Thermostable Protease from Thermophilic Bacteria

Abstract

The effect of temperature on the enzyme activity of the protease produced by Bacillus thermoproteolyticus was examined in the temperature range between 25° and 88°. The kinetic analysis of the enzyme which was heated for 1 hour at 80° indicates that the heated enzyme has the same maximum velocity but lower affinity for substrate than the native one. Molecular weight of the protease was found to be 37,500. The value of intrinsic viscosity was 3.3 ml per g. The ionization behavior of the tyrosine residue of the native and the heat-treated enzyme was examined by spectrophotometric titration. The titration curves indicated that nearly two-thirds of the tyrosine residues in the native enzyme ionized abnormally above pH 10.5. The analysis of the fluorescence spectra of tryptophan residues in the native enzyme was carried out in comparison with those of tryptophan in various solvents. These experiments suggest that some tryptophan residues of the enzyme in the native state are buried in a nonpolar environment with high refractive index rather than exposed to water. From the measurement of the optical rotatory dispersion in the 300 to 600 mµ spectral zone and the ultraviolet region, it appeared that there was a low helix content in the native enzyme. The Cotton effect caused by tyrosine residues was observed by the measurement of the rotatory dispersion and circular dichroism. Amino acid composition of the enzyme was determined by column chromatography with the use of the automatic amino acid analyzer. The structure of the thermostable protease was discussed based on the study of the physicochemical properties of the enzyme.