Alain Gaunand

Lysozyme inactivation under mechanical stirring: effect of physical and molecular interfaces

This article focuses on the role of interfaces on lysozyme inactivation and aggregation process in stirred reactor. The first order inactivation constant of this process has found to be proportional not only to the power imparted by the impeller but also to the area of glass-liquid, air-liquid and PTFE-liquid interfaces in three reactors. Both area and type of interfaces act on inactivation: PTFE and air are four more efficient than glass to promote lysozyme inactivation because of their hydrophobicity. As well as physical interfaces, molecular surfaces of inactivated enzymes -more hydrophobic than native enzymes- enhance lysozyme inactivation and aggregation. This enhancement has been found to be correlated with the properties of aggregates of inactivated enzymes, especially their number. Then, under mechanical stirring, inactivation-aggregation process is induced by physical interfaces and self-catalyzed by increasing hydrophobic surfaces of inactivated enzymes.

Lysozyme inactivation by inert gas bubbling: kinetics in a bubble column reactor

Abstract The article focuses on lysozyme inactivation at gas–liquid interfaces. Bubbling nitrogen in a lysozyme aqueous solution strongly enhances enzyme inactivation. At a 150-ml min −1 flow rate, the half-life of lysozyme is 12 min while in the same conditions without bubbling no activity loss is observed after 8 h. Reported effects vary linearly with the interfacial area which shows that the inactivation is an interfacial mechanism. The inactivation induced by nitrogen bubbling strongly depends on temperature and pH acting on the adsorption process. This study points out on the one hand the importance of controlling gas–liquid interfaces in bioreactors and on the other hand the potentialities of an inactivation process using gas–liquid interfaces.

Lysozyme inactivation and aggregation in stirred-reactor

Abstract Mechanisms of enzyme inactivation and aggregation are still poorly understood. In this work, we are considering the characterisation of both inactivation and aggregation in stirred tank reactor, with lysozyme as the model enzyme. The inactivation kinetics are first order. For stirring speeds in the range of 0–700 rpm, the kinetic constant is found to be proportional to the power brought by the impeller. It suggests that inactivation depends on collisions between enzyme molecules. Efficient collisions between native and inactive molecules induce native molecules to turn into inactive molecules and lead to lysozyme aggregation. During inactivation, enzymes are found to aggregate as shown by light scattering measurements. The structure of aggregates was studied on samples treated for chemical denaturation and reduction. The aggregates are supramolecular edifices, mainly made up of inactivated enzymes linked by weak forces. But aggregates are also made up of dimers and trimers of lysozyme, linked by disulfide bridges. Dimers and trimers are 18% and 5%, respectively, of the total amount of lysozyme aggregates. Whatever the stage of aggregate formation and the initial enzyme concentration are, these aggregates are irreversibly inactivated. Enzyme activity is definitely lost even if stirring is stopped and/or temperature decreased. This study points out the importance of hydrodynamics in bioreactors and highlights the nature of the aggregates resulting from the interactions between native and inactive enzymes.

Enzyme inactivation by inert gas bubbling

Abstract Nitrogen bubbling strongly enhances the inactivation of enzymes such as lysozyme, lipase or pectinmethylesterase.The nitrogen bubbling efficiency depends on the enzyme. The inactivation kinetics are first order except for lipase. A rapid initial phase is followed by a decrease of the inactivation rate which has been explained by a competition between active and inactive lipase species at the gas-liquid interface. The inactivation kinetics of lysozyme and lipase increase linearly with the specific interfacial area. For pectinmethylesterase the inactivation kinetics do not depend on the specific interfacial area. For the three enzymes, physico-chemical parameters like pH and ionic strength act on the inactivation efficiency by probably modifying the adsorption mechanisms. This study points out the importance of hydrodynamics in bioreactors (loss of enzyme activity during their production by fermentation or loss of enzyme efficiency during biocatalysis reactions). This work also highlights the potentialities of an inactivation process using gas-liquid interfaces.

Oxidation of Cu(I) by oxygen in concentrated NaCl solutions. III: Kinetics in a stirred two-phase and three-phase reactor

Abstract A whole set of data on the oxidation of cuprous chloride in a standard stirred reactor is treated using the recent work by Papassiopi and co-workers ( Chem. Engng Sci. 40 , 1527 and thesis, Ecole des Mines de Paris) on homogeneous kinetics. The reaction regime is found to be a slow one and HCl and NaCl concentrations, temperature, volume of solution and stirring speed only influence the quantity k L a (mass-transfer coefficient × specific interfacial area) or the oxygen solubility or both. The system Cu(I)/O 2 is then adequate to measure k L a in gas—liquid contactors. The improvement of oxidation when an immiscible organic phase is added may be attributed either to the decrease of the water surface tension or to the formation of “two-phase bubbles“ or “gas—liquid drops”, more unlikely at low stirring speed.

Oxidation of Cu(I) by oxygen in concentrated NaCl solutions—I. Homogeneous kinetics of oxidation by molecular oxygen in solution

Abstract The homogeneous oxidation of cuprous chloride in 2 M NaCl solutions by dissolved molecular oxygen was studied, using a batch reactor, at constant pH and temperature. The rate of homogeneous reaction is represented by the expression: where square brackets indicate concentrations, in the conditions: temperature 22°C and concentrations 0.0625 M for HCl and between 0.9 × 10−3 M and 6.2 × 10−3 M for Cu(I).

Pectinmethylesterase inactivation by bubbling with N2

The half-life of pectinmethylesterase decreased from 8.5 h in an aqueous solution to 3.5 h when 100 mL/N2 min−1 was bubbled through it at 50°C. Inactivation strongly depended on the pH and the ionic strength and was enhanced at a pH close to the pI of the enzyme.

Oxidation of Fe (II) by oxygen in concentrated NaCl solutions: Prediction of stirred gas-liquid reactor performance from homogeneous kinetic data

Abstract The oxidation of 0.25 M ferrous solutions carried out in a standard gas-liquid reactor is found to proceed in the very slow reaction regime and can be predicted from the expression of the rate R of the homogeneous oxidation: 4Fe(II) + 4H + + O 2 (in solution)→ 4 Fe(III)+ 2H 2 O established for one hundred times lower ferrous concentrations. The rate expression: R = exp 25.336− 82.464×10 3 R T [Fe (II)][O 2 ] (mol −1 L −1 s −1 ) where T is the absolute temperature (K), R the gas constant (8.314 J K −1 mol −1 ) and square brackets indicate concentrations (mol L −1 , is proposed in the range 1.25 mM–0.25 M for ferrous concentration, 18–45°C for temperature, and with concentrations of 0.02 M HCl and 4 M chloride ions, introduced as HCl, NaCl and FeCl 2 .

Iron elimination in cupric chloride hydrometallurgical processes by oxidizing extraction

Abstract Iron elimination from chloride hydrometallurgical solutions by solvent extraction is strongly enhanced by the simultaneous oxidation of cuprous and ferrous species, which creates an important depletion of acidity. Accordingly, it is not necessary to add any neutralizing agent to shift the extraction equilibrium Fe(III) + 3 RH ⇌ FeR 3 + 3H + to the right. Most of the extraction kinetics are controlled by ferrous oxidation, and are represented with the aid of a previous kinetic study of this oxidation catalysed by copper chloride.

Oxidation of Cu(I) by oxygen in concentrated NaCl solutions. II: Kinetics in a known interfacial area cell

The oxidation of cuprous chloride in 2 M NaCl solutions by oxygen was studied using a known interfacial area cell. Each test was carried out at constant pH and temperature. The effect of stirring speed, oxygen partial pressure, cuprous concentration were evaluated, as well as the influence of the presence of an organic layer between the gas and the aqueous phase. The oxidation of 0.9 M Cu(I) solutions was found in a fast or intermediate regime; on the whole range of concentrations covered rates of oxidation were found consistent with homogeneous kinetics data reported by Papassiopi et al. (1985) and Papassiopi (1985) and especially with the order 1.5 with respect to cuprous concentration.