Thermostable Esterase from a Thermoacidophilic Archaeon
2297
reported. A similar phenomenon was also forward in the
recombinant esterase from S. acidocadarius, reported by
Arpigny et al. The thermostability of the recombinant
enzyme appears to be very different from that of the
native esterase directly purified from S. acidocadar-
novel biocatalyst for the kinetic resolution of racemic
compounds. The carboxylesterase from S. solfataricus
P1 (Sso Est1) showed a specific reaction toward the S-
2
0)
2
5)
naproxen ester in co-solvent reaction conditions. But
Est3 displayed a different catalytic pattern toward an ꢀ-
arylpropionic methylester substrate, ketoprofen methyl-
ester, containing the same chiral center. The selectivity
toward substrates observed in previous literature might
be elucidated by the fact that the change in the side-
chain structure of the substrate leads to a drastic change
1
1,20)
ius.
The activity of a recombinant enzyme decreas-
ꢀ
ed to half at 80 C after 45 min while the native enzyme
did not lose activity for 1 h. The unstable characteristics
of recombinant enzymes expressed in E. coli might be
due to a lack of the unique mechanisms as compatible
solutes, heat inducible molecular chaperones, and post-
synthetic modifications for the thermostability of pro-
teins discovered in hyperthermophiles. Further studies of
the thermostability of Est3 are necessary to identify
2
6)
in reaction rate and enantioselectivity. On the basis of
the data presented here, we are now investigating the
enatioselectivity of this enzyme toward substrates and
reaction media such as organic solvents.
ꢀ
accurate mechanisms at high temperatures above 80 C.
A substrate profile test of Est3 showed relative
activity above 50% toward substrates with medium acyl
chain lengths (C5–C10). This suggests that this enzyme
is a typical esterase with broad substrate specificity. This
characteristic of Est3, which has relatively high activity
against medium chain ester substrates, is similar to those
of esterases reported in prokaryotes, Bacillus lichen-
iformis,2 and B. stearothermophilus. This feature of
Est3 might be due to a hydrophobic binding pocket
which might be suitable for binding the substrates of
short or medium chains at the active site.
References
1
)
Junge, W., Esterase. In ‘‘Methods of Enzymatic Analy-
sis’’ 3rd ed. Vol. 4, ed. Bergmeyer, H. U., Verlag
Chemie, Weinheim, pp. 1–143 (1984).
2
)
Chen, Y.-R., Usui, S., Queener, S. W., and Yu, C.-A.,
Purification and properties of a p-nitrobenzyl esterase
from Bacillus subtilis. J. Ind. Microbiol. Biot., 15, 10–18
(1995).
1)
22)
3) Broekhizen, C. P., and Quax, W. J., Development of a
new Bacillus carboxyl esterase for use in the resolution
of chiral drugs. Appl. Microbiol. Biotechnol., 41, 425–
431 (1994).
Esterases can be classified based on substrate specific-
1
1,23,24)
ity and sensitivity toward various inhibitors.
4
)
Demirjian, D. C., Moris-Varas, F., and Cassidy, C. S.,
Enzymes from extremophiles. Curr. Opin. Chem. Biol.,
Although this categorization has limitations due to
overlapping substrate specificities or inhibitor patterns,
this classification is generally used to characterize the
biochemical properties of esterases. Phenylmethylsul-
fonyl fluroride (PMSF) and diethyl p-nitrophenyl phos-
phate (paraoxon) inhibited Est3 activity in the above
experiment. This suggests that serine residue might be
involved at the catalytic site of the enzyme. Activity was
also inhibited by mercuric chloride, indicating that
sulfide (SH) groups are involved in the enzyme activity.
Diethylpyrocarbonate or eserine, which modify histidine
residue, did not significantly affect enzyme activity.
EDTA also did not nearly inhibit the enzymatic activity,
and hence non-metals might not be involved in the
catalytic mechanism of Est3. On the basis of these
inhibition patterns, the Est3 esterase from S. solfataricus
can be classified as a B-type esterase, carboxylesterase
5, 144–151 (2001).
5
)
Niehaus, N., Bertoldo, C., K a¨ hler, M., and Antranikian,
G., Extremophiles as a source of novel enzymes for
industrial application. Appl. Microbiol. Biotechnol., 51,
7
11–729 (1999).
6) Sellek, G. A., and Chaudhuri, J. B., Biocatalysis in
organic media using enzymes from extremophiles.
Enzyme Microb. Technol., 25, 471–482 (1999).
7
)
Hough, D. W., and Danson, M. J., Extremozymes. Curr.
Opin. Chem. Biol., 3, 39–46 (1999).
8
)
Manco, G., Giosu e´ , E., D’Auria, S., Herman, P., Carrea,
G., and Rossi, M., Cloning, overexpression, and proper-
ties of a new thermophilic and thermostable esterase
with sequence similarity to hormone-sensitive lipase
subfamily from the archaeon Archaeoglobus fulgidus.
Arch. Biochem. Biophys., 373, 182–192 (2000).
9
)
Ikeda, M., and Clark, D. S., Molecular cloning of an
extremely thermostable esterase gene from hyperther-
mophilic archaeon Pyrococcus fuiosus in Escherichia
coli. Biotechnol. Bioeng., 57, 624–629 (1998).
(EC 3.1.1.1).
The Est3 esterase showed higher stability toward
relatively high concentrations of solvents or detergents
compared with other esterases. In particular, the resist-
ance of Est3 to mild detergents or polar solvents is a
very attractive property for use as a biocatalyst in
organic media for the synthesis of chiral compounds. It
suggests the possibility that the catalytic properties of
Est3 might be improved by reaction media engineering.
In the above experimental data on kinetic resolution
of ꢀ-arylpropionic acid, the enzyme hydrolyzed the (R)-
ester of racemic ketoprofen methylester. These data led
us to consider the enzyme from S. solfataricus as a
1
1
1
0) Hotta, Y., Ezaki, S., Atomi, H., and Imanaka, T.,
Extremely stable and versatile carboxylesterase from a
hyperthermophilic archaeon. Appl. Environ. Microbiol.,
6
8, 3925–3931 (2002).
1) Sobek, H., and G o¨ risch, H., Purification and character-
ization of a heat-stable esterase from the thermoacido-
philic archaebacterium Sulfolobus acidocaldarius. Bio-
chem. J., 250, 453–458 (1988).
2) Huddleston, S., Yallop, C. A., and Charalambous, B. M.,
The identification and partial characterization of a novel
inducible extracellular thermostable esterase from the