Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production

A novel protease, hydrolyzing azocasein, was identified, purified, and char acterized from the culture supernatant of the fish pathogen Yersinia rucker i. Exoprotease production was detected at the end of the exponential growth phase and was temperature dependent. Activity,vas detected in peptone but not in Casamino Acid medium. Its synthesis appeared to be under catabolite repression and ammonium control. The protease was purified in a simple two- step procedure involving ammonium sulfate precipitation and ion-exchange ch romatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SD S-PAGE) analysis of the purified protein indicated an estimated molecular m ass of 47 kDa. The protease had characteristics of a cold-adapted protein, i.e., it was more active in the range of 25 to 42 degrees C and had an opti mum activity at 37 degrees C. The activation energy for the hydrolysis of a zocasein was determined to be 15.53 kcal/mol, and the enzyme showed a rapid decrease in activity at 42 degrees C. The enzyme had an optimum pH of arou nd 8. Characterization of the protease showed that it required certain cati ons such as Mg2+ or Ca2+ for maximal activity and was inhibited by EDTA, 1, 10-phenanthroline, and EGTA but not by phenylnethylsulfonyl fluoride. Two N -methyI-N-nitro-N-nitrosoguanidine mutants were isolated and analyzed; one did not show caseinolytic activity and lacked the 47-kDa protein, while the other was hyperproteolytic and produced increased amounts of the 47-kDa pr otein. Azocasein activity, SDS-PAGE, immunoblotting by using polyclonal ant i-47-kDa-protease serum, and zymogram analyses showed that protease activit y was present in 8 of 14 strains tested and that two Y. ruckeri groups coul d be established based on the presence or absence of the 47-kDa protease.