Physiochemical Properties of Thermomycolase, the Thermostable, Extracellular, Serine Protease of the Fungus Malbranchea pulchella

Abstract

The physicochemical properties of the extracellular protease of the fungus Malbranchea pulchella, for which we have adopted the name thermomycolase, were investigated. The molecular weight of diisopropylphosphorylthermomycolase was found to be 32 000–33 000 by sedimentation equilibrium and sodium dodecyl sulfate (SDS) gel electrophoresis. Its sedimentation coefficient (s020, w = 2.97 S), intrinsic viscosity ([η] = 3.0 cc/g), and frictional ratio (f/f0 = 1.09) characterize the enzyme as a typical globular protein. The circular dichroism spectrum of the protein is also consistent with its globular structure. Active thermomycolase autolyzes extensively, especially at low calcium ion concentrations, producing low molecular weight peptide material. In the presence of SDS, a different autolytic degradation is observed, resulting in much higher molecular weight polypeptide products (16 500, 12 500,11 000, 8 500). The results indicate that, in the presence of SDS, thermomycolase has three sites that are highly susceptible to autolysis. At calcium ion concentrations of 10−3 M and 10−2 M the enzyme undergoes a sharp thermal denaturation with transition temperatures at 69 °C and 75 °C, respectively, and with complete loss of enzyme activity. At 70 °C the enzyme appeared to be maximally thermostable at a calcium ion concentration of 10−2 M.