Heat stable proteinase fromPseudomonas fluorescensAH-70: purification by affinity chromatography on cyclopeptide antibiotics

Abstract

A heat stable extracellular proteinase from the psychrotroph Pseudomonas fluorescens AH-70 was purified to electrophoretic homogeneity by affinity chromatography on a gramicidin S-Sepharose-4B column. Bacitracin linked to Sepharose-4B was unable to retain any proteolytic activity, whereas the same antibiotic bound to AH-Sepharose-4B retained ~ 25 % of the total activity. The purification procedure on the gramicidin S-Sepharose-4B column was easy to perform, fast and reproducible; it resulted in a 207-fold increase in the specific activity and a yield of 41 % of the original activity. The purified enzyme was a monomer with a mol. wt of 33000. The enzyme hydrolysed whole casein and its fractions whereas no activity was observed against bovine serum albumin. The enzyme was a metalloproteinase. It was heat stable, having D-values at 121, 135 and 150 °C of 3-8, 1-9 and 06 min respectively.

Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0

“…Under denaturing conditions, the purified enzyme had a molecular mass of 47 kDa according to analysis by SDS-PAGE (data not shown). These data indicate that the enzyme is similar to proteases characterized from other isolates of P. fluorescens [14,15,17,29,30].…”

A sandwich enzyme‐linked immunosorbent assay (ELISA) for detection of Pseudomcnas fluorescens and related psychrotrophic bacteria in refrigerated milk

“…The The Ps. Jluorescens strain AH-70 from Spanish refrigerated milk was previously isolated and identified in our laboratory (Azcona et al 1988). The micro-organisms were usually grown at 24°C in a medium containing (g I-' in distilled water): tryptone, 10; yeast extract, 10; NaCI, 5. isolation of outer membrane proteins from Pseudomonas spp.…”

Detection of Pseudomonas fluorescens and Related Psychrotrophic Bacteria in Refrigerated Meat by a Sandwich ELISA

“…The P.fluorescens strains DC-5, DC-7 and NT-19 from meat origin were supplied by B. G. Shaw, Food Research Institute, Bristol, United Kingdom. The P. fluorescens strain AH-70 was previously isolated and identified in our laboratory (3). The microorganisms were usually grown at 24°C in a medium containing 1% tryptone, I% yeast extract and 0.5% sodium chloride (NaCl) (Oxoid, Unipath, Ltd., Basingstoke, Hampshire).…”