Kinetic analysis of a general model of activation of aspartic proteinase zymogens.
ABSTRACT Starting from a simple general reaction mechanism of activation of aspartic proteinase zymogens involving an uni- and a bimolecular simultaneous route, the time course equation of the concentration of the zymogen and of the activated enzyme have been derived. From these equations, an analysis quantifying the relative contribution to the global process of the two routes has been carried out for the first time. This analysis suggests a way to predict the time course of the relative contribution as well as the effect of the initial zymogen and activating enzyme concentrations, on the relative weight. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed. Finally, we apply some of our results to experimental data obtained by other authors in experimental studies of the activation of some aspartic proteinase zymogens.
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ABSTRACT: Chymosin is an aspartic proteinase found in the stomach of neonatal mammals and is used as milk coagulant in the cheese industry. In this study, preprochymosin cDNA from the abomasums of Iranian natives' kid goats was cloned and characterized. This cDNA has an open reading frame of 1143 bp and is predicted to code for a preproenzyme of 381 amino acids with an N-terminal 16 amino acid signal peptide that is followed by a 42 amino acid proenzyme. The deduced amino acid sequence revealed 98.7% and 94% identity with corresponding lamb and cattle sequences, respectively. The cDNA encoding for prochymosin was then subcloned into pET-28a and expressed with and without N-terminal His-tag in E. coli BL21 (DE3). Inclusion bodies were isolated and solubilized in 8 M urea at pH 10.7. Solubilized prochymosion molecules were successfully refolded and active recombinant goat chymosin was recovered after subsequent activation. Milk clotting activity (416 U/mg and 192 U/mg) was observed in (His)-prochymosin and (His)-prochymosin enzymes, respectively, after activation.Food Biotechnology - FOOD BIOTECHNOL. 01/2012; 26(2):143-153.