Characterization of calcium ion sensitive region for β-mannanase from Streptomyces thermolilacinus

Despite the widespread industrial applications of β-mannanase, the relations between the enzymatic properties and metal ions remain poorly understood. To elucidate the effects of metal ions on β-mannanase, thermal stability and hydrolysis activity were characterized. The stman and tfman genes encoding β-mannanase (EC.3.2.1.78) from Streptomyces thermolilacinus NBRC14274 and Thermobifida fusca NBRC14071 were cloned and expressed in Escherichia coli. The thermal stability of each enzyme shifted to the 7-9°C high temperature in the presence of Ca(2+) compared with that in the absence of Ca(2+). These results show that the thermal stability of StMan and TfMan was enhanced by the presence of Ca(2+). StMan, but not TfMan, required Ca(2+) for the hydrolysis activity. To identify the Ca(2+) sensitive region of StMan, we prepared eight chimeric enzymes. Based on the results of the relationship between Ca(2+) and hydrolysis activity, the region of amino-acid residues 244-349 of StMan was responsible for a Ca(2+) sensitive site.