One thermophilic bacterium producing thermostable D-Hydantoinase was isolated from nature. D-Hydantoinase from isolated strian had a high thermostability and activity. Isolated strain was identified to be Thermus sp. GH-1 according to morphological and physiological characteristics.
Culture conditions for the production of D-hydantoinase from Thermus sp. GH-1 were investigated. Meat extract and glucose were effective to produce enzyme as a nitrogen source and carbon source, respectively. The production of D-hydantoinase was inducible. Hydantoin was effective as a inducer. The D-hydantoinase of Thermus sp. GH-1 was synthesized at the late exponential phase and the production of enzyme was decreased at the late stationary phase.
The thermostable D-hydantoinase of Thermus sp. GH-1 was purified to homogeneity by using immuno-affinity chromatography. The immunoaffinity chromatography using polyclonal antibody immobilized on Sepharose 4B gave a purification yield of 10 % at one step operation.
Optimal temperature and pH of the purified D-hydantoinase of Thermus sp. GH-1 were 65℃ and 7.5, respectively. The molecular mass of D-hydantoinase of Thermus sp. GH-1 was determined to be 250 kDa and subunit was determined to be 56 kDa. So, D-hydantoinase of Thermus sp. GH-1 was expected to be organized as a tetramer. Stokes' radius was calculated to be 53.4 Å. Isoelectric point of the D-hydantoinase of Thermus sp. GH-1 was observed to be 4.3. Manganese ion was necessary for enzyme activity. TheD-hydantoinase of Thermus sp. GH-1 had a high thermal stability and the half-life at 80℃ was 43 min. The $K_m$ value of HPH as substrate was 30.67 mM.
