Brigitte Lindet

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.

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.