title: Purification and properties of genetic expressing product of thermostable protease from Bacillus stearothermophilus HY-69.
abstract: 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.
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: C
authors.initials: C
authors.lastname: Sun
authors.forename: C
authors.initials: C
authors.lastname: Jin
authors.forename: S
authors.initials: S
authors.lastname: Yang
authors.forename: S
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000737:chemistry*; D006361:Heating; D008970:Molecular Weight; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
chemical_list: D000596:Amino Acids; D010450:Endopeptidases