Sylviane Pulvin

Enzymatic synthesis of geraniol esters in a solvent-free system by lipases

Geraniol esters were synthesised by direct esterification catalysed by esterases and lipases (five enzymes were tested) in a solvent-free system at 37°C. The best conversions yields, about 85%, on geranyl butyrate and valerate obtained with esterase 30 000 from Mucor miehei. The effect of substrate molar ratio alcohol/acid variation was studied. A study of the water production was made in parallel during the esterification reaction.

Hemoglobin immobilized on mesoporous silica as effective material for the removal of polycyclic aromatic hydrocarbons pollutants from water

Free hemoglobin (Hb) in water at pH 5 is able to oxidize 11 polycyclic aromatic hydrocarbons (PAH) (300 nM each) in the presence of H2O2 amounting to 75% PAH removal. PAH are carcinogenic, mutagenic and xenobiotic pollutants found in wastewaters of oil refineries. However Hb is pH sensitive and is effective only at pH 5. In order to use Hb for real wastewater treatment (6.5 2500) and of Hb ([Hb]/[PAH] > 2) is necessary to reach an optimal PAH removal. The excess of Hb depends on the PAH physico-chemical characteristics. At pH = 7, the activity of free Hb decreased to 47% of PAH removal, whereas the Hb-immobilized silica allowed 82% of PAH removal. Immobilization of Hb in silicas leads to a protection of Hb towards pH, solvent, temperature and inactivation by H2O2. These results open the perspective for a new biotechnology process aimed at cleaning contaminated wastewaters by a reactive adsorption process followed by filtration.

Ennoblement of stainless steel in the presence of glucose oxidase: Nature and role of interfacial processes

The ennoblement of the free corrosion potential (E(corr)) of AISI 316L stainless steel which did not occur in synthetic fresh water (SFW), was observed after introduction of glucose oxidase (Gox) and glucose, or of hydrogen peroxide (H(2)O(2)). The composition of the surface was monitored using AFM and XPS, a detailed XPS analysis being based on the discrimination between oxygen of organic and inorganic nature proposed in a previous study. In H(2)O(2) medium, the main changes regarding the inorganic phase were the increase of the oxygen concentration in the passive film, the increase of the molar concentration ratio of oxidized species Fe(ox)/Cr(ox) and the growth of nanoparticles, presumably made of ferric oxide/hydroxide. In Gox medium, no significant changes were observed in both oxygen concentration and Fe(ox)/Cr(ox) ratio, but the density of colloidal particles decreased, indicating a dissolution of Fe oxide/hydroxide under the influence of gluconate. In contrast with H(2)O(2), in SFW and Gox the amount of organic compounds increased due to the accumulation of polysaccharides and proteins. The influence of glucose oxidase on the ennoblement of stainless steel is not due to indirect effects of H(2)O(2) through the change of surface composition. The E(corr) ennoblement seems to be directly due to the presence of H(2)O(2) and to the electrochemical behavior of H(2)O(2) and related oxygen species. This consideration is important for understanding and controlling microbial influenced corrosion.

Hemoglobin, horseradish peroxidase, and heme-bovine serum albumin as biocatalyst for the oxidation of dibenzothiophene

Hemoglobin, horseradish peroxidase, and bovine serum albumin incubated heme-catalyzed the oxidation of dibenzothiophene into sulfoxide in the presence of hydrogen peroxide. This reaction was carried out in an aqueous buffer containing 25% of water-miscible organic solvents. The observation of this transient state of hemoproteins during sulfoxidation showed heme degradation. None of the compounds usually involved in a classical peroxidative activity mechanism were detected. Furthermore, this activity did not appear to be based on a Fenton-type reaction. The highest degrees of sulfoxidation were obtained with hemoglobin. Under the best conditions of reaction, 100% of dibenzothiophene were converted into dibenzothiophene sulfoxide by hemoglobin. Heat-denatured hemoproteins did keep their sulfoxidation activity. With hemoglobin, a kcat of 0.22 min-1 was determined. Nearly the same values were obtained with heat-denatured hemoglobin and bovine serum albumin-adsorbed heme. With horseradish peroxidase, only 4% of conversion was attained. This percentage could be slightly increased by using a less pure peroxidase or heat-denatured peroxidase.

Catalase-like activity of bovine met-hemoglobin: Interaction with the pseudo-catalytic peroxidation of anthracene traces in aqueous medium

Hemoglobin is a member of the hemoprotein superfamily whose main role is to transport O(2) in vertebrate organisms. It has two known promiscuous enzymatic activities, peroxidase and oxygenase. Here we show for the first time that bovine hemoglobin also presents a catalase-like activity characterized by a V(max )of 344 microM/min, a K(M )of 24 mM and a k(cat) equal to 115/min. For high anthracene and hemoglobin concentrations and low hydrogen peroxide concentrations, this activity inhibits the expected oxidation of anthracene, which occurs through a peroxidase-like mechanism. Anthracene belongs to the polycyclic aromatic hydrocarbon (PAH) family whose members are carcinogenic and persistent pollutants found in industrial waste waters. Our results show that anthracene oxidation by hemoglobin and hydrogen peroxide follows a typical bi-bi ping-pong mechanism with a V(max) equal to 0.250 microM/min, K(M(H2O2) )of 80 microM, K(M(ANT)) of 1.1 microM and k(cat) of 0.17/min. The oxidation of anthracene is shown to be pseudo-catalytic because an excess of hemoglobin and hydrogen peroxide is required to make PAH completely disappear. Thus, bovine hemoglobin presents, in different degrees, all the catalytic activities of the hemoprotein group, which makes it a very interesting protein for biotechnological processes and one with which structure-activity relationships can be studied.

Role of water activity on the synthesis of geranyl butyrate by a Mucor miehei esterase in a solvent-free system

The initial activity rate for the synthesis of geranyl butyrate by an esterase from Mucor miehei increased with increasing water activity and decreased above 0.5 water activity. The conversion yield after 75 h was about 75% for different, but low, initial water activities and decreased above 0.5 water activity. This behaviour is related to water-enzyme interactions.

Coenzyme based synthesis of silver nanocrystals.

In this work we have carried out systematic studies to identify the critical role of a coenzyme (β-NADPH) to synthesize silver nanoparticle. Interestingly, both roles of reducing and stabilizing agents are played by β-NADPH. Nanoparticles obtained by this route exhibit a good crystallinity, a narrow size distribution and excellent stability in aqueous solution. The most advantageous points of this single-step environmentally friendly approach are that it takes place at nearly room temperature (20 °C), overcomes many limitations encountered in other biological methods (such as the restricted concentration of AgNO₃, maintenance and manipulation of microorganisms, preparing extracts and contamination from residual reactants), bypasses the use of surfactants or capping agents and does not necessitate pH adjustment. The nano-Ag were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), dynamic light scattering (DLS), zeta potential, UV-vis, and energy-dispersive X-ray spectroscopy (EDX). DLS, TEM and XRD measurements showed the formation of nano-Ag with an average diameter of 20.77±0.67 nm. XRD studies confirmed the nanocrystalline nature of the silver particles. Zeta potential measurements revealed that the particles are surrounded with negatively charged groups (-41±5 mV) making them stable in an aqueous medium. The EDX spectrum of the silver nanoparticles confirmed the presence of elemental silver signal in high percentage. In addition to the easy and ecofriendly method of synthesis, β-NADPH can be regenerated by enzymatic means through glucose 6-phosphate dehydrogenase, potentially making the synthesis more cost effective.

Silanization with APTES for Controlling the Interactions Between Stainless Steel and Biocomponents: Reality vs Expectation

Jessem Landoulsi1, Michel J. Genet2, Karim El Kirat3, Caroline Richard4, Sylviane Pulvin5 and Paul G. Rouxhet2 1Laboratoire de Reactivite de Surface, Universite Pierre & Marie Curie -Paris VI, 2Institute of Condensed Matter and Nanosciences – Bio & Soft Matter, Universite Catholique de Louvain, 3Laboratoire de Biomecanique et Bioingenierie, 4Laboratoire Roberval, 5Genie Enzymatique et Cellulaire, Universite de Technologie de Compiegne, 1,3,4,5France 2Belgium

Substrate inhibition or activation kinetics of the β-galactosidase from the extreme thermoacidophile archaebacterium Caldariella acidophila

The kinetics of the hydrolysis of p-nitrophenyl-beta-D-galactopyranoside (pNPG) by a thermophile, beta-galactosidase, was studied at different temperatures. This enzyme was isolated from the thermophilic microorganism archaebacterium Caldariella acidophila. The hydrolysis of pNPG by beta-galactosidase does not follow Michaelis-Menten law. This enzyme is inhibited by excess substrate at low temperatures and it is activated by excess substrate at high temperatures. A minimum mechanistic model is proposed to explain the behaviour. This model assumes the binding of an additional substrate molecule on the glycosidyl enzyme intermediate. This model is in good agreement with the postulated mechanism for beta-galactosidase from Escherichia coli. The kinetic parameters are calculated at six different temperatures.

Development of immobilization technique for liver microsomes.

In the present report, physically adsorbed rat liver microsomes were used in order to optimize the immobilization of membrane proteins on solid surfaces for use in biosensing and microreactor applications. Physical adsorption was used to form thin films on solid supports (gold, mica, macroporous aluminum oxide membrane). The characterization of the films was performed by surface plasmon resonance (SPR), atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM). Commercially available macroporous aluminium oxide membranes with a high surface area, allow the retention of a high amount of microsomal membranes in the form of a thin film. Microsomal film functionality was tested by monitoring the activities of several enzymes of phases I and II. Microsomal modified supports can be re-utilized for the same or different substrate after washing with appropriate buffer.