115NISR RESEARCH GRANT
2003 Research Grant

Development of Highly Soluble Enzymes Adopting the Structural Characteristics of Halophilic Enzymes
Masao TOKUNAGA
Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University
I. Research aims:
Some specific microorganisms can grow under extreme
environmental conditions, such as high temperature, extreme pH and high salt concentrations. We have attempted
to characterize halophilic enzymes, aiming at expansion of
their industrial applications. Extremely halophilic archaea
and moderately halophilic bacteria require NaCl above 2.5
M and 0.5 M for growth, respectively and accumulate highconcentrations of ions and compatible solutes inside thecells. These enzymes can function under the extreme conditions where most of the “normal” enzymes cannot, whichmakes industrial applications of these enzymes very attractive. The strategy of halophilic enzymes to adapt high saltconditions is a large excess content of acidic amino acids intheir amino acid composition. These acidic amino acidresidues are localized on the protein surface and contributeto bind excess amounts of water molecules and cationicions. We have studied the properties of nucleoside diphosphate kinases (HsNDK) from extremely halophilic archaea,
Halobacterium salinarum, and moderately halophilic bacteria, Halomonas sp., and b -lactamase (BLA), porin, and
HrdC from moderately halophilic bacteria. We propose thatthe highly acidic molecular nature of halophilic proteins
generates a high solubility without aggregation, and ensures
the several interesting characteristics of halophilic proteins,
such as highly efficient renaturation after denaturation treatment. We are now developing the applications of this interesting “halophilic nature” to microbiological industries andmolecular biology.
II. Methods & Results
1. Nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaea, H. salinarum.
Most halophilic enzymes isolated from extremely
halophilic archaea generally require at least 12 M NaClfor their activity and stability. However, we have observed
for the first time that HsNDK isolated from H. salinarum isstable and active without NaCl and then cloned ndk genefor its further characterization. Its amino acid composition
deduced from the nucleotide sequence indicates high content of acidic amino acid residues, typical characteristics ofhalophilic proteins. The ndk gene was cloned into E. coli
expression vector (pET3a) and was expressed in E. coli.
The HsNDK protein expressed in E. coli was localized insoluble fraction, but in an inactive form. This inactive

HsNDK was activated (folded) properly by dialysis against
4 M NaCl, and once folded under high salt conditions,HsNDK was stable and active even without NaCl, having
the same properties as the native enzyme.
The native enzyme purified from H. salinarum was denatured by heat-treatment at 90°C or 6 M urea, and its renaturation (refolding) was examined in the presence of varioussolutes, such as salts and a compatible solute, trimethylamine N-oxide (TMAO). Denatured HsNDK was refoldedin the presence of either high concentration of salts orTMAO. These results indicate that HsNDK does not require
high salt concentrations for its stability and activity, but requires high salts for its native refolding. TMAO is not anelectrolyte, meaning that the presence of concentrated salts
is not an absolute requirement and hence that charge shielding or ion binding is not a sole factor for the folding and activation of HsNDK. TMAO is a strong protein structure stabilizer and forces the conformation equilibrium to a morecompact structure, suggesting that increasing hydrophobic
interactions alone can induce refolding of HsNDK.
To further characterize this enzyme, we have attempted
to express HsNDK in E. coli with hexa-His-tag. When an
extra sequence containing His-tag (derived from pET15bvector) was fused to the N-terminus of HsNDK, unexpectedly the fusion resulted in a fully active enzyme with increased expression. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. Several lines of evidence suggest that
positive charges in fused N-terminal extension are effective
in suppressing the negative charge repulsion of halophilic
enzyme and thus, facilitate folding and assembly ofHsNDK. These observations open a prospect that small
modification of halophilic protein structures can change
their halophilic properties, and thus generate novel molecules from halophilic enzymes for biological industries.
2. Highly efficient renaturation of b -lactamase isolated
from moderately halophilic bacteria.
Most b -lactamases reported to date are irreversibly denatured at 6070°C. Here, we found that a halophilic b -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 minin the presence of 0.2 M NaCl, suggesting that the protein
either incompletely denatures during the boiling process orreadily renatures upon cooling to the assay temperature.
Circular dichroism showed a complete unfolding at 60°C

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and its full reversibility, indicating that the observed activity after boiling is due to efficient refolding following heatdenaturation. The gene bla was cloned, and the primarystructure of the enzyme deduced from the gene sequence
showed highly abundant acidic amino acid residues, one of
the characteristics of halophilic proteins. These observations suggest that the highly acidic molecular nature of
halophilic proteins generates a high solubility without aggregation, and ensures highly efficient renaturation after
heat or urea treatment. We are now developing an “application” of this protein to molecular biology.

III. References
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Ishibashi, M. et al., FEBS Lett. 493, 134–138 (2001)
Ishibashi, M. et al., FEMS Microbiol. Lett. 216, 235–241 (2002)
Ishibashi, M. et al., J. Protein Chem. 22, 345–351 (2003)
Ishibashi, M. et al., Protein Pept. Lett. 10, 575–580 (2003)
Tokunaga, H. et al., FEBS Lett. 558, 7–12 (2004)
Ishibashi, M. et al., FEBS Lett. 570, 87–92 (2004)
Tokunaga, H. et al., Appl. Environ. Microbiol. 70, 4424–4431 (2004)
Arakawa, T. & Tokunaga, M. Protein Pept. Lett. 11, 125–132 review(2004)
9. Tokunaga, M. et al., Seikagaku (2004) Dec. issue, review, in press