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Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes

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Abstract

A protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa MN7. The strain was found to produce proteases when it was grown in media containing only shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. The use of 60 g/l SWP resulted in a high protease production. Elastase, the major protease produced by P. aeruginosa MN7, was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration, and ultrafiltration using a 10-kDa cut-off membrane, with a 5.2-fold increase in specific activity and 38.4% recovery. The molecular weight of the purified elastase was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for protease activity were 60°C and 8.0, respectively. The activity of the enzyme was totally lost in the presence of ethylene glycol tetraacetic acid, suggesting that the purified enzyme is a metalloprotease. The purified enzyme was highly stable in the presence of organic solvents, retaining 100% of its initial activity after 60 days of incubation at 30°C in the presence of dimethyl sulfoxide and methanol. The lasB gene, encoding the MN7 elastase, was isolated and its DNA sequence was determined.

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Acknowledgment

This work was supported by grants from the « Ministère de l’Enseignement Supérieur, de la Recherche Scientifique et de la Technologie, Tunisie ».

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Authors and Affiliations

  1. Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d’Ingénieurs de Sfax, B.P. « W » 3038, Sfax, Tunisia

    Kemel Jellouli, Ahmed Bayoudh, Laila Manni, Rym Agrebi & Moncef Nasri

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  1. Kemel Jellouli
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  2. Ahmed Bayoudh
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  3. Laila Manni
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  4. Rym Agrebi
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  5. Moncef Nasri
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Correspondence to Kemel Jellouli.

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Kemel Jellouli and Ahmed Bayoudh contributed equally to this work.

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Jellouli, K., Bayoudh, A., Manni, L. et al. Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes. Appl Microbiol Biotechnol 79, 989–999 (2008). https://doi.org/10.1007/s00253-008-1517-z

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  • DOI: https://doi.org/10.1007/s00253-008-1517-z

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