Highly efficient renaturation of beta-lactamase isolated from moderately halophilic bacteria.
Most, if not all, beta-lactamases reported to date are irreversibly denatured at 60-70 degrees C. Here, we found that a halophilic beta-lactamase from the moderately halophilic bacterium Chromohalobacter sp. 560 was highly stable against heat inactivation: it retained approximately 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 degrees 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 degrees 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.
Tokunaga
Hiroko
H
Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan. tokunaga@chem.agri.kagoshima-u.ac.jp
Ishibashi
Matsujiro
M
Arakawa
Tsutomu
T
Tokunaga
Masao
M
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D018698:Glutamic Acid / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006216:Halobacteriaceae / Q000201:enzymology*; D044129:Halomonadaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D001618:beta-Lactamases / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D012965:Sodium Chloride; D005982:Glutathione Transferase; D001618:beta-Lactamases