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Highly efficient renaturation of β‐lactamase isolated from moderately halophilic bacteria
Author(s) -
Tokunaga Hiroko,
Ishibashi Matsujiro,
Arakawa Tsutomu,
Tokunaga Masao
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)01508-4
Subject(s) - halophile , denaturation (fissile materials) , enzyme , circular dichroism , bacteria , biochemistry , chemistry , boiling , enzyme assay , chromatography , biology , nuclear chemistry , organic chemistry , genetics
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.