A. Manjón

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.

A method for assaying the rhamnosidase activity of naringinase

The use of the p-nitrophenyl-alpha-L-rhamnopyranoside for the specific measurement of the alpha-rhamnosidase activity of naringinase, by colorimetrically following the appearance of p-nitrophenolate anion, is proposed. Use of this synthetic substrate did not change the pH, temperature, or ionic strength optima of the enzyme. It did, however, result in (a) a decrease of the Michaelis constant of the enzyme, allowing the Vmax to be measured, this being impossible to accomplish with naringin, (b) an increase in the sensitivity of the assay to the presence of inhibitors in the reaction media, (c) an increase in the sensitivity which enabled measurement of low levels of naringinase due to the high absorptivity of p-nitrophenolate, and (d) a quick and cheap method of evaluating the alpha-rhamnosidase activity of naringinase.

A recyclable enzymatic biodiesel production process in ionic liquids

Immobilized Candida antarctica lipase B suspended in ionic liquids containing long alkyl-chain cations showed excellent synthetic activity and operational stability for biodiesel production. The interest of this process lies in the possibility of recycling the biocatalyst and the easy separation of the biodiesel from the reaction mixture. The ionic liquids used, 1-hexadecyl-3-methylimidazolium triflimide ([C(16)MIM][NTf(2)]) and 1-octadecyl-3-methylimidazolium triflimide ([C(18)MIM][NTf(2)]), produced homogeneous systems at the start of the reaction and, at the end of the same, formed a three-phase system, allowing the selective extraction of the products using straightforward separation techniques, and the recycling of both the ionic liquid and the enzyme. These are very important advantages which may be found useful in environmentally friendly production conditions.

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.

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.

2,3,5-triphenyltetrazolium chloride as a viability assay for immobilized plant cells

Grape (V. vinifera L. cv Gamay Freaux) cells were entrapped in calcium alginate beads. The effect of bead diameter and gel concentration on the viability of the cells was checked. The reduction of 2,3,5-triphenyltetrazolium chloride as well as O2 consumption were used as viability test, and their results compared. The existence of diffusional limitations for O2 introduces an unaccuracy as high as 25% for the O2 consumption method. The reduction assay is more simple, precise and reliable.

A cross-flow reactor with immobilized pectolytic enzymes for juice clarification

SummaryA new system for continuous juices clarification is presented. The bioreactor combines microporous plates commercially available and industrial pectinases immobilized on nylon membranes in a cross-flow configuration. The kinetic behaviour of the reactor for different recirculation flow rates has been determined. Fresh apricot juice has been continuously clarified in the bioreactor with excellent results.

Acid proteinase activity in fish II. Purification and characterization of cathepsins B and D from Mujil auratus muscle

Two cathepsins were detected in Mujil auratus muscle extracts. They were classified as a thiol- and aspartyl-proteinase (cathepsins B and D, respectively) on the basis of their catalytic behaviour in presence of specific inhibitors. Following extraction in 1% KCl, the proteinases were purified by autolysis, acetone fractionation, affinity chromatography, and gel permeation chromatography. The haemoglobin-agarose column chromatography allowed us to separate the two activities. Sephadex G-75 column chromatography resulted in apparent molecular weights of 25,000 (cathepsin B) and 35,000 (cathepsin D). The molecular size, together with pH-activity profiles and kinetic parameters are similar to those reported for mammalian cathepsins B and D. This was not the case with the temperature-activity profiles, the optimum temperature as well as the heat stability being higher for fish cathepsins than for those obtained from other sources. Cathepsin B was characterized by its ability to inactivate aldolase. Fluorescence quenching experiments showed that tryptophyl residues of cathepsin B were less occluded and located in a more electronegative microenvironment that those pertaining to cathepsin D.

Kinetic and operational study of a cross-flow reactor with immobilized pectolytic enzymes

The kinetics and operational behavior of a nylon membrane derivative with immobilized pectolytic enzymes in a cross-flow reactor were analyzed. A high dependence on the recycling flow rate was observed. A design equation of the system has been proposed by taking into account both the shear stress deactivation and the control of the external diffusional limitations. Integration of the resulting design equation allowed us to study the effect of different operational parameters on substrate conversion. The catalytic efficiency of the immobilized derivative in a cross-flow reactor showed the highest pectin hydrolysis capability when it was compared with two different configurations of packed-bed reactors.