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Biophysical characterization of a recombinant leucyl aminopeptidase from Bacillus kaustophilus

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Abstract

The biophysical properties of Bacillus kaustophilus leucyl aminopeptidase (BkLAP) were examined in terms of analytical ultracentrifugation, fluorescence spectroscopy, and circular dichroism. By using the analytical ultracentrifuge, we demonstrated that tetrameric BkLAP exists as the major form in solution at protein concentration of 1.5 mg/ml at pH 8.0. The native enzyme started to unfold beyond ∼1 M GdnHCl and reached an unfolded intermediate with [GdnHCl]1/2 at 1.8 M. Thermal unfolding of BkLAP was found to be highly irreversible and led to a marked formation of aggregates.

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Abbreviations

BkLAP:

B. kaustophilus LAP

CD:

circular dichroism

GdnHCl:

guanidine hydrochloride

LAP:

leucyl aminopeptidase

L-Leu-p-NA:

L-leucine-p-nitroanilide

Ni2+-NTA:

nickel nitrilotriacetate

p-NA:

p-nitroaniline

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Authors and Affiliations

  1. Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, Taiwan

    Meng-Chun Chi, Hui-Ping Chang & Long-Liu Lin

  2. Department of Life Sciences and Institute of Genome Science, National Yang-Ming University, Taipei, Taiwan

    Gu-Gang Chang

  3. Department of Life Sciences and Institute of Molecular Biology, National Chung Cheng University, Chiayi, Taiwan

    Tzu-Fan Wang & Hsien-Bin Huang

Authors
  1. Meng-Chun Chi
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  2. Hui-Ping Chang
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  3. Gu-Gang Chang
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  5. Hsien-Bin Huang
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Correspondence to Long-Liu Lin.

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Chi, MC., Chang, HP., Chang, GG. et al. Biophysical characterization of a recombinant leucyl aminopeptidase from Bacillus kaustophilus . Biochemistry Moscow 75, 642–647 (2010). https://doi.org/10.1134/S0006297910050159

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  • DOI: https://doi.org/10.1134/S0006297910050159

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