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Purification and biological characterization of a halophilic thermostable protease from Haloferax lucentensis VKMM 007

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Abstract

An extremely halophilic archaeon Haloferax lucentensis VKMM 007, isolated from a solar saltern, was found to produce a protease. This extracellular enzyme consisted of a single polypeptide chain of 57.8 kDa as determined by SDS–PAGE and was purified by a combination of ultrafiltration, bacitracin–Sepharose affinity chromatography and Sephadex G-100 gel filtration. The purified protein was stable in a wide range of temperatures (20–70°C), NaCl concentrations (0.85–5.13 M) and pH (5.0–9.0) with maximal activity observed at 60°C, 4.3 M NaCl and pH 8.0. Proteolytic activity was enhanced by Ca2+, K+, Mg2+, Na+, and Fe2+ ions and the protein was classified as a trypsin-like serine protease. Further assays indicated highest degree of specificity when hemoglobin was used as an enzyme substrate. Most importantly, the proteolytic activity remained stable or only marginally inhibited in the presence of various polar and non-polar solvents, surfactants and reducing agents thus emphasizing the biotechnological potential of this novel halophilic protease.

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Acknowledgments

This work was supported by a grant from Government of India Ministry of Science and Technology, Department of Biotechnology to Centre for Advanced studies in Botany, University of Madras (MM, VK) and Slovenian Research Agency research programme P1-0198 (LP).

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Authors and Affiliations

  1. Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, 600 025, India

    Muthu Manikandan & Vijayaraghavan Kannan

  2. Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000, Ljubljana, Slovenia

    Lejla Pašić

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  1. Muthu Manikandan
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  2. Lejla Pašić
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  3. Vijayaraghavan Kannan
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Correspondence to Lejla Pašić.

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Manikandan, M., Pašić, L. & Kannan, V. Purification and biological characterization of a halophilic thermostable protease from Haloferax lucentensis VKMM 007. World J Microbiol Biotechnol 25, 2247–2256 (2009). https://doi.org/10.1007/s11274-009-0132-1

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  • DOI: https://doi.org/10.1007/s11274-009-0132-1

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