Thermal Inactivation Parameters for cod pepsin, cod pepsinogen and porcine pepsin MAT AMIN AMIZA Department of Food Science, Faculty of Science and Technology Kolej Universiti Sains dan Teknologi Malaysia 21030 Mengabang Telipot, Kuala Terengganu, Terengganu MALAYSIA http://www.kustem.edu.my AND R.K. OWUSU APENTEN Department of Food Science The Pennsylvania State University College of Agriculture Sciences 111 Borland Laboratory, University Park, PA 16802 USA http://www.cas.psu.edu Abstract: - The thermal inactivation parameters of cod pepsin, cod pepsinogen and porcine pepsinis were compared at pH 2, 40-65 o C. Cod pepsinogen and cod pepsin were less stable than porcine pepsin mainly as a result of large value of activation entropy. Key-Words: - pepsin, pepsinogen, inactivation, rate constant, porcine, activation free energy, activation enthalpy, activation entropy, cod 1 Introduction Proteases are the most extensively used enzymes in industrial application. At present, industrial proteases are mainly derived from microbial, plant and animal sources. During the last two decades, aquatic organisms have been recognised as a new sources of proteases [10,3]. Cold-adapted fish proteases are of interest owing to their greater proteolytic activity towards native protein substrates and a lower activation energy for enzymatic compared with proteases from mammalian or microbial sources [9]. Thermal inactivation is perhaps the most common reason for inactivation. From a biotechnological view point, thermal inactivation is by far the most frequently encountered cause of enzyme inactivation in industry [1]. Knowledge about thermal inactivation is important in order to minimise enzyme inactivation during isolation, application and storage [8]. Such a study is also important for the understanding of the structure- function relationship of enzymes. Most enzyme inactivations occur as first order reactions. Many studies have been reported on purification and characterization of fish pepsin [5], but only few studies reported its thermal inactivation. This paper reports thermal inactivation parameters for cod pepsin, cod pepsinogen and its comparison with porcine pepsin. 2 Methodology 2.1 Materials and Method Purification of cod pepsin was carried out on ion exchange of Amberlite-50 as described in Amiza and Owusu Apenten [2]. Inactivation of pepsin activity as a function of time were performed at four different temperatures. Cod pepsin and pepsinogen were studied in the temperature range of 40-55 o C, while porcine pepsin was studied in the temperature range of 50-65 o C. The concentration of protein was 0.25 mg/ml and 0.8 mg/ml for cod pepsin and cod pepsinogen, respectively (determined by the modified Lowry method [8]). For porcine pepsin, a stock solution of 0.1 mg/ml was prepared. The protein concentrations were chosen in a way that after 5-fold dilution in the buffer, the activity was in the range of 0.4-0.5 U (1 U is defined as the amount of enzyme causing an increase of 1.000 in absorbance at 280 nm per 30 min reaction time for 1-cm pathlength). Enzyme solution (1 ml) was added to a heated buffer solution (4 ml of 50 mM KCl/HCl buffer, pH 2) in a 50-ml stoppered conical flask. Samples (100 µl) were transferred to an