39 research outputs found
Biotransformation of halogenated compounds by lyophilized cells of Rhodococcus erythropolis in a continuous solid-gas biofilter
The irreversible hydrolysis of 1-chlorobutane to 1-butanol and HCl by lyophilized cells of Rhodococcus erythropolis NCIMB 13064, using a solidâgas biofilter, is described as a model reaction. 1-Chlorobutane is hydrolyzed by the haloalkane dehalogenase from R. erythropolis. A critical water thermodynamic activity (aw ) of 0.4 is necessary for the enzyme to become active and optimal dehalogenase activity for the lyophilized cells is obtained for a aw of 0.9. A temperature of reaction of 40 ⊠C represents the best compromise between stability and activity. The activation energy of the reaction was determined and found equal to 59.5 kJ/mol. The absence of internal diffusional limitation of substrates in the biofilter was observed. The apparent MichaelisâMenten constants Km and Vmax for the lyophilized cells of R. erythropolis were 0.011 (1-chlorobutane thermodynamic activity, aClBut ) and 3.22 ”moles/min g of cell, respectively. The activity and stability of lyophilized cells were dependent on the quantity of HCl produced. Since possible modifications of local pH by the HCl product, pH control by the addition of volatile Lewis base (triethylamine) in the gaseous phase was employed. Triethylamine plays the role of a volatile buffer that controls local pH and the ionization state of the dehalogenase and prevents inhibition by Clâ . Finally, cells broken by the action of the lysozyme, were more stable than intact cells and more active. An initial reaction rate equal to 4.5 ”moles/min g of cell was observed
Bioremediation of halogenated compounds: comparison of dehalogenating bacteria and improvement of catalyst stability
Five bacterial strains were compared for halogenated compounds conversion in aqueous media. Depending on the strain, the optimal temperature for dehalogenase activity of resting cells varied from 30 to 45 degrees C, while optimal pH raised from 8.4 to 9.0. The most effective dehalogenase activity for 1 chlorobutane conversion was detected with Rhodococcus erythropolis NCIMB13064 and Escherichia coli BL21 (DE3) (DhaA). The presence of 2-chlorobutane or propanal in the aqueous media could inhibit the 1-chlorobutane transformation
Dépollution d'effluents gazeux halogénés par des microorganismes déshydratés en réacteur solide/gaz (étude de la stabilité du biocatalyseur)
L objectif de ce travail Ă©tait de dĂ©terminer et quantifier les phĂ©nomĂšnes impliquĂ©s dans la perte de stabilitĂ© d un catalyseur prĂ©parĂ© Ă partir de cellules entiĂšres dĂ©shydratĂ©es et utilisĂ© pour la dĂ©halogĂ©nation directe et en continu de COV gazeux. L Ă©tude de l instabilitĂ© du biocatalyseur a rĂ©vĂ©lĂ© que (i) les bactĂ©ries ne rĂ©sistent que 6 heures aux conditions de rĂ©action (tempĂ©rature, aw) mais queLa perte de vitesse catalytique n est pas liĂ©e Ă cette forte mortalitĂ© (ii) les substrat et produit organiques de la rĂ©action modĂšle (1 chlorobutane et 1 butanol) ne recondensent pas sur le lyophilisat et n inactivent pas l hydrolyse du 1 chlorobutane (iii) l importante rĂ©hydratation du lyophilisat entraĂźne la dĂ©naturation thermique d une partie des DhaA (iv) et le HCl produit et accumulĂ© lors de la rĂ©action inactive rĂ©versiblement le biocatalyseur. L Ă©tude dĂ©taillĂ©e du biocatalyseur a permis de mettre en Ă©vidence que le comportement du biocatalyseur peut ĂȘtre expliquĂ© par des diffĂ©rences de micro-environnement entre enzymes au sein d un mĂȘme lyophilisat. En effet, ce biocatalyseur complexe est composĂ© de sels de tampon borate servant de support aux bactĂ©ries. AprĂšs rĂ©hydratation une partie des enzymes est fortement maintenue dans la matrice cellulaire et subit des dĂ©naturations thermiques, et une autre est stabilisĂ©e au contact des sels du tampon borate.L utilisation d extraits cellulaires permet d obtenir un lyophilisat stable durant plus de 1800 heures (75 jours). Le contact direct des DhaA avec les sels de tampon borate dĂ©s le dĂ©but de la rĂ©action permet d Ă©viter les dĂ©naturations thermiques, mais aussi l inhibition par le HCl.The aim of this work was to determine and quantify phenomena involved in the loss of stability of a catalyst prepared from whole dehydrated cells and used for the direct and continuous dehalogenation of gaseous VOCs. The study of the biocatatalyst instability revealed that (i) whole cells stay alive only few hours with selected operational conditions (temperature, aw, salt buffer borate concentration) but that the dehalogenases performances are not linked to this strong mortality (ii) the organic substrate and product of the reaction (1 chlorobutane et 1 butanol) do not sorb on the catalytic bed and do not inactivate the hydrolysis in the gas phase (iii) the important rehydration of the catalyst is responsible of thermal denaturation of a part of DhaA at 40C (iv) all the HCl producted is retained by the biocatalyst and consequently reversibly inactivate it. Observation of the biocatalyst highlights that the behaviour of the biocatalyst can be explained by the existence of two different micro-environments for the enzymes (DhaA). Indeed this complex biocatalyst contains 50% of borate buffer salts that act as support for dehydrated cells.During rehydration, part of DhaA remains in the cellular matrix environment and is denaturated and another part is stabilized by a direct contact with borate buffer salts. The lyophilised cellular extract show a stability of 1 800 hours (75 days). The direct contact between salt and DhaA allow to avoid thermal denaturations and inactivation by HCl accumulation.LA ROCHELLE-BU (173002101) / SudocSudocFranceF
Biocatalyse sous irradiation micro-ondes
Dans cette Ă©tude, nous avons utilisĂ© l'irradiation micro-ondes comme source d'Ă©nergie pour rĂ©aliser diffĂ©rentes biosynthĂšses catalysĂ©es par des b-galactosidases ou la lipase B de Candida antarctica. L'influence de ce mode de chauffage alternatif sur l'activitĂ© et la stabilitĂ© enzymatiques est Ă©tudiĂ©e. Trois types de milieux rĂ©actionnels (aqueux fortement concentrĂ© en substrat, sans solvant et avec solvant organique) sont testĂ©s pour la mise en Ćuvre de biocatalyseurs sous irradiation micro-ondes. Dans chaque cas, le profil rĂ©actionnel obtenu est comparĂ© Ă celui observĂ© sous chauffage classique par convection, Ă la mĂȘme tempĂ©rature macroscopique. En milieux aqueux, nous avons Ă©tudiĂ© la biosynthĂšse de galacto-oligosaccharides Ă 40C par transgalactosidation. La vitesse initiale de la rĂ©action, son Ă©quilibre, ainsi que la rĂ©action d'hydrolyse secondaire sont identiques sous irradiation micro-ondes et chauffage classique. En milieu organique, l'alcoolyse du butyrate d'Ă©thyle par le butanol, catalysĂ©e par la lipase B de Candida antarctica, sous forme libre et immobilisĂ©e, est Ă©tudiĂ©e en systĂšme sans solvant et dans diffĂ©rents solvants de logP compris entre -0,15 et 3,29. En systĂšme sans solvant, de 40 Ă 100C, le mode de chauffage n'a pas d'influence sur la vitesse initiale et l'Ă©quilibre de la rĂ©action d'alcoolyse. Dans le systĂšme avec solvant, la vitesse initiale de la rĂ©action augmente d'un facteur 1,8 sous irradiation micro-ondes, tandis que le mĂȘme Ă©quilibre rĂ©actionnel est obtenu sous les deux modes de chauffage. Le facteur d'accĂ©lĂ©ration de la rĂ©action, sous irradiation, semble ne pas dĂ©pendre de la polaritĂ© du solvant utilisĂ©. La stabilitĂ© de la lipase, en stockage dans le butanol ou le butyrate d'Ă©thyle, est influencĂ©e par le mode de chauffage. Le processus d'inactivation enzymatique peut ĂȘtre ralenti ou accĂ©lĂ©rĂ© dans le champ Ă©lectromagnĂ©tique, en fonction de la tempĂ©rature d'incubation.LA ROCHELLE-BU (173002101) / SudocSudocFranceF
Transestérification énantiosélective catalysée par la lipase de Candida antarctica immobilisée (application à la résolution de précurseurs d'analogues d'hormones d'insectes)
LA ROCHELLE-BU (173002101) / SudocSudocFranceF
Kinetic studies of fusarium solani pisi cutinase used in a gas/solid system: Transesterification and hydrolysis reactions
Fusarium solani cutinase supported onto Chromosorb P was used to catalyze transesterification (alcoholysis) and hydrolysis on short volatile alcohols and esters in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrates and removed reaction products simultaneously. A kinetic study was performed under differential operating conditions in order to get initial reaction rates. The effect of the hydration state of the biocatalyst on the kinetics was studied for 3 conditions of hydration (aw = 0.2, aw = 0.4 and aw = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from aw = 0.2 to aw = 0.6) for the hydrolysis of propionic acid methyl, ethyl or propyl esters. F. solani cutinase was found to have an unusual kinetic behavior. A sigmoid relationship between the rate of transesterification and the activity of methyl propionate was observed, suggesting some form of cooperative activation of the enzyme by one of its substrate. For the hydrolysis of short volatile propionic acid alkyl esters, threshold effects on the reaction rate, highly depending on the water activity and the substrate polarity, are reported. \ufffd 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 1-8, 1997.NRC publication: Ye
D.: Study of vitamin ester synthesis by lipase-catalyzed transesterification in organic media
Immobilized lipase from Candida antarctica (Novozym 435) was used in organic media to catalyze esterifications of vitamins (ascorbic acid and retinol) from hydroxy acid. We described the synthesis of retinyl L-lactate by transesterification between retinol and L-methyl lactate with yield reaching 90% and the synthesis of ascorbyl L-lactate by transesterification between ascorbic acid and L-methyl lactate with yield reaching 80%. The kinetic study of the esterification of vitamins with L-methyl lactate in organic media has been carried out and agrees with ping-pong-ordered Bi-Bi when the initial vitamin concentration is low. When initial vitamin concentration is high, the kinetic is similar to a hybrid ping-pong-ordered Bi Bi or hybrid ping-pong-random Bi Bi mechanism. However, with high initial substrate concentration, change of the kinetic by other phenomena, such as interaction of substrates with molecular sieves, adsorption of the methanol formed, and decreases of substrate diffusion, could be considered. It is obvious that in these conditions, classical enzymology (i.e., Michaelian enzymology) cannot be used for the interpretation of results