H. Stamatis

Purification and characterization of a feruloyl esterase from Fusarium oxysporum catalyzing esterification of phenolic acids in ternary water–organic solvent mixtures

An extracellular feruloyl esterase (FAE-II) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by SP-Sepharose, t-butyl-HIC and Sephacryl S-200 column chromatography. The protein corresponded to molecular mass and pI values of 27 kDa and 9.9, respectively. The enzyme was optimally active at pH 7 and 45 degrees C. The purified esterase was fully stable at pH 7.0-9.0 and temperature up to 45 degrees C after 1 h incubation. Determination of k(cat)/K(m) revealed that the enzyme hydrolysed methyl sinapinate 6, 21 and 40 times more efficiently than methyl ferulate, methyl coumarate and methyl caffeate, respectively. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 but inactive to the C-2 positions of arabinofuranose such as 4-nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside. In the presence of Sporotrichum thermophile xylanase, there was a significant release of ferulic acid from destarched wheat bran by FAE-II, indicating a synergistic interaction between FAE-II and S. thermophile xylanase. FAE-II by itself could release only little ferulic acid from destarched wheat bran. The potential of FAE-II for the synthesis of various phenolic acid esters was tested using as a reaction system a surfactantless microemulsion formed in ternary mixture consisting of n-hexane, 1-propanol and water.

Lipase-catalyzed esterification of rutin and naringin with fatty acids of medium carbon chain

Abstract Flavonoids rutin and naringin were acylated with fatty acids of medium carbon chain (with 8–12 carbon atoms on their molecule) in a reaction catalyzed by immobilized lipase from Candida antarctica (Novozyme) in various solvent systems. The reaction parameters affecting the acylation rate and the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity ( a w ) of the system, as well as the kinetic of the reaction have been investigated. In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective. The enzymatic acylation of flavonoids seems to follow Michaelis–Menten kinetics.

A comparison of different strategies for lipase-catalyzed synthesis of partial glycerides

SummaryFour different approaches for the synthesis of monolaurylglycerol (MLG) by non specificPseudomonas cepacia lipase in a crude and purified form have been studied: a. The direct esterification of glycerol by lauric acid in bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsion systems; b. the transesterification of glycerol by vinyl laurate in the presence or not of any solvent; c. solid-phase glycerolysis of trilaurin; and, d. transesterification of protected glycerol, 1,2-O-isopropylidene glycerol, by vinyl laurate, in the presence or not of any solvent. It was found that in the two latter cases (d and c) the formation of pure MLG was occurred, while in the first two cases (a and b) apart of MLG the formation of DLG was also observed.

Enantiomeric selectivity of a lipase from Penicillium simplicissimum in the esterification of menthol in microemulsions

SummaryLipase from Penicillium simplicissimum catalyzes the stereospecific esterification of menthol with fatty acids. The studies on the specificity of this new lipase were carried out using (+), (-) and racemic menthol with water soluble enzyme entrapped in microemulsion systems stabilized with sodium(bis-2-ethylhexyl)sulfosuccinate (AOT) as surfactant, in isooctane. Microemulsions appear to be an effective and fast system for racemic resolution of alcohols.

Pseudomonas cepacia lipase: Esterification reactions in AOT microemulsion systems

SummaryThe activity of purifiedPseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis(2ethylhexyl)sulfosuccinate sodium salt (AOT). The optima pH, T and water content (wo) for the enzyme activity in this type of microemulsions have been determined. Studies on the effect of various fatty acids and alcohols on the enzyme specificity have shown a preference of this lipase for palmitic and caprylic acid as well as for propanol, while reactions involving cyclic alcohols can not be catalyzed at all. The differences on the behavior of this lipase as compared to other lipases studied in microemulsion systems as well as in other systems are discussed.

Reverse micelles, a system for antibody-catalysed reactions

This work presents some aspects of the application of catalytic antibodies in water-in-oil microemulsions (reverse micelles) based on sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) in isooctane. The monoclonal antibody (mAb) 9A8 used in this study is a fully characterised acetylcholinesterase-like antibody produced by the anti-idiotypic approach. The effect of various parameters, such as, the size and the concentration of reverse micelles, as well as the concentration and the nature of substrates on abzyme catalytic activity were investigated.

Lipase immobilization on microemulsion-based polymer gels

Lipase from Pseudomonas cepacia has been immobilized in lecithin or AOT microemulsion-based gels formed with two natural gelling agents such as gelatin and agar. The results presented in this work provide information concerning the utility of these solid gels as lipase immobilization matrices. Lipase keeps its catalytic activity after entrapment in the microemulsion-based gels, catalyzing the esterification reaction of propanol with lauric acid in isooctane. High yields (80%) were obtained with agar organogels after 72 h of incubation at room temperature. Gelatin and agar microemulsion-based gels exhibited good stability with regard to both retention of lipase catalytic activity and gel integrity.

Unexpected reaction profile observed in the synthesis of propyl laurate when using Candida rugosa lipases immobilized in microemulsions based organogels.

Abstract1-Propyl laurate synthesis should not be used as standard reaction test of immobilized enzymes in microemulsion-based organogels (MBGs) prepared using lecithin/1-propanol as surfactant when extremely active enzymes with high load are used. In these cases, an anomalous kinetic reaction constant value is observed over short reaction times. Such an anomalous profile is strongly dependent on the concentration of catalyst in the crude powder and, consequently, is not appreciated when either commercial or low activity lipase samples are employed.