Guido Greco

Dephenolisation of olive mill waste-waters by olive husk

Olive mill waste-waters (OMWW) are a by-product of olive-oil production and a major environmental problem in the Mediterranean area. The use of a polyphenol-oxidase from olive husk is proposed for the enzymatic removal of low-molecular phenolics in OMWW. For comparison purposes, a purified microbial polyphenol-oxidase (Trametes versicolor laccase) was also employed. Though both enzymatic systems show relevant activity towards phenol polymerization, olive husk is much more appealing in view of possible applications because of its availability and extremely low cost, associated with excellent enzymatic activity and specificity.

Production of glucose and bioactive aglycone by chemical and enzymatic hydrolysis of purified oleuropein fromOlea Europea

Pure-grade oleuropein, a bitter, hypotensive, phenolic glucoside, was obtained from organic extracts of olive plant leaves by two Chromatographic steps. The purified compound was characterized by spectroscopic NMR and FAB-MS methods. The glucoside underwent chemical and enzymatic hydrolysis. Aglycone was characterized by spectroscopic methods (1H-NMR and FAB-MS). Glucose was measured by enzymatic methods. The enzymatic hydrolysis of oleuropein was carried out by a soluble β-glucosidase. The reaction was characterized in terms of kinetic parameters, optimal pH value, activation energy, inhibition constant by glucose, and thermal stability. Preliminary experiments were also performed in a continuousflow ultrafiltration membrane reactor. The cut-off of the membrane was lower than the molecular-weight of the enzyme, thus determining β-glucosidase confinement within the reactor. Under these conditions, β-glucosidase had a good long-term stability. This is an encouraging result in view of possible industrial applications.

Oxidative polymerisation of phenols by a phenol oxidase from green olives

Abstract The phenol oxidase activity of green olive drupae was characterised in view of possible applications for the de-phenolisation of polluted waters. For experimental convenience, activity measurements were performed on acetone powders and raw-extracts from drupae tissues. Phenols were oxidised in the presence of dissolved molecular oxygen. Oxidation products were o-quinones undergoing non-enzymatic polymerisation. Final products were insoluble polymers that could be easily removed from the reaction mixture by centrifugation. The kinetics of activity loss was analysed and a possible “suicide substrate” deactivation mechanism, analogous to that of mushroom tyrosinase, has been proposed. Substrate specificity was particularly high towards o-diphenols and p-methyl substituted phenols. For real applications, the observed, low operational stability discourages the use of purified and immobilised enzyme in lieu of minced tissues. Furthermore, the seasonal availability of olive suggests an on-site use for polyphenol removal from vegetation waters resulting from the milling process.

Enzyme stabilization towards thermal, chemical and proteolytic deactivation

Abstract An enzyme stabilization technique which consists of entrapping protein within a polymeric network has been discussed. The high macromolecular concentration levels which lead to formation of the network are produced as a consequence of polarization phenomena which take place within an unstirred ultrafiltration membrane reactor. Increases in enzyme half-life were generally produced in connection with simple and complex deactivation phenomena of widely different natures (thermal, chemical and proteolytic). Experimental tests have been carried out on the following enzymes: β- d -glucosidase (β- d -glucoside glucohydrolase, EC 3.2.1.21), β- d -fructofuranosidase (β- d -fructofuranoside fructohydrolase, EC 3.2.1.26), acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] and β- d -galactosidase (β- d -galactoside galactohydrolase, EC 3.2.1.23).

Soil-catalyzed polymerization of phenolics in polluted waters

Some biotic and abiotic soil components are able to catalyze phenol oxidation, producing water-insoluble polymers. In phenol-polluted water bodies, this phenomenon could be exploited to prevent phenol dispersion. The reaction kinetics of phenol polymerization catalyzed by soil samples drawn from unsaturated and aquifer layers was measured in slurry, aerated batch reactors. Catechol was used as a model phenol. The observed catalytic activity is essentially abiotic and can be attributed to inorganic soil components. The rate of phenol removal is first-order with respect to both catechol and soil concentration. Soil activity towards other phenolic compounds was tested, as well. Diphenols show the highest reactivity. Comparisons were performed with the enzymatic activity of phenol oxidases-containing mushroom tissues whose use has been envisaged in the treatment of phenol-polluted waters. The use of phenol oxidases can complement the intrinsic activity of soil for the removal of recalcitrant phenols.

The stabilizing effect of soluble macromolecules on enzyme performance

A new enzyme stabilization method is proposed which is associated with dynamical immobilization within an unstirred ultrafiltration membrane reactor. Upon injection of suitable amounts of macromolecular solutions, the very polarization phenomena that yield the immobilization produce high macromolecular concentration levels in the reactor region immediately upstream from the ultrafiltration membrane where the enzymes operate. This procedure results in considerable improvements in enzyme stability that seem to be quite independent of the nature of the stabilizing macromolecule adopted. Stabilization effects of the same order of magnitude have been obtained for different enzymatic systems.

Enzyme inactivation and stabilization studies in an ultrafiltration reactor

SummaryAn ultrafiltration membrane enzymatic reactor is used in connection with different reacting systems.The experimental conditions are such that the enzyme, which operates at fairly high concentration levels because of the concentration polarization phenomena taking place in the reactor, is still in soluble form.The analysis of the system unsteady-state response enables the identification of the mechanism of enzyme deactivation and the extraction of the kinetic parameters of both the deactivation and the main reaction.The stabilizing effect observed in connection with enzyme entrapment within an inert gel deposited onto the U.F. membrane active surface is also discussed.

Purified penicillin acylase performance in a stabilized ultrafiltration membrane reactor

SummaryA new simplified technique is discussed for quantitative penicillin acylase extraction from mutant Escherichia coli cells which is based on freezing and thawing followed by an osmotic pressure shock. The purification steps are also briefly illustrated. Purified acylase was employed in an unstirred ultrafiltration membrane reactor. Different linear-chain soluble polymers were tested as potentially stabilizing macromolecules. Reference runs were performed both with homogeneous native enzyme when operating in the homogeneous phase and with the membrane reactor with one addition of other macromoleculars. Promising results were obtained in terms of enzyme stabilization, although considerable reduction in activity levels also occurred.

Lipase-catalyzed transformations for the synthesis of butyl lactate: a comparison between esterification and transesterification.

The α‐hydroxy esters are increasingly employed in cosmetic, food, and pharmaceutical formulations as they determine reduced skin‐irritant effects in comparison with the respective acids, offering similar hygroscopic, emulsifying, and exfoliating properties. The enzymatic synthesis of lactate esters in nonaqueous systems was studied as regards the influence of the critical process parameters, to enable a comparison between the most commonly used synthetic routes, namely, esterification and transesterification. The experimental results showed that the direct esterification of lactic acid with butanol may be limited by the reduced lipase stability in the presence of the acid (substrate) and of the water (product), in particular when solvent‐free media are used. The stability of the enzyme is further reduced as polar solvents are required as a result of the polarity of the lactic acid. Therefore, the use of the lactic acid as substrate is of practical interest only when the acid is significantly cheaper in comparison with its short‐chain esters. If this is not the case, the transesterification of the ethyl lactate with butanol is to be preferred for the higher flexibility in the choice of the experimental conditions, the operability of solvent‐free systems, and the simplicity of the product removal assembly.

Kinetic behaviour of immobilized enzyme membrane reactors

Abstract Enzyme immobilization techniques which are founded on protein gelification onto the active surface of an ultrafiltration membrane are discussed in connection with p -nitro-phenyl-phosphate hydrolysis by acid phosphatase (E.C.3.1.3.2.) Two different immobilized enzyme membrane reactor layouts have been considered. In both the enzyme has been confined within a gel layer onto the active surface of the U.F. membrane. The gel formation has been obtained by means of a concentration polarization technique. In the first reactor the enzyme has been cogelified with albumin, in the second it has been previously co-cross-linked with the same albumin and subsequently ultrafiltered. The reduction in activation energy which is shown by both immobilized enzyme configurations as compared to the corresponding soluble enzyme tests, clearly indicates that a combined substrate mass transfer/reaction step is rate controlling.