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Purification and Identification of a Novel Leucine Aminopeptidase from Bacillus thuringiensis israelensis

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Abstract

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65°C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65°C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase’s effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.

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Acknowledgments

The authors thank Hen Friman, Hadas Friman, Sasi Sigawi, and Yafit Sigawi, students of the Department of Chemical Engineering and Biotechnology at the College of Judea and Samaria, for assistance with this research.

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Authors and Affiliations

  1. Department of Chemical Engineering and Biotechnology, College of Judea and Samaria, Ariel, 44837, Israel

    Rivka Cahan, Efrat Hetzroni & Marina Nisnevitch

  2. The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel

    Efrat Hetzroni & Yeshayahu Nitzan

Authors
  1. Rivka Cahan
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  2. Efrat Hetzroni
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  3. Marina Nisnevitch
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  4. Yeshayahu Nitzan
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Correspondence to Rivka Cahan.

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Cahan, R., Hetzroni, E., Nisnevitch, M. et al. Purification and Identification of a Novel Leucine Aminopeptidase from Bacillus thuringiensis israelensis . Curr Microbiol 55, 413–419 (2007). https://doi.org/10.1007/s00284-007-9004-9

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  • DOI: https://doi.org/10.1007/s00284-007-9004-9

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