Mn2+-Dependent Thermophilic Protease of Thermoactinomyces Vulgaris Tsiklinsky

Abstract

Protease of Thermoactinomyces vulgaris was highly thermophilic, as it exhibited its optimal catalytic activity at 65^oC, which was much higher than the growth temperature (50^oC) of this obligate thermophile. Among the metal ions tested, Mn²⁺ was most stimulatory, enhancing the specific activity of protease by about 4.4-fold over the control, and broadened the range of its temperature optimum (i.e. 60–65^oC). A heavy metal (Hg²⁺) inhibited the enzyme activity by about 15%. The increase in slope of Arrhenius plot as well as the energy of activation (E_A) of T. vulgaris protease due to Mn²⁺ indicated that this divalent cation enhanced the rate of high-temperature enzyme catalysis at the expense of E_A. The increasing concentration of the substrate (casein) increased the specific activity of protease gradually in a dose-dependent manner, both in the absence as well as in the presence (10 mM) of Mn²⁺. This divalent cation increased the V_max of this enzyme without affecting K_m for the substrate. The thermophilic protease of T. vulgaris appeared to be a metalloenzyme, in which Mn²⁺ is firmly bound to it, as even 10 mM of EDTA was not able to chelate/remove Mn²⁺ completely to inhibit its activity.