Purification and Characterization of an Extracellular Protease from the Fish Pathogen Yersinia ruckeri and Effect of Culture Conditions on Production

Abstract

A novel protease, hydrolyzing azocasein, was identified, purified, and characterized from the culture supernatant of the fish pathogenYersinia ruckeri. Exoprotease production was detected at the end of the exponential growth phase and was temperature dependent. Activity was 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 chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein indicated an estimated molecular mass 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°C and had an optimum activity at 37°C. The activation energy for the hydrolysis of azocasein was determined to be 15.53 kcal/mol, and the enzyme showed a rapid decrease in activity at 42°C. The enzyme had an optimum pH of around 8. Characterization of the protease showed that it required certain cations such as Mg2+ or Ca2+ for maximal activity and was inhibited by EDTA, 1,10-phenanthroline, and EGTA but not by phenylmethylsulfonyl fluoride. TwoN-methyl-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 protein. Azocasein activity, SDS-PAGE, immunoblotting by using polyclonal anti-47-kDa-protease serum, and zymogram analyses showed that protease activity was present in 8 of 14 strains tested and that two Y. ruckeri groups could be established based on the presence or absence of the 47-kDa protease.

Modelling and optimization of fermentation factors for enhancement of alkaline protease production by isolated Bacillus circulans using feed-forward neural network and genetic algorithm

“…These results suggest that fermentation parameters greatly influence the extracellular production of proteases in this microbial strain and their interaction plays an important role in the synthesis of this enzyme because of their regulatory role in the induction or repression of the enzyme production. A similar catabolic control mechanism for extracellular enzyme production has been described for Pseudomonas maltophilia (Boethling 1975), Yersinia ruckeri (Secades and Guijarro 1999) and Pseudoalteromonas sp. SM9913 (Li et al.…”

Profiling acylated homoserine lactones in Yersinia ruckeri and influence of exogenous acyl homoserine lactones and known quorum-sensing inhibitors on protease production

“…The last four AHLs detected were C8‐HSL, C6‐HSL, N ‐(3‐hydroxyoctanoyl)‐homoserine lactone (3‐hydroxy‐C8‐HSL) and N ‐(3‐oxododecanoyl)‐homoserine lactone (3‐oxo‐C12‐HSL). Further experiments on exoprotease production was done using Y. ruckeri strain 150 as this was typical of the group and has been used in other studies of virulence factor production (Secades and Guijarro 1999; Fernández et al. 2002).…”

“…It has previously been shown that protease production of Y. ruckeri is temperature‐dependent with high protease production at 4, 18 and 20°C and low or no caseinolytic activity at 28°C (Secades and Guijarro 1999; Fernandez et al. 2003).…”

“…1990), siderophores (Fernández et al. 2004) and the exoprotease Yrp1 (Secades and Guijarro 1999; Fernández et al. 2002).…”

The detrimental impact of extracellular bacterial proteases on wound healing

“…However, a fortuitous by-product of protease production for the microorganism is the degradation of host proteins, growth factors and receptors, which can impede the immune response and contribute towards tissue degradation, enabling further microbial dissemination into the underlying soft tissue (19,23,(30)(31)(32)(33). Arguably, microbial proteases are considered to be among the most important type of microbial virulence factor influencing wound healing (20,34,35).…”