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Heat stable proteinase from Pseudomonas fluorescens AH-70: purification by affinity chromatography on cyclopeptide antibiotics

  • Juan I. Azcona (a1), Rosario Martín (a1), Miguel A. Asensio (a1), Pablo E. Hernández (a1) and Bernabé Sanz (a1)...


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 0·6 min respectively.



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