Dehalogenation of gaseous 1-chlorobutane by dehydrated whole cells: Influence of the microenvironment of the halidohydrolase on the stability of the biocatalyst.

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.

Effects of hydrostatic pressure on horse liver alcohol dehydrogenase (HLADH): a new way of analyzing kinetic study.

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.

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system obeys a Ping Pong Bi Bi mechanism with competitive inhibition by the alcohol substrate and water.

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.

Identification of an indigo precursor from leaves of Isatis tinctoria (Woad).

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.

Gas phase biotransformation reaction catalyzed by baker's yeast.

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.

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.

The kinetic behaviour of a two-enzyme system in biphasic media: coupling hydrolysis and lipoxygenation.

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.

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 (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.

Investigation of behavior of an enzyme in a biphasic system: soybean lipoxygenase-1.

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.

NMR on-line monitoring of esterification catalyzed by cutinase.

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.

The pressure-dependence of two beta-glucosidases with respect to their thermostability.

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.

Hydrostatic pressure induces conformational and catalytic changes on two alcohol dehydrogenases but no oligomeric dissociation.

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.

Working at controlled water activity in a continuous process: the gas/solid system as a solution.

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.

Enzymatic synthesis of alkyl beta-D-xylosides by transxylosylation and reverse hydrolysis.

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.

Biocatalysis in the gas phase.

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-oligosaccharide synthesis by free and immobilized beta-glucosidase.

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.

Gas phase transesterification reactions catalyzed by lipolytic enzymes.

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.

Modulation of lipase hydrolysis and synthesis reactions using carbohydrates.

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.