Purification and characterization of thermostableD-hydantoinase from thermophilicbacillus stearothermophilus SD-1 |
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Authors: | Seung-Goo Lee Dong-Cheol Lee Hak-Sung Kim |
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Institution: | (1) Department of Biotechnology, Korea Advanced Institute of Science and Technology, 3731 Kusung-Dong, Yusung-Gu, 305701 Taejon, Korea |
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Abstract: | A thermostable D-hydantoinase of thermophilicBacillus stearothermophilus SD-1 was purified to homogeneity using an immuno-affinity chromatography. The affinity chromatography that employed polyclonal
antibody immobilized on Sepharose 4B was simple to operate and gave a purification yield of 60% of enzyme activity. Molecular
mass of the enzyme was determined to be about 133.9 kDa by gel filtration chromatography and the molecular mass of the subunit
was 54 kDa on SDS-PAGE. Mass spectrometric analyses were also performed for the determination of the molecular mass of the
native enzyme and its subunit. The apparent molecular masses were 51.1 and 102.1 kDa for the subunit and native enzyme, respectively.
Based on the molecular masses determined by these two methods, it is suggested that the D-hydantoinase exists as a dimeric
conformation in the cell. Isoelectric pH of the enzyme was observed to be 4.47. It was found that the enzyme requires one
manganese ion per molecule of enzyme for the activity. The optimal pH and temperature for the catalytic activity were about
8.0 and 65‡C., respectively. The half-life of the enzyme was estimated to be 30 min at 80‡C., confirming that the enzyme purified
is one of the most thermostable D-hydantoinase reported so far. Kinetic constants of the enzyme for different substrates were
also determined. |
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Keywords: | Thermostability D-hydantoinase" target="_blank">D-hydantoinase Bacillus stearothermophilus immuno-affinity chromatography |
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