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Biochemistry (Moscow)

, Volume 75, Issue 5, pp 642–647 | Cite as

Biophysical characterization of a recombinant leucyl aminopeptidase from Bacillus kaustophilus

  • Meng-Chun Chi
  • Hui-Ping Chang
  • Gu-Gang Chang
  • Tzu-Fan Wang
  • Hsien-Bin Huang
  • Long-Liu LinEmail author
Article

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.

Key words

Bacillus kaustophilus leucyl aminopeptidase analytical ultracentrifuge thermal unfolding chemical denaturation 

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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Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • Meng-Chun Chi
    • 1
  • Hui-Ping Chang
    • 1
  • Gu-Gang Chang
    • 2
  • Tzu-Fan Wang
    • 3
  • Hsien-Bin Huang
    • 3
  • Long-Liu Lin
    • 1
    Email author
  1. 1.Department of Applied ChemistryNational Chiayi UniversityChiayiTaiwan
  2. 2.Department of Life Sciences and Institute of Genome ScienceNational Yang-Ming UniversityTaipeiTaiwan
  3. 3.Department of Life Sciences and Institute of Molecular BiologyNational Chung Cheng UniversityChiayiTaiwan

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