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A novel extracellular subtilisin-like protease from the hyperthermophile Aeropyrum pernix K1: biochemical properties, cloning, and expression

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Abstract

A novel extracellular serine protease designated Pernisine was purified to homogeneity and characterized from the archaeon Aeropyrum pernix K1. The molecular mass, estimated by SDS-PAGE analysis and by gel filtration chromatography, was about 34 kDa suggesting that the enzyme is monomeric. Pernisine was active in a broad range of pH (5.0–12.0) and temperature (60–120 °C) with maximal activity at 90 °C and between pH 8.0 and 9.0. In the presence of 1 mM CaCl2 the activity, as a function of the temperature, reached a maximum at 90 °C but at 120 °C the enzyme retained almost 80% of its maximal activity. Activity inhibition studies suggest that the enzyme is a serine metalloprotease and biochemical data indicate that Pernisine is a subtilisin-like enzyme. The protease gene, identified from the sequenced genome of A. pernix, was amplified from total genomic DNA by PCR technique to construct the expression plasmid pGEX-Pernisine. The Pernisine, lacking the leader sequence, was expressed in Escherichia coli BL21 strain as a fusion protein with glutathione-S-transferase. The biochemical properties of the recombinant enzyme were found to be similar to those of the native enzyme.

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Acknowledgements

This work was supported by the U E grant number BIO 4-98-6065.

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Authors and Affiliations

  1. Institute of Protein Biochemistry, CNR, Via Marconi 10, 80125, Naples, Italy

    G. Catara, G. Ruggiero, F. La Cara, F. A. Digilio, A. Capasso & M. Rossi

  2. Department of Biological Chemistry, University of Naples Federico II, Via Mezzocannone 16, 80134, Naples, Italy

    M. Rossi

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  1. G. Catara
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  2. G. Ruggiero
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  3. F. La Cara
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  4. F. A. Digilio
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  5. A. Capasso
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  6. M. Rossi
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Correspondence to M. Rossi.

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Communicated by G. Antranikian

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Catara, G., Ruggiero, G., La Cara, F. et al. A novel extracellular subtilisin-like protease from the hyperthermophile Aeropyrum pernix K1: biochemical properties, cloning, and expression. Extremophiles 7, 391–399 (2003). https://doi.org/10.1007/s00792-003-0337-4

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  • DOI: https://doi.org/10.1007/s00792-003-0337-4

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