Partial Purification, Characterization, and Thermal and High-Pressure Inactivation of Pectin Methylesterase from Carrots (Daucus carrota L.) BINH LY-NGUYEN,ANN M. VAN LOEY,DIANA FACHIN,ISABEL VERLENT, INDRAWATI, AND MARC E. HENDRICKX* Laboratory of Food Technology, Department of Food and Microbial Technology, Faculty of Agricultural and Applied Biological Sciences, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B-3001 Leuven, Belgium Pectin methylesterase (PME) from carrots (Daucus carrota L.) was extracted and purified by affinity chromatography on a CNBr-Sepharose 4B-PME inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters of carrot PME was performed. In a second step, the thermal and high- pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified carrot PME could be described by a fractional-conversion model. KEYWORDS: Daucus carrota L.; carrots; pectin methylesterase; purification; thermal inactivation; high- pressure inactivation INTRODUCTION Pectin methylesterase (pectinesterase, PME, PE, EC 3.1.1.11) is widely distributed in plants and microorganisms (1). PME catalyzes the de-esterification of methyl ester of polygalacturonic acid polymer to form pectic acid, methanol, and hydrogen ions, as part of an array of pectin hydrolyzing enzymes, and leads to the formation of a calcium pectate gel (2-6). Detrimental effects of PME on cloud stability of juices and nectars have been reported in detail (7-11). In contrast, beneficial effects of PME, including (i) enhancement of firmness of thermally processed fruit and vegetable products (2, 3, 12-14), (ii) effective increase of extraction yield of juices by conventional methods (15), and (iii) promotion of water removal from tissues on drying (16), have also been reported. In plants, PME is bound to the cell wall by electrostatic interaction, and high-ionic-strength solutions are required in order to solubilize the enzyme and obtain a good yield of extraction (17-25). PME has been extracted and/or purified from different sources: e.g., tomatoes (23, 26-33), oranges (18, 21, 34-40), apples (17, 24, 41-46), grapefruits (22, 47, 48), and bananas (49-51). Biochemical properties, thermal stability and food applications of PME from these sources were reported in more or less details by several authors. PME from carrot (Daucus carrota L.) has been investigated by Polacsek and Pozsar (52), Markovic (53), Tijskens et al. (54), Stratilova et al. (55), Vora et al. (56), and Hyeon et al. (57). Polacsek and Pozsar (52) found that in all cases, carrot PME activities were much lower than those found in tomatoes. Markovic (53) extracted and partially purified carrot PME by ion-exchange chromatography on DEAE-Sephadex A 50 and subsequent chromatography on Sephadex G 75 and found three multiple forms of PME. However, no further studies on purified carrot PME have been reported yet. The use of carrots as a base product for the formulation of mixed juices and baby foods is presently increasing. The conventional thermal processing schedule has a deteriorative effect on texture, flavor, and nutritional attributes. High-pressure processing, in contrast to high-temperature treatment, is specific so far as not or slightly affecting covalent bonds in the pressure range studied. Hence, high-pressure processing does not destroy natural flavors or colors (58). To obtain a stable product and eliminate detrimental effects caused by pressure stable enzymes, we recommended a thermal treatment combined with high- pressure processing. In this paper, PME from carrots was purified using a single-step affinity chromatography. Purified carrot PME was characterized in terms of biochemical properties and thermal and high-pressure stability. MATERIALS AND METHODS Materials. Carrots (Daucus carrota L.) were purchased from a local supermarket. Apple pectin [degree of esterification (DE) 75%] was obtained from Fluka Chemical Co. (Switzerland). CNBr-activated Sepharose 4B and commercial orange pectin methylesterase were purchased from Sigma (USA). Other chemicals were of analytical grade. Kiwi PME inhibitor (PMEI) Extraction. The extraction of kiwi PMEI was performed according to the method of Giovane et al. (59, 60). About 0.75 kg of ripe kiwi fruits was peeled and homogenized in water (1:1 w/v) at 4 °C. The suspension was centrifuged at 20 000g,4 °C, for 20 min. The supernatant containing the glycoprotein inhibitor (PMEI) was separated from the pellet and adjusted to pH 6.0 using 1 * To whom correspondence should be addressed. Tel: +32-16-321585. Fax: +32-16-321960. E-mail: marc.hendrickx@agr.kuleuven.ac.be. J. Agric. Food Chem. 2002, 50, 5437-5444 5437 10.1021/jf011666v CCC: $22.00 © 2002 American Chemical Society Published on Web 08/09/2002