Purification and properties of genetic expressing product of thermostable protease from Bacillus stearothermophilus HY-69.

The thermostable metal protease gene from Bacillus stearothermophilus HY-69 had been cloned and expressed in Bacillus subtilis MI113. The genetic expressing product of the enzyme was purified by CM-cellulose chromatography. The product shows homogeneity on PAGE. Its molecular weight is 27,000 +/- 1000 by SDS-PAGE and Sephadex G100 filtration, respectively. The alpha-helix content of the protease is estimated to be about 66%, the beta-turn about 28%, the random coil about 6%, but not beta-sheet, calculated from the circular dichroism data. The optimal temperature of the enzyme was 70 degrees C. When the enzyme was denatured in 3 mol/L of Gdn--HCl in phosphate buffer pH6.0 for 20 min, it remained about 40% of original activity. It shows that it is rather resistant to heat and Gdn-HCl denaturation. Its conformational variety coursed by Gdn-HCl was investigated by the far UV circular dichroism and fluorescence spectra. The results show that the enzyme has more compact conformation and internal hydrophobility.