Thermostable D-hydantoinase from thermophilic Bacillus stearothermophillus SD-1 : characteristics of purified enzyme
S-G Lee, D-C Lee, S-P Hong, M-H Song, and H-S Kim
One thousand thermophiles isolated from soils were screened for hydantoinase and its thermostability. One thermophilic bacterium that showed the highest thermostability and activity of hydantoinase was identifiedto be Bacillus stearothermophilus SD-1 according to morphological and physiological characteristics. The hydantoinase of B. stearothermophilus SD-1 was purified to homogeneity via ammonium sulfate fractionation, anion-exchange chromatography, heat treatment, hydrophobic-interaction chromatography and preparative gel electrophoresis. The relative molecular mass of the hydantoinase was determined to be 126kDa by gel-filtration chromatography, and a value of 54kDa was obtained as a molecular mass of the subunit on analytical sodiumdodecylsulfate/polyacrylamide gel electrophoresis. The hydantoinase was strictly D-specific and metal-dependent. The optimal pH and temperature were about 8.0 and 65C respectively, and the half-life of the D-hydantoinase was estimated to be 30min at 80C, indicating the most thermostable enzyme so far.