Lysozyme inactivation by inert gas bubbling: kinetics in a bubble column reactor Mylene Caussette, Alain Gaunand, Henri Planche, Sophie Colombie, Pierre Monsan, ` ´ and Brigitte Lindet Biotechnology Laboratory, Ecole Nationale Superieure des Mines de Paris, Paris, France ´ The article focuses on lysozyme inacti  ation at gas  liquid interfaces. Bubbling nitrogen in a lysozyme aqueous solution strongly enhances enzyme inacti  ation. At a 150-ml min 1 flow rate, the half-life of lysozyme is 12 min while in the same conditions without bubbling no acti  ity loss is obser  ed after 8 h. Reported effects  ary linearly with the interfacial area which shows that the inacti  ation is an interfacial mechanism. The inacti  ation 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 inacti  ation process using gas  liquid interfaces.  1999 Else  ier Science Inc. All rights reser  ed. Keywords: Lysozyme; gas; inactivation; interface; bubble column; kinetics Introduction The enzymes and microorganisms cause undesirable deterioration in the quality of food products. Gener- ally, they are inactivated by thermal treatment, but the product quality may also be altered. Food industry is therefore looking for an improvement of inactivation processes. Most globular proteins such as enzymes have a strong tendency to accumulate at interfaces. 1 Protein adsorp- tion on interfaces may induce conformational changes. For solid liquid interfaces, several authors have corre- lated the adsorption process to an increase of protein -sheets. 2  4 Studies on protein adsorption also report protein irreversible denaturation on hydrophobic sur- faces. 5 The environmental changes induced by the presence of interfaces may destabilize the electrostatic, hydrophobic and hydrogen bonds of the protein. 6,7 The Address reprint requests to Prof. B. Lindet, Biotechnology Labora- tory, Ecole Nationale Superieure des Mines de Paris, 60, Bd St. ´ Michel, 75006 Paris, France Received 22 April 1998; accepted 27 October 1998 more hydrophobic the surface is, the more important the conformational changes are. 8  10 In the case of liquid  liquid interfaces, Ghatorae et al. showed that enzymes like urease, lipase, ribonuclease and chy- motrypsin were inactivated by organic solvent and the inactivation was found to be proportional to the amount of liquid  liquid interfaces. 11,12 No general mechanism has been proposed to account for the inactivation in- duced by organic solvents and especially to discrimi- nate between physical and chemical influences. For gas  liquid interfaces, the literature mainly reports pro- tein diffusion fluxes on subsurface regions of a stabilized air  water interface. 13  15 These studies highlight the structural protein modifications induced by the adsorp- tion process on a static gas  liquid interface. Few works correlate the adsorption process to a loss of enzyme biological activity. 16  18 This article focuses on lysozyme inactivation at dy- namic gas  liquid interfaces. Inactivation studies have been performed by measuring residual enzymatic activ- ity after nitrogen bubbling treatment In order to characterize the lysozyme inactivation at gas  liquid interfaces, a bubble column contactor has been designed. The gas  liquid area in a bubble column Enzyme and Microbial Technology 24:412 418, 1999  1999 Elsevier Science Inc. All rights reserved. 0141-0229 / 99 / $ - see front matter ( ) 665 Avenue of the Americas, New York, NY 10010 PII S0141-0229 98 00151-3