Role of Calcium Ions in the Thermostability of Thermolysin and Bacillus subtilis var. amylosacchariticus Neutral Protease

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Abstract
The stabilizing effect of Ca2+ on thermolysin and B. subtilis var. amylosacchariticus neutral protease was investigated. Ca2+ and Zn2+ were removed from the proteases by gel filtration over Sephadex G-25 equilibrated with metal chelating agents. Using these enzymes with different metal content, heat inactivation kinetics were studied at various temperatures. Removal of Ca2+ caused a sharp decrease in thermostability and diminished the values of the activation enthalpy (.DELTA.H*) and entropy (.DELTA.S*) for heat inactivation. There was little difference in stability between thermolysin containing 0.3 g-atom/mol and B. subtilis neutral protease containing 1.4 g-atoms/mol. Ca binding isotherms of the proteases were obtained by equilbrium gel chromatography with various concentrations of free Ca2+. Thermolysin had 4 independent Ca binding sites with an identical intrinsic binding constant (K) of 2.0 .times. 104 M-1. B. subtilis neutral protease had 4 independent sites. The K value for 3 sites was 1.1 .times. 105 M-1 and the binding constant for the other site was 1.5 .times. 103 M-1. There was little difference in total free energy change for Ca binding between these proteases. The stabilizing effect of Ca on these enzymes is apparently almost equal and the extra thermal stability of thermolysin is likely to come from its polypeptide chain structure.

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