PURIFICATION AND SOME PROPERTIES OF PROTEINASE FROM PSEUDOMONAS-FLUORESCENS NO-33

The extracellular proteinase from Pseudomonas fluorescens No. 33 was purified to electrophoretic homogeneity by a procedure including precipitation with HCl and (NH4)2SO4, and column chromatography. The enzyme was purified 170-fold giving a yield of 7% of the original activity. The molecular mass of the purified enzyme was 48000 by SDS-PAGE. The optimum pH and temperature for the hydrolysis of casein were 8.0-9.8 and 30-35-degrees-C respectively. The enzyme was more thermostable in synthetic milk salts solution than in 0.1 M-sodium phosphate buffer, but was heat-labile at 50-degrees-C in both buffer systems. The activity was inhibited by o-phenanthroline, Hg2+, Cu2+, Fe2+ and, to a lesser extent, Ni2+. Caseins were susceptible to the proteinase, but degradation patterns were dependent on the form of the casein.