in continuous operation cycles showed a half-life time 2300 times greater than that observed when the enzyme was incubated in the absence of substrate (3.2 h), and a selectivity higher than 90%.

SHORT-CHAIN FLAVOUR ESTER SYNTHESIS BY IMMOBILIZED LIPASE IN ORGANIC MEDIA

SummaryMucor miehei lipase has been adsorbed on Celite and covalently bound to nylon. The obtained derivatives have been studied regarding their ability for synthetize several flavouring esters in biphasic aqueous/organic media. The influence of the immobilization procedure on the synthetic activity of the derivatives was considered. Solvent hydrophobicity and water content in the biphasic system influenced both enzyme stability and equilibrium displacements. In this way, solvents with log P>3.5 and less than 1% water were optimal. It was important to consider pH effects on enzyme microenvironment when using acidic substrates. Optimum temperature and reuse of the catalyst were also checked.

Kinetic study of the pathway of melanizationn between l-dopa and dopachrome

Abstract The first part of the melanization pathway from l -dopa to dopachrome has been studied as a system of various chemical reactions coupled by an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system. Rate constants for the implicated chemical steps at different pH and temperature values can be evaluated from measurement of the lag period arising from the accumulation of dopachrome that takes place when l -Dopa was oxidized at acid pH. The thermodynamic parameters of the chemical steps, the deprotonation of dopaquinone-H + into dopaquinone and the internal cyclization of dopaquinone into leukodopachrome, have been obtained. From the results presented, an alternative series of chemical reactions to the Raper-Mason scheme are proposed and discussed.

A quantitative high-performance liquid chromatographic method to analyse commercial saffron (Crocus sativus L.) products.

An HPLC method to check components and purity in commercial saffron by photodiode array detection has been developed. The method was suitable for the standard analysis of commercial saffron. Therefore, 10 saffron metabolites responsible for the taste, flavour and colour were identified and quantified with high selectivity, precision and accuracy. Also, some artificial colorants, which can be used as adulterants, were also detected and identified. Three different saffron types were studied and their metabolite concentrations determined at different wavelengths.

Effect of polyols on α-chymotrypsin thermostability: a mechanistic analysis of the enzyme stabilization

The influence of the synthetic substrate (N-acetyl-L-tyrosine ethyl ester) and the different polyols (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of alpha-chymotrypsin at 60 degrees C have been studied. The results obtained showed an important stabilizing effect in the presence of both additives. In order to describe the kinetics of enzyme stabilization, the experimental results were analyzed by a four-parameters deactivation model with excellent agreement. In all cases, alpha-chymotrypsin exhibited non-first-order deactivation kinetics, corresponding to a two-step unimolecular mechanism, where the main protective effect of polyols was observed in the first-step of the deactivation profile. Thus, the presence of polyols increased the level of activity stabilization (alpha 1), and decreased the first-order deactivation rate constant (k1). Additionally, the experimental results were analyzed as a function of both, the change in the standard free energy of denaturation (delta(delta Gzero)), and a protective effect, defined as the ratio of alpha-chymotrypsin half-lives (with and without polyols), showing in both cases a clear stabilizing effect of these polyhydroxylic cosolvents for the enzyme. The overall protective effect of polyols was also simultaneously related to their concentration and their water-activity depressing power.

Immobilization of naringinase on glycophase-coated porous glass

SummaryNaringinase from Penicillium sp. was covalently linked to Glycophasecoated controlled-pore glass. The parameters of the immobilization process were characterized with respect to both the coupling method as well as to support pore size. The efficient kinetic parameters shown by the most active and stable derivative enables it to be used for debittering of naringin-containing juices.

High-density Escherichia coli cultures for continuous L(-)-carnitine production.

Abstract The use of a biological procedure for l-carnitine production as an alternative to chemical methods must be accompanied by an efficient and highly productive reaction system. Continuous l-carnitine production from crotonobetaine was studied in a cell-recycle reactor with Escherichia coli O44 K74 as biocatalyst. This bioreactor, running under the optimum medium composition (25 mM fumarate, 5 g/l peptone), was able to reach a high cell density (26 g dry weight/l) and therefore to obtain high productivity values (6.2 g l-carnitine l−1 h−1). This process showed its feasibility for industrial l-carnitine production. In addition, resting cells maintained in continuous operation, with crotonobetaine as the only medium component, kept their biocatalytic capacity for 4 days, but the biotransformation capacity decreased progressively when this particular method of cultivation was used.

Preparative high-performance liquid chromatographic purification of saffron secondary metabolites

Abstract A preparative HPLC procedure to isolate picrocrocin, the compound responsible for the taste of saffron and precursor of the aromatic safranal, and the mixture of yellow pigments from a saffron hydroalcoholic extract has been developed. A reversed-phase C 18 column was employed as the stationary phase. The best separation was obtained with 45% methanol, plus a 90% acetonitrile pulse 3 min after starting the run, as mobile phase. To obtain the highest yield from the system, sample size was increased up to 2 ml of 200 mg ml −1 saffron extract; under such conditions a good resolution was obtained and picrocrocin and saffron pigments were separated with a high purification yield and purity.

Comparative thermostability of glucose dehydrogenase from Haloferax mediterranei. Effects of salts and polyols

The effect of temperature and pH on thermoinactivation kinetics of glucose dehydrogenase from Haloferax mediterranei has been studied in the presence of different monovalent salts (LiCl, LiBr, NaCl, NaBr, KCl, KBr, NH4Cl, and NH4Br) and polyols (glycerol, erythrytol, xylitol, and sorbitol) concentrations. The stabilization degree of salts followed the rank of the Hofmeister series, and the product of the Setchenov constant (Ks) times the concentration of solute (Cs) was useful to predict the enzyme stability in the presence of salt solutions. Polyols stabilized the halophilic enzyme as much as salts. For an equal polyol concentration, the thermostability increased in the range glycerol < erythritol < xylitol < sorbitol. The overall hydroxyl group concentration proved to be a good parameter for correlating the protective effect of polyols with the polyol nature. Thermoinactivation of the halophilic glucose dehydrogenase in the presence of NaCl and sorbitol was compared with that of a nonhalophilic glucose dehydrogenase in terms of the transition state theory. The free activation energy was, in all cases, enthalpy driven, and hydrogen-bond and/or ionic-binding interactions are the main forces involved in protein stabilization. The halophilic enzyme showed, in general, lower free activation energies for the deactivation process. The adaptation of the enzyme to a halophilic environment led to an enzyme with higher activity at high salt concentrations, but such an increase in enzyme activity was not related to an enhancement in enzyme thermostability.

Biotransformation of D(+)–carnitine into L(−)–carnitine by resting cells of Escherichia coli O44 K74

l(−)‐carnitine was produced from d(+)‐carnitine by resting cells of Escherichia coli O44 K74. Oxygen did not inhibit either the carnitine transport system or the enzymes involved in the biotransformation process. Aerobic conditions led to higher product yield than anaerobic conditions. The biotransformation yield depended both on biomass and initial substrate concentrations used; the selected values for these variables were 4·30 g l−1 cells and 100 mmol l−1d(+)‐carnitine. Under these conditions the l(−)‐carnitine production rate was 0·55 g l−1 h−1, the process yield was 44%, and the productivity was 0·22 g l−1 h−1 after a 30 h incubation period. Crotonobetaine production, besides l(−)‐carnitine, showed that the action of more than one enzyme occurred during the biotransformation process. On the other hand, the addition of fumarate at high substrate concentrations (250 and 500 mmol l−1) led to a higher metabolic activity, which meant an increment of l(−)‐carnitine production.