Hervé Adenier

Electrically assisted extraction of soluble matter from chardonnay grape skins for polyphenol recovery.

The objective of this study was to investigate the effects of pulsed electric field (PEF) and high-voltage electrical discharges (HVED) application on the efficiency of aqueous extraction of total soluble matter and polyphenols from grape skins ( Vitis vinifera L.) at different temperatures within 20-60 degrees C. The highest level of polyphenol concentration C was reached after about 60 min of extraction for HVED treatment: C(HVED) = 21.4 +/- 0.8 micromol of gallic acid equivalent (GAE)/g of dry matter (DM). Almost the same level of C was reached after 180 min of extraction for the PEF-treated skins. These levels exceeded the value C = 19.1 +/- 0.5 micromol of GAE/g of DM for the untreated samples. The difference between degrees Brix values for HVED-treated and untreated systems decreased with temperature increase (from 40 to 60 degrees C), but a large difference in the total amount of polyphenols was observed for HVED-treated and untreated systems. The activation energies were W(u) = 31.3 +/- 3.7 kJ/mol and W(PEF) = 28.9 +/- 5.5 kJ/mol for untreated and PEF-treated systems, respectively.

Structure-function relationship in glycosylated α-chymotrypsin as probed by IMAC and IMACE

Abstract Chemical glycosylation of bovine α-chymotrypsin, by a glucosamine adduct on the carboxyl group, results in the modification of its catalytic activity. The structural alterations of α-chymotrypsin resulting from its glycosylation are studied by immobilized metal-ion affinity chromatography (IMAC) and immobilized metal-ion affinity capillary electrophoresis (IMACE). The chemical glycosylation of α-chymotrypsin generates two distinct subpopulations of the protein: one which totally loses the initial affinity for IDA-Cu(II) and another which exhibits an increased affinity for the metal chelate ligand.

Activity of immobilised rat hepatic microsomal CYP2E1 using alumina membrane as a support

Porous alumina membranes are attractive materials for the construction of biosensors and also have utility for the production of immobilised enzyme bioreactors. Microsomes from rat liver were adsorbed onto alumina membrane activated by silane. Microsomal membranes were pumped through the channels where they became immobilised by binding to amine groups on the surface of the alumina membrane. In an effort to gain a quantitative understanding of the effects of microsomal film growth on enzyme activity, we compared the para-nitrophenol (pNP) hydroxylase activity of the microsomes by varying the amount of microsomes fixed in alumina microchannels. The alumina membrane was placed in a fluidic device at a fast flow that afforded short residence time (seconds) to obtain transformation of pNP to 4-nitrocatechol (pNC), which was detected by LC-MS/MS. This enabled the use of this bioreactor where CYP2E1 activity is low and tissue sources are limiting. The microsomes, successfully immobilised on the alumina membranes, were used to produce stable biocatalytic reactors that can be used repeatedly over a period of 2 months.

Analysis of sporamin forms expressed in different subcellular compartments of transgenic tobacco plants by IMAC and ESI-MS

Abstract Various forms of sporamin (spo, Δpro, ΔproHDEL and spoHDEL) corresponding to different targetings within the plant cell, respectively, to the vacuole, the extracellular compartment and the endoplasmic reticulum (ER) for the last two were used as model proteins to study further structural modifications. These proteins were expressed in tobacco plants ( Nicotiana tabacum L. cv. PB D6), purified by immobilized metal ion affinity chromatography (IMAC) and then analyzed by electrospray ionization mass spectrometry (ESI-MS). Conformational and post-translational modifications were observed between these different forms of sporamin, extracted from leaves. By the combination of these two techniques, detection of discrete intermediate species was achieved. Three distinct entities were detected for the vacuolar form of sporamin (spo) indicating a two-step processing of the protein, while only one entity, shorter by two amino-acids at its N-terminus, was detected for the extracellular form of sporamin (Δpro). The analysis of sporamin forms with an HDEL amino-acid extension can not be deduced easily from MS data and may reflect post-translational modifications distinct from proteolytic processing. Thus post-translational modifications appear to be closely related to targeting within the plant cell.