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It was observed that a biocatalyst prepared from dehydrated whole cells of a recombinant Escherichia coli (initially suspended in borate buffer) was able to hydrolyze gaseous 1-chlorobutane in a solid/gas reactor. Nevertheless, at 40 degrees C and for a 0.7 water activity, it rapidly lost its activity. The explanation of this phenomenon was first investigated by observing the biocatalyst structure at the microscopic level and by studying the localization of the dehalogenase involved in catalysis (intracellular/extracellular). The behavior of this biocatalyst was then compared with that of a preparation made from cells extracts. The reasons of the inactivation are discussed in terms of thermal denaturation and protective effect of buffer salts., ((c) 2009 Wiley Periodicals, Inc.)

Although microwave-assisted reactions are widely applied in various domains of organic chemistry, their use in the area of enzyme chemistry has been rather limited, due to the high temperatures associated with the microwave heating: Because current technology, allows a good control of reaction parameters, several examples of microwave-assisted enzyme chemistry have been reported, using stable and effective biocatalysts (modified enzymes). The purpose of this review is to highlight the applications and studies on the influence of microwave irradiation on enzymatic properties and their application in enzyme chemistry.

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.

The influence of microwave heating on free Candida antarctica lipase B activity and stability was studied over the temperature range from 40 to 110 degrees C. Concerning the lipase activity, identical initial rate and conversion yield were obtained under microwave radiation and classical thermal heating for the alcoholysis between ethyl butyrate and butanol in a solvent-free system. On the other hand, the kinetics of the free lipase inactivation in butanol appears to be influenced by the heating mode. The Arrhenius plot obtained under classical heating was linear over all the temperature range studied whereas a biphasic Arrhenius plot was obtained under microwaves. The non-classical effect of the microwave heating on the initial rate of the enzymatic inactivation was thus dependent on the temperature of incubation.

A new approach to galacto-oligosaccharides and galacto-conjugates synthesis performed by the beta-galactosidase from Kluyveromyces lactis is reported. The enzymatic galactosylation of eight kinds of adsorbed aromatic primary alcohols, in particular the two drugs guaifenesin and chlorphenesin, gave the corresponding beta-D-galacto-pyranosides in yields ranging between approximately 10% and 96%. For the first time, we have showed that the adsorption of acceptor substrates onto solid supports such as silica gel influences the yield and the selectivity of galacto-conjugates synthesis. In particular, we observed that adsorption of acceptor favored the synthesis of digalactosylated compounds.

Rhodococcus erythropolis NCIMB 13064 and Xanthobacter autotrophicus GJ10 are able to catalyze the conversion of halogenated hydrocarbons to their corresponding alcohols. These strains are attractive biocatalysts for gas phase remediation of polluted gaseous effluents because of their complementary specificity for short or medium and for mono-, di-, or trisubstituted halogenated hydrocarbons (C2-C8 for Rhodococcus erythropolis and C1-C4 for Xanthobacter autotrophicus). After dehydration, these bacteria can catalyze the hydrolytic dehalogenation of 1-chlorobutane in a nonconventional gas phase system under a controlled water thermodynamic activity (a(w)). This process makes it possible to avoid the problems of solubility and bacterial development due to the presence of water in the traditional biofilters. In the aqueous phase, the dehalogenase activity of Rhodococcus erythropolis is less sensitive to thermal denaturation and the apparent Michaelis-Menten constants at 30 degrees C were 0.4 mM and 2.40 micromol min(-1) g(-1) for Km and Vmax, respectively. For Xanthobacter autotrophicus they were 2.8 mM and 0.35 micromol min(-1) g(-1). In the gas phase, the behavior of dehydrated Xanthobacter autotrophicus cells is different from that observed with Rhododcoccus erythropolis cells. The stability of the dehalogenase activity is markedly lower. It is shown that the HCl produced during the reaction is responsible for this low stability. Contrary to Rhodococcus erythropolis cells, disruption of cell walls does not increase the stability of the dehalogenase activity. The activity and stability of lyophilized Xanthobacter autotrophicus GJ10 cells are dependant on various parameters. Optimal dehalogenase activity was determined for water thermodynamic activity (a(w)) of 0.85. A temperature of 30 degrees C offers the best compromise between activity and stability. The pH control before dehydration plays a role in the ionization state of the dehalogenase in the cells. The apparent Michaelis-Menten constants Km and Vmax for the dehydrated Xanthobacter autotrophicus cells were 0.07 (1-chlorobutane thermodynamic activity) and 0.08 micromol min(-1) g(-1) of cells, respectively. A maximal transformation capacity of 1.4 g of 1-chlorobutane per day was finally obtained using 1g of lyophilized Xanthobacter autotrophicus GJ10 cells., (Copyright 2005 Wiley Periodicals, Inc.)

The main objective of this work was to improve the selective synthesis of a volatile compound: aldehyde or alcohol using a coupled-enzyme system. A novel method of synthesis of C6-aldehyde or alcohol was carried out in the presence of hydroperoxide lyase (HPLS) activity coupled to alcohol dehydrogenase (ADH) activity. After cleavage of the initial substrate, hydroperoxy fatty acid catalyzed by HPLS, the second enzyme, ADH, can catalyze the reduction of the aldehyde to the corresponding alcohol, or the oxidation of contaminating alcohol into aldehyde, depending on the cofactor present in the medium (oxidized or reduced form). We succeeded in improving the synthesis of one of the products. When coupling HPLS to NADP, the selectivity of hexanal production from 13-hydroperoxy linoleic acid was improved, and hexanol production was reduced 5 to 10 times after 15 min of reaction at 15 degrees C and pH 7.0. In another experiment, HPLS was coupled to ADH in the presence of NADH. The production of alcohol (hexenols) was then favored especially when using 13-hydroperoxy linolenic acid as substrate at concentrations >15 mM, reaching 95% of the products. Coupling of the enzymatic reactions (cleavage reduction) not only reduced the number of steps but also allowed us to increase the conversion rate of the initial substrate (hydroperoxy fatty acid). Structures of the compounds produced in this work were confirmed using gas chromatography-mass spectroscopy analysis. Each of these products has its own delicately different fresh odor that can be used in various applications.

The extraction of 13-hydroperoxide-lyase activity from mint leaves as well as its use for C6-aldehyde production was studied in this work. The enzyme cleaves 13(S)-hydroperoxy-C18 fatty acids into C6-aldehyde and C12-oxo-acid. Two mint species were tested: Mentha veridis and Mentha pulegium. The headspace injection method coupled to gas chromatography was used for volatile compound analysis. The optimal conditions for temperature and pH were, respectively, 15 and 7 degrees C. We also studied the specific synthesis of hexanal and hexenals respectively from 13(S)-hydroperoxy-linoleic acid and 13(S)-hydroperoxy-linolenic acid. Considerable quantities of aldehyde (up to 2.58 micromol) were produced after 15 min of cleavage reaction in 2 ml stirred at 100 rpm, especially in presence of extract of M. veridis. The conversion yields decreased from 52.5% as maximum to 3.3% when using initial hydroperoxide concentrations between 0.2 and 15 mM. An unsaturated aldehyde, the 3(Z)-hexenal was produced from 13(S)-hydroperoxy-linolenic acid. The 3(Z)-isomer was unstable and isomerized in part to 2(E)-hexenal. In this work, we observed a very limited isomerization of 3(Z)-hexenal to 2(E)-hexenal, since the reaction and the volatile purge were carried out successively in the same flask without delay or any contact with the atmosphere. These aldehydes contribute to the fresh green odor in plants and are widely used in perfumes and in food technology. Their importance increases especially when the starting materials are of natural biological origin as used in this work. GC-MS analysis allowed the identification of the products.

Oxidation of ethanol by horse liver alcohol dehydrogenase (HLADH) is monitored under pressure (0.1 MPa - 225 MPa). The pressure-induced modifications of catalytic activity are followed by plotting reaction velocities as a function of substrates concentrations in the traditional double reciprocal form: then, pressure is treated as an activator (p < 100 MPa) or an inhibitor (p<225 MPa). Surprising typical patterns of Lineweaver-Burk curves are observed and interpreted. These results suggest that this approach could be a powerful tool to study enzyme's structure-activity relationship.

The influence of microwave heating on the stability of immobilized Candida antarctica lipase B was studied at 100 degrees in an organic medium. The microwave radiation was carried out before enzymatic reaction (storage conditions) or during the enzymatic catalysis (use conditions). In both cases, enzymatic stability was higher under microwave heating than under conventional thermal heating, in strictly identical operating conditions. Furthermore, the gain of enzymatic stability under microwave heating appears to be higher in a more polar solvent, which interacts strongly with the microwave field. Our results suggest that microwave radiation has an effect, not related to temperature, on the process of enzymatic inactivation.

Biofiltration of air polluted by volatile organic compounds is now recognized by the industrial and research communities as an effective and viable alternative to standard environmental technologies. Whereas many studies have focused on solid/liquid/gas biofilters, there have been fewer reports on waste air treatment using other biological processes, especially in a solid/gas biofilter. In this study, a comparison was made of the hydrolysis of halogenated compounds (such as 1-chlorobutane) by lyophilized Rhodococcus erythropolis cells in a novel solid/gas biofilter and in the aqueous phase. We first determined the culture conditions for the production of R. erythropolis cells with a strong dehalogenase activity. Four different media were studied and the amount of 1-chlorobutane was optimized. Next, we report the possibility to use R. erythropolis cells in a solid/gas biofilter in order to transform halogenated compounds in corresponding alcohols. The effect of experimental parameters (total flow into the biofilter, thermodynamic activity of the substrates, temperature, carbon chain length of halogenated substrates) on the activity and stability of lyophilized cells in the gas phase was determined. A critical water thermodynamic activity (a(w)) of 0.4 is necessary for the enzyme to become active and optimal dehalogenase activity for the lyophilized cells is obtained for an a(w) of 0.9. A temperature of reaction of 40 degrees C represents the best compromise between stability and activity. Activation energy of the reaction was determined and found equal to 59.5 KJ/mol. The pH effect on the dehalogenase activity of R. erythropolis cells was also studied in the gas phase and in the aqueous phase. It was observed that pH 9.0 provided the best activity in both systems. We observed that in the aqueous phase R. erythropolis cells were less sensitive to the variation in pH than R. erythropolis cells in the gas phase. Finally, the addition of volatile Lewis base (triethylamine) in the gaseous phase and the action of the lysozyme in order to permeabilize the cells was found to be highly beneficial to the effectiveness of the biofilter., (Copyright 2004 Wiley Periodicals, Inc.)

A domain of epiglucan was synthesized by beta-glucosidases. Two beta-glucosidases, an extracellular beta-glucosidase derived from Sclerotinia sclerotiorum grown on xylose, and a commercial lyophilized preparation of beta-glucosidase from Aspergillus niger, were used to synthesize gluco-oligosaccharides from cellobiose and, specially, beta-(1-6) branched beta-(1-3) gluco-oligosaccharides, corresponding to the structure of epiglucan. Gentiobiose, cellotriose, cellotetraose, beta-Glc-(1-3)-beta-Glc-(1-4)-Glc, beta-Glc-(1-6)-beta-Glc-(1-4)-Glc and beta-Glc-(1-6)-beta-Glc-(1-3)-Glc were synthesized from cellobiose by both enzymes. The latter compound was preferentially synthesized by the beta-glycosidase from Sclerotinia sclerotiorum. Under the best conditions, only 7 g l(-1) of beta-Glc-(1-6)-beta-Glc-(1-3)-Glc was synthesized by the beta-glycosidase from Aspergillus niger compared to 20 g l(-1) synthesized with beta-glycosidase from Sclerotinia sclerotiorum.

The influence of the addition of an extra component in a gaseous reaction medium, on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample, which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates and additional components to the enzyme while removing reaction products. The system permits to set thermodynamic activity of all gaseous components (substrates or not) independently at the desired values. This allows in particular to study the influence of an extra added component at a constant thermodynamic activity value, contrary to classical solid/liquid system, which involves large variations of thermodynamic activity of added solvent, when performing full kinetic studies. Alcohol inhibition constant (K(I)) and methyl propionate and propanol dissociation constants (K(MP) and K(P)) have been determined in the solid/gas reactor in the presence of 2-methyl-2-butanol, and compared with values previously obtained in the absence of added component and in the presence of water. Complementary experiments were carried out in the presence of an apolar compound (hexane) and led to the conclusion that the effect of added organic component on lipase-catalyzed alcoholysis is related to their competitive inhibitory character towards first substrate methyl propionate. The comparison of data obtained in liquid or with gaseous 2-methyl-2-butanol shows that lower K(MP) and K(I) are found in gaseous medium, which would correspond on the one hand to a lower acylation rate k(2), and on the other hand to a higher binding rate k(1) between substrate and free enzyme in gaseous medium.

The influence of water on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates to the enzyme while removing reaction products. In this system, interactions between the enzyme and nonreacting molecules are avoided, since no solvent is present, and it is thus more easy to assess the role of water. To this end, alcohol inhibition constant, substrates dissociation constants as well as acylation rate constant and ratio of acylation to deacylation rate constants have been determined as a function of water activity (a(w)). Data obtained highlight that n-propanol inhibition constant and dissociation constant of methyl propionate are a lot affected by a(w) variations whereas water has no significant effect on the catalytic acylation step nor on the ratio of acylation to deacylation rate constants. These results suggest the water-independent character of the transition step.

Galacto-oligosaccharides (GOS) were synthesized from lactose by immobilized and free beta-galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP-G) using either focused microwave irradiation or conventional heating. Immobilization of the beta-galactosidase on to Duolite A-568 increased the synthesis of GOS. GOS selectivity (GOS synthesis/lactose hydrolysis ratio) increased when the water activity of the media was reduced, notably with a high initial lactose concentration but also by using co-solvents in the media. The advantage of microwave heating on GOS formation was also examined. Addition of solvent and carrying out the reaction under microwave irradiation resulted an increase in the production of GOS. The selectivity for GOS synthesis can be increased by 217-fold under microwave irradiation, using immobilized beta-glucosidase and with added co-solvents such as hexanol.

The effect of water on the alcoholysis of methyl propionate and n-propanol catalyzed by immobilized Candida antarctica lipase B (CALB) has been compared in a continuous solid-gas reactor and in an organic liquid medium. The enthalpic and entropic contributions of water to the Gibbs free energy of activation in the gas phase were different from the ones in the organic phase, the inverse trends being observed for the variation of both DeltaH* and DeltaS* with water activity. Different phenomena were identified for their influence on the thermodynamic parameters. When increasing a(w), the enhanced flexibility of the enzyme was predominant in the gas phase whereas substrate-solvent interactions due to an increased polarity of the solvent affected mainly the thermodynamic parameters in the organic phase. The observed variations of DeltaG* with water activity were in accordance with kinetics results previously obtained in both reaction media.

The process of pressure-induced modification of horse liver alcohol dehydrogenase (HLADH) was followed by measuring in situ catalytic activity (up to 250 MPa), intrinsic fluorescence (0.1-600 MPa) and modifications of FTIR spectra (up to 1000 MPa). The tryptophan fluorescence measurements and the kinetic data indicated that the pressure-induced denaturation of HLADH was a process involving several transitions and that the observed transient states have characteristic properties of molten globules. Low pressure (< 100 MPa) induced no important modification in the catalytic efficiency of the enzyme and slight conformational changes, characterized by a small decrease in the centre of spectral mass of the enzyme's intrinsic fluorescence: a native-like state was assumed. Higher pressures (100-400 MPa) induced a strong decrease of HLADH catalytic efficiency and further conformational changes. At 400 MPa, a dimeric molten globule-like state was proposed. Further increase of pressure (400-600 MPa) seemed to induce the dissociation of the dimer leading to a transition from the first dimeric molten globule state to a second monomeric molten globule. The existence of two independent structural domains in HLADH was assumed to explain this transition: these domains were supposed to have different stabilities against high pressure-induced denaturation. FTIR spectroscopy was used to follow the changes in HLADH secondary structures. This technique confirmed that the intermediate states have a low degree of unfolding and that no completely denatured form seemed to be reached, even up to 1000 MPa.

In this article, a HPLC method to identify and quantify the dyes and the indigo precursors produced in Polygonum tinctorium is described. Using this technique, indican has been positively identified in extracts of P. tinctorium. Our work with two cultivars of P. tinctorium has confirmed that the quantity of indican is dependent on the cultivars, harvest period, and age of the leaves. Two enzymes, Novozym 188 (cellobiase) and Novarom G (beta-glucosidase), are compared on the basis of their activities to hydrolyze the indican at several pH values. We observed that Novarom G is more active than Novozym 188 whatever the pH and that optimum pH of both enzymes for indican hydrolysis is 3. Liberated indoxyl can be oxidized in alkaline media and transformed into indigo and indirubin.

Retinoids (vitamin A and derivatives) are of great commercial potential in cosmetics and pharmaceuticals such as skin care products. However, the clinical effectiveness of these retinoids is limited by skin irritation, water insolubility, and except for retinyl-esters, extreme instability. In this paper, an enzymatic method for preparing water-soluble retinol derivatives catalyzed by immobilized lipase is described. The synthesis is based on a unique strategy of two-step enzymatic acylation. Among the different synthesized compounds, the most water-soluble are the disaccharide derivatives such as saccharose retinyl adipate (nonionic water-soluble retinol derivative) and the sodium salt of retinyl diacids such as retinyl succinate sodium salt (ionic water-soluble retinol derivative).

The unsaturations and groups bound to the ring and to the lateral chain of lactones give a large diversity in this class of molecules. In this work we produced enzymatically a macrolactone in organic media. The substrate used was a hydroxy-fatty acid: (+)-coriolic acid and the enzymes tested were free or immobilized microbial lipases. The immobilized lipase from Candida antarctica seems to be the most adequate catalyst offering a high reaction yield. The intra-esterification was studied as a function of temperature and type of solvent. Higher yields were obtained when using diisopropyl-ether at 35 degrees C. This reaction, involving an alcohol group on an internal position on the carbon chain of the substrate hydroxy-acid, produces an original lactone: 13S-octadeca-(9Z,11E)-dienolide. The product was purified and characterized using (1)H nuclear magnetic resonance spectroscopy, mass spectrometry and infrared spectroscopy.

The kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by Candida antarctica lipase B supported onto silanized Chromosorb P was studied in a continuous solid/gas reactor. In this system the solid phase is composed of a packed enzymatic sample and is percolated by nitrogen as carrier gas, which simultaneously carries substrates to the enzyme while removing reaction products. In this reactor the thermodynamic activity of substrates and effectors can be perfectly adjusted allowing kinetic studies to be performed under different operating conditions. The kinetics obtained for alcoholysis were suggested to fit a Ping Pong Bi Bi mechanism with dead-end inhibition by the alcohol. The values of all apparent kinetic parameters were calculated and the apparent dissociation constant of enzyme for gaseous ester was found very low compared with the one obtained for liquid ester in organic medium, certainly due to the more efficient diffusion in the gaseous phase. The effect of water thermodynamic activity was also investigated. Water was found to act as a competitive inhibitor, with a higher inhibition constant than n-propanol. Thus alcoholysis of gaseous methyl propionate and n-propanol catalyzed by C. antarctica lipase B was found to obey the same kinetic mechanism as in other non-conventional media such as organic liquid media and supercritical carbon dioxide, but with much higher affinity for the substrates.

Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we describe an HPLC method dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and isoindirubin) and indigo precursors produced in I. tinctoria (Woad). This work, carried out with two cultivars of I. tinctoria, has confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-ketogluconate) and indican (indoxyl-beta-D-glucoside). We suggest the name "isatan C" for this new indigo precursor in I. tinctoria. Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan C reacts with isatan B increasing the red pigment production.

The gas phase continuous production of acetaldehyde from ethanol and hexanol from hexanal using dried baker's yeast was studied as an alternative approach to conventional processes. The effects of water activity, activity of substrates, and amount of yeast on the performance of the continuous bioreactor were investigated. The extent of yeast hydration and ethanol activity are the most important factors affecting yeast activity and stability., (Copyright 2001 John Wiley & Sons, Inc.)

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.

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 (a(w) = 0.2, a(w) = 0.4 and a(w) = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from a(w) = 0.2 to a(w) = 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.

Analysis of the kinetic behaviour of a two-enzyme-system carrying out two consecutive reactions was investigated in macroheterogeneous biphasic media (octane/buffer pH 9.6, v/v = 1:1). The lipase-catalysed hydrolysis of trilinolein and the subsequent lipoxygenation of the liberated linoleic acid, were coupled in a modified Lewis cell with a well-defined liquid/liquid interfacial area. Trilinolein was dissolved in the organic phase and hydrolysed in the presence of Mucor javanicus lipase at the organic/aqueous interface. Linoleic acid, liberated after hydrolysis was transferred to the aqueous phase and reacted with lipoxygenase. This reaction consumed linoleic acid and produced hydroperoxides, which favoured the transfer of residual linoleic acid, since they possess surface active properties. Catalysis and transfer influenced each other reciprocally. At low substrate concentrations, cooperativity phenomena were observed in the experimental and also the modelled two-enzyme systems. When the initial substrate concentration was high, the kinetic behaviour of the two-enzyme system in a compartmentalised medium, seemed to be independent of the substrate concentration, unlike that observed in homogeneous monophasic enzymology. The numerical integration program used to model the two-enzyme system was based on results obtained in separate studies of the following three phenomena: (1) trilinolein hydrolysis in biphasic medium. (2) linoleic acid transfer across a liquid/liquid interface and (3) lipoxygenation in an aqueous media. Results obtained by modelling were similar to the results observed experimentally.

A nuclear magnetic resonance (NMR) method has been developed to monitor on-line lipase-catalyzed esterification reactions without the need to sample the reaction medium. The technique, through (1)H NMR, measures the concentrations of alcohol, ester, hydroxylic hydrogens in the organic phase, and hydroxylic hydrogens in the aqueous phase, if any. Also, the chemical shift evolution of the two types of hydroxylic hydrogens has been followed, providing information on water content of the organic phase and on the appearance of a distinct aqueous phase. As far as (13)C NMR is concerned, it has been possible to measure, first the acid and the ester concentrations in the carbonyl region, and second, the alcohol and the ester concentrations in the methylene region. All (1)H and (13)C results are in agreement with one another. Furthermore, NMR allows for the choice of detection zone. Preliminary studies on the solid phase proved the presence of much more water in the solid phase than in the organic phase, and also gave evidence of the existence of two types of esters, one in the organic phase, mainly associated with the acid, and the other one not associated with the acid, most probably entrapped within the solid enzyme.

Soybean lipoxygenase-1 (EC 1.13.11.12) reaction with linoleic acid as substrate was used to study the biocatalysis in a biphasic system when the reactants have surface-active properties. The poorly water-soluble substrate was initially dissolved in an apolar solvent (octane). The hydroperoxide produced was water soluble and remained in the aqueous phase (borate buffer). The bioreactor was a modified Lewis cell with a well-defined interfacial area between the two phases. Two phenomena were studied separately: the reactant transfer between the two phases and the biocatalyzed reaction in an aqueous medium. This allowed determination of the transfer and the reaction constants. Substrate transfer was found to be affected by the progress of the reaction, because linoleic acid and the hydroperoxy acid have an influence on the interfacial tension. Inactivation of the biocatalyst at the interface was observed in the bioreactor. These results indicate that it is impossible to analyze the system behavior with the method proposed in the literature, which is based on the sequential study of the substrate transfer to the aqueous phase and its biocatalysis by lipoxygenase. The interaction between transfer phenomena and reaction kinetics was studied in the biphasic system. The kinetics were different from those obtained in the aqueous medium. Catalysis and transfer influence each other reciprocally. In this compartmentalized system, cooperativity phenomena were obtained using a nonallosteric enzyme. The evolution of the system was modeled (Runge-Kutta algorithm). The curves obtained were very close to those determined experimentally.

A comparative study of temperature and pressure effects were carried out by using two homologous enzymes exhibiting different thermostability and oligomery: almond beta-glucosidase and Sulfolobus solfataricus beta-glucosidase. Both the activity and stability were studied using an in-house built bioreactor allowing injection, stirring, sampling and on-line spectrophometric monitoring with retention of pressure up to 2.5 kbar and temperature control possible up to 150 degrees C. Almond beta-glucosidase, the most pressure sensitive enzyme of the two was continuously affected by pressure up to 1.5 kbar. Activation volume changes revealed that the inactivation of almond beta-glucosidase was due to both catalytic step inactivation and enzyme-substrate binding inactivation. Structural modifications generated by pressure, related to a loss of activity did not affect the global conformation of almond beta-glucosidase, after depressurization. In contrast, Sulfolobus solfataricus beta-glucosidase was a highly barostable enzyme. It maintained a half-life of 91 h at 60 degrees C and 2.5 kbar. Moreover, this enzyme appeared to be activated by pressure between atmospheric pressure and 2.5 kbar with a maximal activity at 1.25 kbar. However, this enzyme still displayed the best catalytic efficiency at atmospheric pressure because of a Km value drastically increased by pressure. Activation volume changes indicated that the hydrolysis reaction catalysed by Sulfolobus solfataricus beta-glucosidase, was alternatively favoured and disfavoured by pressure due to the catalytic step activation or inactivation associated with the enzyme-substrate binding step being continuously inactivated by pressure.

A comparison between the pressure effects on the catalysis of Thermoanaerobium brockii alcohol dehydrogenase (TBADH: a thermostable tetrameric enzyme) and yeast alcohol dehydrogenase (YADH: a mesostable tetrameric enzyme) revealed a different behaviour. YADH activity is continuously inhibited by an increase of pressure, whereas YADH affinity seems less sensitive to pressure. TBADH activity is enhanced by pressure up to 100 MPa. TBADH affinity for alcoholic substrates increases if pressure increases, was TBADH affinity for NADP decreases when pressure increases. Hypothesis has been raised concerning the dissociation of oligomeric enzymes under high hydrostatic pressure ( < 200 MPa) [1]. But in the case of these two enzymes, unless the oligomers reassociate very quickly (< 1 min), the activity inhibition of YADH at all pressures and TBADH for pressures above 100 MPa is not correlated to subunit dissociation. Hence we suggest that enzymes under pressure encounter a molecular rearrangement which can either have a positive or a negative effect on activity. Finally, we have observed that the catalytic behaviour of alcohol dehydrogenases under pressure is connected to their thermostability.

Fusarium solani cutinase and Candida cylindracea lipase were used to catalyze a transesterification reaction 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 substrate and removed reaction products simultaneously. Different conditions of immobilization were used and compared to the results obtained with a nonsupported enzyme. The enzymatic activity was found to be highly dependent of a key parameter: water activity (a(w)). Biocatalyst stability was greatly influenced by water activity and the choice of immobilization technique for the enzymatic material. For free and adsorbed enzymes, water requirements exhibited optima which corresponded to the complete hydration coverage of the protein. These optima presented a good correlation with the isotherm sorption curves obtained for the different preparations. In this work are reported the results concerning the possibility of using a continuous system able to operate at controlled water activity in a heterogeneous medium. Lipolytic enzyme in such a system appears to be a new process for the biotransformation of volatile esters.

The Trichoderma reesei beta-xylosidase (EC 3.2.1.37) is used to catalyze the production of alkyl beta-D-xyloside. Two general methods of production are tested and compared using the same enzyme: transglycosylation and reverse hydrolysis. Using both methods, primary, secondary, and tertiary alcohols are studied as acceptors. In kinetically controlled process (transglycosylation), the chosen donor is methyl beta-D-xyloside and primary, secondary, and tertiary alkyl alcohols are accepted. In the equilibrium-controlled synthesis, the donor is xylose whereas acceptors are only primary and secondary alcohols. The influence of the donor concentration is investigated in both processes. The yields of the kinetically controlled reactions are higher compared with those of the equilibrium-controlled synthesis. The specificity of the beta linkage is confirmed by proton nuclear magnetic resonance ((1)H NMR) analysis. (c) 1994 John Wiley & Sons, Inc.

The biocatalysis of substrates in the gas phase may offer advantages over many conventional solution-based reactions, both in analytical devices and in bioreactors designed to accommodate this new technology. To date, however, the range of substrates for which gas-phase biocatalysis has been shown to be suitable is limited. Further research is required to establish the parameters that affect the kinetics and productivity of such systems.

Gluco-oligosaccharides were synthesized through the enzymatic condensation of D-glucose at high concentration using a commercial almond beta-glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (a(w)) in the presence of different glucose concentrations. The yield and the composition of the gluco-oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di-, tri-, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL . mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated., ((c) 1993 John Wiley & Sons, Inc.)

Porcine pancreatic lipase and Fusarium solani cutinase were used to catalyze transesterification reactions between methyl propionate, ethyl propionate, and a series of primary alcohols at high temperatures in a continuous packed-bed gas-solid reactor, in which the solid phase is composed of the enzyme and the substrates and products are in a gaseous form. In this type of system, enzyme activity was found to depend essentially on the water activity (A(w)) of the enzyme preparation.

A novel method for modulation of lipase hydrolysis and synthesis lipase was investigated by using carbohydrates in the microenvironment of the Candida rugosa enzyme. The influence of the addition of different sugars to the previously dialysed enzyme was tested on the two reactions. Rates of hydrolysis were lowered by using dialysed enzyme but were increased after sugar addition, regardless of the identity of the added sugar. In contrast, synthesis reaction rates depended on the nature of the carbohydrate. Rates were increased by adding lactose, which is not a water activity depressor, but were lowered by adding fructose, glucose, sucrose or sorbitol, which are all water activity depressors.

Ester synthesis catalyzed by Candida cylindracea lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was investigated in solid/liquid biphasic media containing the enzyme preparation and reactants without addition of organic solvents not participating in the reaction. Although the effects of water on enzyme kinetics have been abundantly studied in nearly anhydrous media, reactions in which water is produced have not been investigated. The effect of water produced by the reaction itself on the enzymatic activity was studied. The dispersion of water in a shaken, nearly anhydrous medium was shown to be responsible for the lack of activity of the enzyme. In contrast, when slowly shaken, the enzyme was fully activated by the water furnished as a product of the reaction. However, when experiments were performed in a two-phase aqueous/organic system with previously solubilized enzyme in water, the enzyme activity was increased by shaking and was of the same order of magnitude as in nearly anhydrous media. Under low water activity conditions, a powerful agitation can lead to slower reaction rate, because water, a product of esterification, is not retained in the microenvironment of the enzyme to activate it. The activation effect of water produced by the reaction was clearly shown using enzyme preparations shaken in an anhydrous medium and previously equilibrated at low water activities (aw = 0.13 and 0.69). This activation did not occur for an enzyme preparation equilibrated at high aw (0.89) or for a preparation gently shaken in a water-saturated medium. The lag time preceding activation of the enzyme increased with the extent of enzyme dehydration.(ABSTRACT TRUNCATED AT 250 WORDS)

The reaction rate of two lipase-catalysed reactions, esterification and transesterification, were studied in a liquid/solid two-phase system in order to investigate the effect of water partition between the enzyme preparation and the liquid phase composed of only the reactants, i.e. without the conventional solvents. Lipase from Candida cylindracea was used for these studies. The enzyme was inactive in dehydrated systems. In the case of monoester synthesis, the reaction rate increased with increasing water activity. The reaction rates of the non-specific C. cylindracea lipase-catalysed reactions were very sensitive to the nature of the substrates in this unusual system. For instance, the transesterification reaction rate of ethyl propionate was 48 times higher with nonanol than heptanol in the case of dehydrated substrates, but only 2.2 times higher in the case of water-saturated substrates. The results presented here demonstrate the absolute necessity to consider the polarity of every substrate, because of its ability to modify the water partition between the solid phase (enzyme preparation) and the liquid phase (substrate and product), which results in drastic changes in enzyme activity. Contrary to esterification, which is known to be activated by the water produced, the rate of transesterification remained constant at the beginning of the reaction. However, when transesterification and esterification were carried out in the same liquid phase, the transesterification reaction rate was controlled by the water produced by the concomitant esterification. Activation effects of the water molecules produced during the enzymatic reaction were of exactly the same order of magnitude for both reactions.

The catalytic activity of extracellular phospholipase A2 was studied in low polarity solvents where hydrolytic enzymes have been demonstrated to catalyze synthesis reactions. It was demonstrated that extracellular phospholipase A2 can catalyze the esterification of lysophosphatidylcholine with oleic acid. Up to 6.5% of lysophosphatidylcholine can be esterified into phosphatidylcholine. This activity requires a preincubation of the enzyme in a pH 9 aqueous solution containing calcium, before the incubation in the non-aqueous solvent. No transfer of fatty acid between a phospholipid and a lysophospholipid or between two phospholipids was observed. These results may be useful in understanding the function of the membrane phospholipase A2 which may catalyze acylation or deacylation depending on the local physico-chemical environment.

One of the limiting steps in the further development of enzyme technology is the regeneration of cofactors, especially the pyridinic nucleotide cofactors. Immobilization of alcohol dehydrogenase and steroid dehydrogenase is described. In the last case stabilized enzymes could work in non aqueous solvents. Co-enzyme molecules are bound in the immediate vicinity of the active site of the enzyme. Cofactor regeneration was performed with an electron carrier (Phenazine methosulfate). Ageing phenomena were observed. The co-immobilization of superoxide dismutase gives rise to an increase of stability.

Unusual reactions of interesterification and synthesis catalyzed by Candida cylindracea lipase have been tested in reverse microemulsions. The microemulsions used are made of fatty acids or triglycerides, the enzyme dissolved in a very low water quantity, Brij 35 used as surfactant and an alcoholic cosurfactant. In such a system, fats and alcohols are both the substrates of the enzyme and the microemulsion components. Incidentally, non specific Candida cylindracea lipase does not catalyze interesterification of short chain triglycerides, revealing a specificity for the chain length. Interesterification reactions tested in the presence of a given water quantity but with varying water activities show that it is the water activity and not the water quantity which is a fundamental parameter of the system. The effect of the surfactant (Brij 35) on the interesterification reaction is studied. Heptyl-oleate synthesis catalyzed by non-specific lipase is obtained in microemulsions at a 98% yield. Synthesis of glycerol esters is also tested in monophasic medium and mono and diglycerides are obtained.

Two biological approaches have been investigated for specific modifications of steroids. The first one uses the purified enzyme for the specific dehydrogenation of androsterone to androstanedione. The enzyme used is 3 alpha-hydroxysteroid dehydrogenase which requires a cofactor (NAD). A cofactor regeneration is needed so that the process could work continuously. The conjugation of two points (immobilization of the enzyme and optimization of the ratio methanol-water) allows a continuous work of the enzyme during 25 days. Moreover, we propose a chemical regeneration of the cofactor using methoxy derivative of phenazine methosulphate. Right now it is the limiting step of the process. The second approach of steroid modification uses a whole mycelium of Aspergillus phoenicis for the specific hydroxylation of progesterone to 11 alpha-hydroxy progesterone. The transformation of 90% of the progesterone is obtained with calcium alginate immobilization and the lowest number of products is obtained at pH lower than 2.5 with carrageenan and polyurethane immobilization. It seems promising to apply immobilized biocatalysts to the bioconversion of hydrophobic compounds in organic solvents system.

3 beta, 17 beta-Hydroxysteroid dehydrogenase (3 beta 17 beta HSDH) is an NAD-dependent dehydrogenase which has a double specificity for the 3- and 17-positions on the steroid skeleton. When dehydroepiandrosterone (DHEA) is used as steroid substrate, and the assay coupled with ketosteroid-isomerase, the two reactions occur alternately and each reaction on the 3-position produces a chromophoric molecule. These two reactions can follow one another without dissociation of the coenzyme from the enzyme binding site. This is confirmed by competition experiments with another dehydrogenase.

A functional NAD molecule was immobilized at the active site of Alcohol dehydrogenase within a proteic membrane. The presence and the functionality of the cofactor was checked by fluorescence analysis. The dehydrogenase NAD membrane does not require addition of soluble cofactor for its activity. The system represents a new worthwhile approach because both problems of retention and regeneration of cofactor are solved. The method can be used not only for industrial and analytical applications but also to try to get a better understanding of the kinetics and mechanisms of the catalytic action of dehydrogenase.