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Biophysical characterization of a recombinant aminopeptidase II from the thermophilic bacterium Bacillus stearothermophilus

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Abstract

In the present study, the biophysical properties of His6-tagged Bacillus stearothermophilus aminopeptidase II (His6-tagged BsAmpII) are characterized in detail by gel-filtration, analytical ultracentrifugation, and various spectroscopic techniques. Using size-exclusion chromatography and analytical ultracentrifugation, we demonstrate that His6-tagged BsAmpII exists predominantly as a dimer in solution. The enzyme is active and stable at pHs ranging from 6.5 to 8.5. Far-UV circular dichroism analysis reveals that the secondary structures of His6-tagged BsAmpII are significantly altered in the presence of SDS, whereas the presence of 5–10% acetone and ethanol was harmless to the folding of the enzyme. Thermal unfolding of His6-tagged BsAmpII was found to be irreversible and led to the formation of aggregates. The native enzyme started to unfold beyond 0.6 M guanidine hydrochloride and had a midpoint of denaturation at 1.34 M. This protein remained active at concentrations of urea below 2.7 M but experienced an irreversible unfolding by >5 M denaturant. Taken together, this work lays a foundation for potential biotechnological applications of His6-tagged BsAmpII.

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Acknowledgements

The authors thank the anonymous reviewers for their valuable comments and suggestions to improve the quality of the manuscript. Financial support (NSC 100-2313-B-415-003-MY3) from the National Science Council of Taiwan is also acknowledged.

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Authors and Affiliations

  1. Department of Chemistry, National Cheng Kung University, Tainan City, 701, Taiwan

    Tzu-Fan Wang

  2. Institute of Molecular Biology, Academia Sinica, Nankang District, Taipei City, Taiwan

    Min-Guan Lin

  3. Department of Food Science and Technology, Hungkuang University, 34 Chungchie Road, Taichung City, 433, Taiwan

    Huei-Fen Lo

  4. Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City, 60004, Taiwan

    Meng-Chun Chi & Long-Liu Lin

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Wang, TF., Lin, MG., Lo, HF. et al. Biophysical characterization of a recombinant aminopeptidase II from the thermophilic bacterium Bacillus stearothermophilus . J Biol Phys 40, 25–40 (2014). https://doi.org/10.1007/s10867-013-9332-x

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  • DOI: https://doi.org/10.1007/s10867-013-9332-x

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