Activity and Process Stability of Purified Green Pepper (Capsicum annuum) Pectin Methylesterase SO Ä NIA MARI ÄLIA CASTRO, ² ANN VAN LOEY, § JORGE ALEXANDRE SARAIVA, ² CHANTAL SMOUT, § AND MARC HENDRICKX* ,§ Department of Chemistry, Universidade de Aveiro, Campus Santiago, 3810-193 Aveiro, Portugal, and Laboratory of Food Technology, Faculty of Applied Bioscience and Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B-3001 Heverlee (Leuven), Belgium Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 °C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 °C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57 °C) and a biphasic model for higher temperatures (58-70 °C). The enzyme showed a stable behavior toward high-pressure/temperature treatments. KEYWORDS: Capsicum annuum; pepper; pectin methylesterase; purification; characterization; thermal and high-pressure stability INTRODUCTION Peppers have become more popular in recent years due to their chemical composition (e.g., vitamins), and a wide variety is nowadays available on the market. Most varieties belong to the Capsicum annuum species, and they can be consumed fresh or processed, as immature (i.e., green) or as mature fruit (i.e., yellow, orange, red), as a spice or as a vegetable, because of their distinct colors, intense taste, and unique flavor. The texture, in particular, the crispness, of peppers is an important quality attribute to consumers. It is known that vegetable texture is closely related to the pectic substances and to activities of pectolytic enzymes (1). Prominent among the enzymes impli- cated in the softening of fruits and vegetables during ripening are polygalacturonase and pectin methylesterase (PME) due to their relationship with the cell-wall pectic content. Although peppers, like tomatoes, belong to the Solanaceae family, there is little information in the literature on the pectolytic enzymes and their relationship with biochemical cell-wall changes of bell peppers and texture (2-4). One reason such information is lacking is probably due to the anticipated low enzyme activities in peppers, as the texture degradation in peppers is a slow process (2). Nevertheless, PMEs play a central role in texture evolution; the control of its activity, through knowledge of its dependence on parameters such as temperature and pH, is of great practical importance in the food industry for protecting and improving the texture and firmness of several processed fruits and vegetables (5). Thermal processing is still one of the most frequently used methods for food preservation, and one approach to optimize the heat treatment of fruits and vegetables in order to maximize the overall quality is to develop a model considering, among other parameters, the (in)activation kinetics for relevant enzymes to predict quality changes during process- ing and subsequent storage. PME has been extracted and purified from many different sources and characterized in terms of biochemical properties and thermal stability (e.g., see refs 5-12). In this work, PME was extracted from green peppers and purified by affinity chromatography. The purified pepper PME obtained was biochemically characterized and submitted to thermal and high-pressure inactivation. MATERIALS AND METHODS Materials. Green bell peppers (C. annuum) were purchased from a local auction (Mechelen, Belgium). Apple pectin [degree of esterifi- cation (DE) ) 75%] was obtained from Fluka Chemicals Co. (Buchs, Switzerland). CNBr-Sepharose 4B resin was purchased from Sigma (St. Louis, MO). All other chemicals were of analytical grade. Methods. Pectin Methylesterase Purification. PME was extracted from peppers with 0.2 M tris(hydroxymethyl)aminomethane buffer (i.e., Tris buffer) (pH 8.0) with 1 M NaCl, followed by purification by affinity chromatography on a CNBr-Sepharose 4B-PME inhibitor column and finally stored at -80 °C in 20 mM Tris buffer (pH 7.5) using the procedure as described by Ly-Nguyen et al. (11). * Author to whom correspondence should be addressed (fax +32 16 32 19 60; telephone +32 16 32 15 72; e-mail Marc.Hendrickx@ agr.kuleuven.ac.be). ² Universidade de Aveiro. § Katholieke Universiteit Leuven. 5724 J. Agric. Food Chem. 2004, 52, 5724-5729 10.1021/jf0352071 CCC: $27.50 © 2004 American Chemical Society Published on Web 08/11/2004 Article 11