Graduate Thesis Or Dissertation
 

Kinetic properties and characterization of purified proteases from Pacific whiting (Merluccius productus)

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  • Kinetic properties of the two proteases, causing textural degradation of Pacific whiting (Merluccius productus) during heating, were compared and characterized with the synthetic substrate, Z-Phe-Arg-NMec. Pacific whiting P-I and P-II showed the highest specificity on Z-Phe-Arg-NMec, specific substrate for cathepsin L. The K [subscript m] of preactivated P-I and P-II were 62.98 and 76.02 (μM), and k [subscript cat], 2.38 and 1.34 (s⁻¹) against Z-Phe-Arg-NMec at pH 7.0 and 30°C, respectively. Optimum pH stability for preactivated P-I and P-II is between 4.5 and 5.5. Both enzymes showed similar pH-induced preactivation profiles at 30°C. The maximal activity for both enzymes was obtained by preactivating the enzyme at a range of pH 5.5 to 7.5. The highest activation rate for both enzymes was determined at pH 7.5. At pH 5.5, the rate to reach the maximal activity was the slowest, but the activity was stable up to 1 hr. P-I and P-II shared similar temperature profiles at pH 5.5 and pH 7.0 studied. Optimum temperatures at pH 5.5 and 7.0 for both proteases on the same substrate were 55°C. Significant thermal inactivation for both enzymes was shown at 75°C. Preactivated P-I and P-II displayed a similar first order thermal inactivation profile at pH 7.0. At 30 and 90°C, half lives, t [subscript 1/2], for Pacific whiting P-I were 49.50 and 0.20 min and for P-II, 32.54 and 0.18 min, respectively. The rate constant of inactivation for both proteases increased about 200-fold between two limits, 30 and 90°C. Half lives at 55°C, optimum temperature, for P-I and P-II were also determined to be 5.29 and 6.75 min. The increase in thermal inactivation rate constants independent of substrates corresponded to an activation energy for heat denaturation of 21.18 kcal/mol for P-I and 19.97 kcal/mol for P-II by Arrhenius plot. These similar kinetic properties, i.e., kinetic parameters, pH profile and thermal inactivation rate constant, suggested that Pacific whiting P-I and P-II are the same enzyme.
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