THERMOSTABLE D-HYDANTOINASE FROM THERMOPHILIC BACILLUS-STEAROTHERMOPHILUS SD-1 - CHARACTERISTICS OF PURIFIED ENZYME

Abstract

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 identified to 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 126 kDa by gel-filtration chromatography, and a value of 54 kDa 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 65 degrees C respectively, and the half-life of the D-hydantoinase was estimated to be 30 min at 80 degrees C, indicating the most thermostable enzyme so far.