Pectinmethylesterase inactivation by bubbling with N 2 Myl ` ene Caussette, Alain Gaunand, Henri Planche, Sophie Colombi ´ e, Pierre Monsan and Brigitte Lindet* Ecole Nationale Sup ´ erieure des Mines de Paris, CEREP, Biotechnology Laboratory, 60, bd St Michel, 75006 Paris, France; Fax: +33143265910; Email: lindet@cerep.ensmp.fr The half-life of pectinmethylesterase decreased from 8.5 h in an aqueous solution to 3.5 h when 100 mL/N 2 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. Introduction Experimental observations in our laboratory have shown that gas bubbling induces enzyme inactivation (Lindet et al., 1995). As enzymes cause undesirable deterioration in the quality of food product, they are generally inactivated by heat treatment. Because losses of product quality may occur, the food industry is therefore looking for an improvement of the enzyme inactivation kinetics. Very few studies refer to enzymes at gas-liquid interfaces and only protein behaviour at static air-water interfaces has been studied. Changes in the secondary structure of peroxidase have been identified after adsorption at the air-water interface (Tronin et al., 1996) and low losses of enzymatic activity have been observed in the case of lysozyme and nuclease (Xu and Damodaran, 1993 ; Inbar and Miller, 1976). Pectinmethylesterase (PME) is a widely present enzyme in fruit plants. It catalyses the hydrolysis of methyl ester groups of pectin. It is a very thermostable enzyme (Rouse et Atkins, 1952). The problem of cloud and consistency loss of fluids of vegetable origin are important in the food juice industry. Many authors have demonstrated that PME activity is responsible for the physico-chemical modifica- tions of the juices (Arreola et al., 1991 ; Balaban et al., 1991 ; Laratta et al., 1995). Techniques allowing a better pectinmethylesterase inactivation in relevant food products could be valuable (Ishikawa et al., 1995). In order to characterize the PME inactivation at gas-liquid interfaces, two different reactors have been used : a bubble column and a stirred gas-liquid reactor. In this paper, the effect of gas-bubbling on PME inactivation is reported in the two reactors as well as the effect of the parameters acting on the adsorption process at the gas-liquid inter- face. Materials and methods Materials Pectinmethylesterase (PME) from Aspergillus niger was kindly provided by Gist Brocades. A complete PME purity is found on agarose electrophoretic gel. Its molecular weight is 40 000. Its isoelectric point is 4.4. Pectin (SBI, France) with a methylation degree 70 % was used as the enzyme substrate. All the other chemicals used in this work were of the highest grade commercially available. N 2 was from Airgaz (France) with a certified purity of 99.995 %. PME activity The PME activity was determined by automatic titration according to the method of Rouse and Atkins (1952). The method is based on the production of acidity during the decomposition of pectin by PME. A 0.5 wt % pectin and 0.1 M NaCl was used. Given volumes of the PME solution and of the pectin solution were contacted at a controlled temperature of 25°C, and the volume of 10 mM NaOH needed to keep the pH constant at 4.5 was recorded. One unit of PME activity was defined as the concentration of PME which released 1 μmol carboxyl groups.min -1 . Reactors Bubble column A 15 mm internal diameter and 0.4 m height column shown in Figure 1 was thermostated with a double envel- ope. Pure nitrogen from a gas cylinder was bubbled in the column through a gas sparger located at its bottom. Three single nozzles 50 μm in diameter were used for the generation of the bubbles. The N 2 flow rate was controlled with a mass flow meter within 0–400 mL.min -1 with an error  0.1 %. N 2 was humidificated and heated to the working temperature before entering the bubble column in order to avoid water evaporation and temperature Biotechnology Techniques, Vol 12, No 7, July 1998, pp. 561–564 © 1998 Chapman & Hall Biotechnology Techniques ⋅ Vol 12 ⋅ No 7 ⋅ 1998 561