Biophysical Characterization of a Recombinant Leucyl Aminopeptidase from Bacillus kaustophilus

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Meng-Chun Chi¹, Hui-Ping Chang¹, Gu-Gang Chang², Tzu-Fan Wang³, Hsien-Bin Huang³, and Long-Liu Lin^1*

¹Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, Taiwan; fax: +886(5)271-7901; E-mail: llin@mail.ncyu.edu.tw

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

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

^* To whom correspondence should be addressed.

Received August 26, 2009; Revision received January 26, 2010

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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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KEY WORDS: Bacillus kaustophilus, leucyl aminopeptidase, analytical ultracentrifuge, thermal unfolding, chemical denaturation

DOI: 10.1134/S0006297910050159

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