Isolation of thermostable D-hydantoinase and its application to the synthesis of D-amino acids

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. Culture conditions for the production of thermostable D-hydantoinase from thermophilic Bacillus stearothermophilus SD-1 were investigated. Yeast extract was most effective as a nitrogen source, and the addition of carbon sources gave no significant increase in enzyme formation. The production of D-hydantoinase was repressed by the presence of glucose during the cultivation, but, when glucose was depleted, the enzyme activity remarkably increased. Manganese ions were found to be essential, but no inducer was required for the production of D-hydantoinase from B. stearothermophilus SD-1. The microorganism showed a linear growth in the medium containing yeast extract, and addition of meat extract enabled an exponential growth. The D-hydantoinase of B. stearothermophilus SD-1 was synthesized at the stationary phase in the medium supplemented with meat extract, and enzyme production level was improved about three times compared to that in the absence of meat extract. The thermostable D-hydantoinase of thermophilic B. stearothermophilus SD-1 was purified to homogeneity by using an immunoaffinity chromatography. The affinity chromatography using polyclonal antibody immobilized on Sepharose 4B was relatively simple to operate and gave a purification yield of 60\%. Molecular mass of the enzyme was determined to be about 133.9 kDa on gel filtration chromatography and the molecular mass of the subunit was 54 kDa on SDS-PAGE. Mass spectrometer was also applied to the determination of molecular mass, and molecular masses of the subunit of enzyme and native enzyme were obtained as 50.1 and 102.1 kDa, respectively. From the molecular mass determined by two methods, the...