ISOLATION AND CHARACTERIZATION OF AN EXTRACELLULAR PROTEINASE FROM PSEUDOMONAS-TOLAASII

The plant pathogen, Pseudomonas tolaasii, produced an extracellular proteinase in 10% (w/v) reconstituted skim milk, which was isolated to homogeneity by ion-exchange chromatography on DEAE-52 cellulose and gel filtration on Sephadex G-150. Ion-exchange chromatography achieved a 65-fold purification and gel filtration increased specific activity a further 2.7-fold. The enzyme was optimally active at pH 7 and 40 degrees; activity decreased rapidly at higher temperatures. The proteinase was almost totally inactivated on heating at 50-55 degrees for 1.5 min but ca 50 and 25% residual activity remained after heating for 1.5 min at 90-120 degrees or 140 degrees, respectively. The Z-value for inactivation of the proteinase in the temperature range 80-140 degrees was ca 34 degrees and the corresponding activation energy (E(a)) was 82 kJ mol(-1). Activity was strongly inhibited by EDTA and o-phenanthroline and partially restored by Zn2+, Co2+, or Mn2+. The M(r) was ca 50 000 (by SDS-PAGE) or ca 45 000 by gel filtration (TSK G3000 SW).