Statistical optimization and biochemical characterization of bioactive Bacillus megaterium 314 caseinase produced on egg shell and molokihya stalks

Abstract

Abstract Egg shell (ES) was a good inducer for caseinase enzyme production by Bacillus megaterium 314 without adding supplements (only distilled water). Mixture of ES with molokihya stalk (MS) was the most suitable substrate that increased enzyme production by 4.7-fold. Optimized HCl pretreatment of ES using Central composite design enhanced enzyme production by 3.3-fold. Moreover, caseinase production was enhanced by 2.5-fold using Plackett-Burman (PB) followed by Central composite (CC) design. Enzyme showed maximum activity at temperature 50 °C and pH 9.0. Activation energy (Ea) and activation energy for the thermal denaturation (Ed) were 11.2 and 135.9 kJ/mol, respectively. Caseinase was highly tolerant to heat inactivation retained 93.6% of its initial activity after 1 h heating at 50 °C. In addition, the calculated half-life time (t0.5) and decimal reduction time (D-value) at 50 °C were 26.9 and 89.3 h, respectively. Moreover, thermodynamic parameters (enthalpy, Gibbs free energy and entropy) indicated that the enzyme was highly tolerant to heat inactivation. Sesame protein was the most suitable for hydrolysis by caseinase. Optimization of Sesame proteolysis using CC design causing 16.9-fold increases compared with the unoptimized. In vitro results showed promising biological activities as anticoagulation and fibrinolytic for caseinase preparation.