Highly efficient renaturation of β‐lactamase isolated from moderately halophilic bacteria

FEBS Letters

Arakawa, Tsutomu²  Tokunaga, Hiroko¹  Ishibashi, Matsujiro¹  Tokunaga, Masao¹ 

[1] Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan;Alliance Protein Laboratories, 3957 Corte Cancion, Thousand Oaks, CA 91360, USA

关键词: Halophilic enzyme;    Halophilic bacterium;    β-Lactamase;    Heat stability;    Renaturation;    Refolding;   

DOI  :  10.1016/S0014-5793(03)01508-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Most, if not all, β-lactamases reported to date are irreversibly denatured at 60–70°C. Here, we found that a halophilic β-lactamase from the moderately halophilic bacterium Chromohalobacter sp. 560 was highly stable against heat inactivation: it retained ∼75% of its activity after boiling for 5 min in the presence of 0.2 M NaCl, suggesting that the protein either incompletely denatures during the boiling process or readily renatures upon cooling to the assay temperature. Circular dichroism showed a complete unfolding at 60°C and a full reversibility, indicating that the observed activity after boiling is due to efficient refolding following heat denaturation. The enzyme showed optimal activity at 50–60°C, indicating that an increase in activity with temperature offsets the thermal denaturation. The gene bla was cloned, and the primary structure of the enzyme was deduced to be highly abundant in acidic amino acid residues, one of the characteristics of halophilic proteins. Despite its halophilic nature, the enzyme refolds in low salt media after heat denaturation.

【 授权许可】

Unknown