Effect of the binding of bivalent ion to the calcium-binding site responsible for the thermal stability of actinomycete mannanase: Potential use in production of functional mannooligosaccharides

In a previous study, we determined that the calcium-binding site in the catalytic domain of actinomycete mannanases is responsible for the thermal stability [18]. To evaluate whether the calcium-binding site could bind to other bivalent ions, we measured the ability of mannanase to bind bivalent ions by using isothermal titration calorimetry (ITC) by employing the catalytic domain mutants StMandC (from Streptomyces thermolilacinus) and TfMandC (from Thermobifida fusca) and the calcium-binding site deletion mutants StDEDAAAdC and TfDEDAAAdC. The calcium-binding site in StMandC and TfMandC bound bivalent ions with a Ka of 0.10 × 104 to 3.02 × 104 M−1 and 0.21 × 104 to 1.52 × 104 M−1, respectively. Among the tested bivalent ions, thermal stability was enhanced in the following order: magnesium, manganese, and calcium. Magnesium barely enhanced the thermal stability in mannanases. On the other hand, StDEDAAAdC and TfDEDAAAdC did not bind to the tested bivalent ions. From these results, we showed that the calcium-binding site is involved in the binding of the other bivalent ions. The association constant comprised of negative enthalpy and low entropy was suitable for bivalent ion binding in actinomycete mannanases.