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

Published online by Cambridge University Press:  01 June 2009

Juan I. Azcona ,
Rosario Martín ,
Miguel A. Asensio ,
Pablo E. Hernández  and
Bernabé Sanz
Juan I. Azcona
Affiliation:
Departamento de Higiene y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
Rosario Martín
Affiliation:
Departamento de Higiene y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
Miguel A. Asensio
Affiliation:
Departamento de Higiene y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
Pablo E. Hernández
Affiliation:
Departamento de Higiene y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
Bernabé Sanz
Affiliation:
Departamento de Higiene y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
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Summary

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.

Type
Original articles
Information
Journal of Dairy Research , Volume 55 , Issue 2 , May 1988 , pp. 217 - 226
Copyright
Copyright © Proprietors of Journal of Dairy Research 1988

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