Preparation of a very stable immobilized Solanum tuberosum epoxide hydrolase

Article abstract of DOI:10.1016/j.tetasy.2007.05.008

The covalent immobilization of the Solanum tuberosum epoxide hydrolase (StEH) was explored using highly activated Sepabeads-epoxy or Glyoxyl-agarose based supports. A Glyoxyl-agarose immobilizate, prepared under optimized experimental conditions, led to a material exhibiting excellent thermal and chemical stability. The key step of this immobilization process was the use of 164 kDa dextran as an additive during immobilization, which prevented the enzyme from inactivation at the high pH (pH 10) necessarily used for performing this immobilization. This afforded a Glyoxyl-agarose-StEH immobilizate with 80% initial enzymatic activity retention and a stabilization factor of at least 300 at 60 °C, as compared to the free enzyme. The high enantio- and regio-selectivity properties of this novel biocatalyst were shown to be nearly identical to those of the free enzyme.

Full text of DOI:10.1016/j.tetasy.2007.05.008

Tetrahedron: Asymmetry 18 (2007) 1233–1238
Preparation of a very stable immobilized Solanum tuberosum
epoxide hydrolase
a
b
a,
*
Cesar Mateo, Roberto Fernandez-Lafuente, Alain Archelas,
b,
a
*
Jose M. Guisan and Roland Furstoss
a
Groupe Biocatalyse et Chimie Fine, FRE CNRS 3005, Universit e´ de la M e´ diterran e´ e, Facult e´ des Sciences de Luminy,
Case 901, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France
b
Departamento de Biocat a´ lisis, Instituto de Cat a´ lisis (CSIC), Campus UAM, Cantoblanco, 28049 Madrid, Spain
Received 17 April 2007; accepted 8 May 2007
Abstract—The covalent immobilization of the Solanum tuberosum epoxide hydrolase (StEH) was explored using highly activated Sepa-
beads-epoxy or Glyoxyl-agarose based supports. A Glyoxyl-agarose immobilizate, prepared under optimized experimental conditions,
led to a material exhibiting excellent thermal and chemical stability. The key step of this immobilization process was the use of
1
64 kDa dextran as an additive during immobilization, which prevented the enzyme from inactivation at the high pH (pH 10) necessarily
used for performing this immobilization. This afforded a Glyoxyl-agarose-StEH immobilizate with 80% initial enzymatic activity reten-
tion and a stabilization factor of at least 300 at 60 °C, as compared to the free enzyme. The high enantio- and regio-selectivity properties
of this novel biocatalyst were shown to be nearly identical to those of the free enzyme.
Ó 2007 Elsevier Ltd. All rights reserved.
1
. Introduction
ee’ goal starting from a racemic mixture. In this context,
some of us have previously described the surprising enan-
tioconvergent behavior of the recombinant plant Solanum
tuberosum epoxide hydrolase (StEH) on differently substi-
Due to the recent release of new FDA marketing
guidelines, biotechnological processes, which are generally
recognized as being environmentally safe since they avoid
production of potentially toxic waste in industrial pro-
cesses, are becoming increasingly important. This in partic-
ular applies to the synthesis of enantiomerically pure
4
,5
tuted styrene oxide derivatives (Scheme 1).
Immobilization of the biocatalyst may constitute as
another step further as far as process cost aspects are
concerned. Hence, with enantioconvergent single enzyme
processes still being very scarce, we pursued the exploration
of the potential of this highly interesting EH and, in partic-
ular, of the possibility to efficiently immobilize it on various
supports. To date, only one study has been reported
regarding this question, which led to poor to moderate
success (only 10% of recovered activity and a stabilization
1
pharmacological building blocks or end products. We
and others have previously amply demonstrated that the
preparation of enantiomerically pure epoxides and/or of
their corresponding vicinal diols, which are very attractive
chirons, can be efficiently performed by running a hydro-
lytic kinetic resolution of a given racemic epoxide. This
implies the use of dedicated enzymes—that is epoxide
hydrolases (EHs), such as the commercially available
2
6
factor of about 7 at 52 °C). Obviously, a more efficient
3
Aspergillus niger epoxide hydrolase (EC 3.3.2.3). An even
immobilization protocol allowing us to obtain higher enzy-
matic activity recovery and improved stability still remains
more stringent industrial demand, triggered by the neces-
sity to set up as cost effective as possible processes, relates
to the possibility to run so-called enantioconvergent strate-
gies, which allow approaching the ideal ‘100% yield 100%
7
–11
highly desirable. As previously documented,
the use of
adequate supports and experimental conditions permitting
the multipoint covalent attachment of an enzyme to a
highly activated support via short spacer arms, has in some
cases been reported to increase enzyme stability. This is
essentially due to the fact that the residues involved in
the immobilization process could preserve their relative
*
Corresponding authors. Tel.: +33 (0)4 91 82 91 58; fax: +33 (0)4 91 82 91
5 (A.A.); e-mail: archelas@luminy.univ-mrs.fr
4
0957-4166/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.05.008

1234
C. Mateo et al. / Tetrahedron: Asymmetry 18 (2007) 1233–1238
HO
(R)
OH
O
O
HO
(R)
OH
(
R)
StEH
StEH
+
resolution
process
enantioconvergent
process
X
X
X
X
rac
X= H, Cl
Scheme 1.
position nearly unaltered during any conformational
change promoted by heat, solvents or any other distorting
modification of the epoxy groups with ethylene diamine
1
7
as previously described) at pH 7 led to almost total activ-
ity disappearance in the supernatant. However, nearly no
activity was recovered on the support either, suggesting
that the enzyme was inactivated after reaction with the sup-
port. Better results were obtained using the Sepabeads-EP-
Cu support (partial modification of the epoxy groups with
1
2,13
agent.
For instance Glyoxyl-agarose (GA), a support
bearing aldehyde functions on its surface, was shown to
direct immobilization of an enzyme to the areas of the
protein bearing the highest primary amino group density
1
4,15
and, as a consequence, to allow high stabilization.
1
7
iminodiacetic acid followed by incubation with CuSO4)
Herein, we explored the possibility to immobilize the StEH
onto different such supports and describe our results
related to the activity, stability, and selectivity of the thus
obtained materials.
at pH 7, where almost 70% of immobilized activity was
recovered. In order to allow further reaction of the enzyme
residues with the epoxide moieties born by this support, the
already immobilized enzyme was re-incubated at pH 9 for
an additional 24 h period. However, the activity then
decreased to about 40%, thus suggesting some enzyme-
support reaction.
2
. Results and discussion
Glyoxyl-agarose supports (GA) are very suitable for pro-
viding multipoint and non-distorting covalent immobiliza-
tion of enzymes, but the immobilization protocol requires
the reaction to be performed at about pH 10 in order to
have enough unprotonated primary amino residues during
2
.1. Immobilization of the StEH onto different supports
Four types of supports were prepared starting either from
commercially available Sepabeads-epoxy support (Sepa-
bead-EP) or from an Agarose based support, and were
used to conduct preliminary experiments aimed at deter-
mining the most promising material. Our results are sum-
marized in Table 1. The amount of immobilization was
estimated by measuring the enzymatic activity remaining
in the aqueous solution after incubation of the enzyme
solution with the support. The amount of enzymatic
activity recovered on the support was measured using our
previously described methodology (UV spectrometry test
against styrene oxide, based on the oxidation of the formed
diol to benzaldehyde using sodium periodate as chemical
1
2,14
the first immobilization process.
Incubation of StEH
with this support at pH 10 led to a considerable loss of
enzymatic activity, with only about 10% being recovered
on the immobilizate. This can be explained by the fact that
the StEH is deactivated in less than 1 h at this pH, at 25 °C.
Even at 4 °C the activity rapidly decreased at this pH.
Very satisfactorily however the same immobilization proce-
dure, conducted using a solution of enzyme containing an
initial 20% (w/v) amount of 164 kDa dextran as addi-
1
8–21
1
6
tive,
on the support after only 1 h incubation, at 4 °C and pH
0, indicating that dextran did efficiently protect the
enzyme from any pH induced inactivation.
led to nearly full recovery of enzymatic activity
oxidant). The results obtained clearly appeared to depend
on the nature of the support.
1
Incubation of the StEH with (commercially available)
hydrophobic Sepabeads-epoxy support (moderately hydro-
phobic polymethacrylate resin containing oxirane groups)
in 1 M sodium phosphate at pH 7 led to nearly total recov-
ery of the enzyme activity in the supernatant, indicating
that no noticeable immobilization occurred. Conversely,
incubation with Sepabeads-EP-amino supports (partial
2.2. Stability of the different immobilizates
An additional parameter to be determined when preparing
such immobilizates is the thermal stability of the material
obtained. The soluble StEH is a reasonably stable enzyme,
its half-life at 60 °C and pH 7 being about 30 min. The sta-
bility of the above prepared immobilizates was therefore
tested using identical experimental conditions (Fig. 1).
The enzymatic activity of the Sepabeads-EP-Cu immobiliz-
ate appeared to be slightly more stable than that of the sol-
uble enzyme and the stabilization factor was even higher
when the immobilized enzyme preparation was re-incu-
bated at pH 9 before the epoxy blocking step, suggesting
an increase of the number of covalent enzyme-support
bonds. The stability reached using these protocols was
Table 1. Immobilization of StEH on different supports
Support
Immobilized
enzyme (%)
Recovered
activity (%)
Sepabead EP
Sepabead-EP-Cu
Sepabead-EP-NH
0
100
100
100
0
70
< 10
>95
2
a
Glyoxyl-agarose (GA)
a
Immobilization carried out in the presence of 20% of 164 kDa dextran.

C. Mateo et al. / Tetrahedron: Asymmetry 18 (2007) 1233–1238
1235
lized protein on Glyoxyl-agarose.24 Our results indicated
that the enzymatic stability of the thus obtained immobiliz-
ate was strongly dependent on the incubation conditions,
as illustrated in Figure 2.
Thus, in two separate experiments, the enzyme was first
incubated at 4 °C for 20 or 180 min. An additional experi-
ment was conducted by first incubating the enzyme at 4 °C
for 180 min, and then for an additional 3 h period at 25 °C.
This last experiment led to a very interesting result, since it
allowed an activity recovery as high as 80% (i.e., about
23 U per gram of support) and to an excellent (thermal)
stabilization factor of at least 300.
2.4. Enantio- and regio-selectivity of the GA immobilizate
Contrary to most enzymatic mechanisms, which are intrin-
sically unambiguous as far as their regioselectivity is con-
cerned, the epoxide hydrolase catalyzed ring opening of
an oxirane ring may imply different regioselectivities, due
to the possible attack of either carbon atom of the oxirane
ring by the nucleophylic enzyme residue. As a consequence,
the stereochemical outcome of such a reaction will be
strongly governed by the intimate mechanism of a given
epoxide hydrolase, which partly implies the optimized posi-
tioning of the substrate into its active site. Some of us have
previously proposed to quantify this behavior by using the
Figure 1. Thermal stability of the enzyme immobilized on different
supports. The different preparations were incubated at pH 7 and 60 °C.
Circles: soluble enzyme; rhombus: enzyme immobilized on Sepabeads-EP-
Cu at pH 7 during 24 h; triangles: enzyme immobilized on Sepabeads-EP-
Cu at pH 7 during 24 h and re-incubated during 24 h at pH 9; squares:
enzyme immobilized on Glyoxyl-agarose support (GA).
about 10-fold as compared to the soluble enzyme. Even
better results were obtained using the GA support for
which a stabilization factor of about 20 was observed
2
5
(
half-life about 10 h).
so-called regioselectivity coefficients a(S) and a(R). Inter-
estingly enough, these coefficients may vary from one enan-
tiomer to the other of a given racemic epoxide, which in
fact is the fundamental reason of the enantioconvergency
observed in the StEH catalyzed hydrolysis of various sty-
2
.3. Optimization of the GA immobilizate stability
Owing to this promising result, we decided to improve the
experimental conditions for the preparation of an optimal
GA immobilizate. Indeed, more finely tuned experimental
conditions could allow us to increase the number of cova-
5
rene oxide derivatives. Therefore, creating covalent bonds
between an enzyme and a given support could eventually,
due to discrete deformation of the enzyme in its immobi-
lized form, alter the positioning of each enantiomer in
the active site and, as a consequence, modify the stereo-
and regiochemical outcome of the reaction. Therefore, a
comparative biohydrolysis between the free StEH enzyme
and its GA immobilizate using rac-styrene oxide as sub-
strate were performed. The results (Fig. 3) indicated that,
in both experiments (a) the (S)-epoxide was preferentially
hydrolyzed to the (R)-diol, (b) the (recovered) less reactive
enantiomer was the (R)-epoxide, (c) the ee of this epoxide
2
2,23
lent bonds of the StEH to the support.
Some of us have
recently showed a direct correlation between the number of
enzyme-support linkages and the stability of an immobi-
(
(
ee ) reached a value of nearly 100% after about 90 min,
s
d) the ee of the produced diol (ee ) only slightly decreased
p
from 93% to 89% after 2 h. Interestingly, overlapping ee
curves were obtained (after fine tuning of the amount of
immobilizate to be used) indicating that both the E value
(E = 30) and the regioselectivity remained unaltered. To
further confirm these observations, we also determined
the regioselectivity of the immobilizate catalyzed reaction
by separately hydrolyzing pure (R) and (S) styrene oxide
enantiomers. The values thus obtained, that is, a(S) =
97% and a(R) = 10% are very similar to those we had
determined previously for the free StEH [a(S) = 97% and
5
a(R) = 8%]. This result further confirmed that the immo-
bilization of StEH on the GA support did not modify the
global stereoselectivity of the enzyme.
Figure 2. Stability optimization of the StEH immobilized on Glyoxyl-
agarose supports (GA). The different preparations were incubated at pH 7
and 60 °C in presence of 20% dextran 164 kDa. Circles: soluble enzyme;
rhombus: incubation during 20 min at 4 °C; squares: incubation during 3 h
at 4 °C; triangles: incubation during 3 h at 4 °C and additional incubation
at room temperature during 3 h.
Finally, we also have controlled that, using this material, it
was possible—as we have previously described using the

1236
C. Mateo et al. / Tetrahedron: Asymmetry 18 (2007) 1233–1238
Figure 3. Biohydrolysis of rac-styrene oxide by the free and immobilized
StEH. The enantiomeric excesses of the residual epoxide (ee ) and the
formed diol (ee ) were monitored over time using a substrate concentra-
tion of 10 mM at 27 °C. Open triangles: ee (free StEH); filled rhombus:
ee (immobilized StEH); open circles: ee (free StEH); filled squares: ee
immobilized StEH).
Figure 4. Repeated-batch reactor conducted with heating of the immo-
bilizate between each resolution cycle. The ee of the residual epoxide (ee
and the formed diol (ee ) were monitored over time using an initial styrene
oxide concentration of 10 mM at 27 °C. First cycle: filled rhombus: ee
white rhombus: ee . Second cycle: filled triangle: ee , white triangle: ee
Third cycle: filled circle: ee , white circle: ee
ee , white hexagon: ee . Fifth cycle: filled squares: ee
s
s
)
p
p
s
s
,
.
s
p
p
p
s
p
(
s
p
. Fourth cycle: filled hexagon:
, white squares: ee
s
p
s
p
.
free enzyme—to perform the enantioconvergent biohydro-
lysis of meta-chlorostyrene. As expected, total (analytical
scale) biohydrolysis of this epoxide exclusively afforded
the corresponding (R)-diol, in a nearly quantitative yield
3
. Conclusion
Immobilization of the recombinant S. tuberosum epoxide
hydrolase was explored using different supports. The use
of a Glyoxyl-agarose support allowed us to obtain an
optimized immobilizate exhibiting excellent thermal and
chemical stability. The key parameter of this successful
immobilization process was the use of 164 kDa dextran
as an additive, which prevented the enzyme from inactiva-
tion at the high pH (pH 10) necessarily used for performing
this immobilization. The enantio- and regio-selectivity
properties of the thus obtained GA–StEH immobilizate
were shown to be very similar to those of the free StEH,
indicating that the stereochemical properties of the enzyme
were not noticeably altered. These results thus clearly pave
the way to the efficient use of this novel biocatalyst for car-
rying out either the biocatalyzed hydrolytic kinetic resolu-
tion of an epoxide or the enantioconvergent preparation of
the corresponding diol, thus obtained in nearly quantita-
tive amount and highly enantiomerically enriched form,
by running a packed bed downflow-column reactor. Work
is currently in progress in our laboratories to further
explore the preparative scale potentialities of this new
biocatalyst.
5
and an excellent ee of 96%. This (R)-diol is an important
building block in the synthesis of various biologically
2
6
active molecules.
2.5. Confirmation of the GA–StEH immobilizate tempera-
ture resistance using repeated-batch reactors
During our stability studies, we showed that the GA–StEH
immobilizate seemed to be a thermally very robust biocat-
alyst (t1/2 >200 h at 60 °C). In order to confirm this result,
we performed ‘repeated-batch’ experiments, using rac-sty-
rene oxide as substrate. After each resolution process (per-
formed at 27 °C in order to avoid chemical hydrolysis,
which would prevent a good understanding of the results)
the recovered biocatalyst was heated at 58 °C for several
hours (20 then 44 then 68 h) before being re-used in the
next experiment. As shown in Figure 4, the kinetic profile
of the different experiments remained nearly identical after
1
20 min (the ee and ee stayed unaltered over the first four
s p
cycles), indicating that no loss of activity, enantio- or
regio-selectivity occurred. After the fourth experiment,
additional heating of the recovered immobilizate was
performed, at 58 °C for 7 days (164 h). Surprisingly, 30%
4. Experimental
4.1. Materials
activity still was present and an ee higher than 98% still
s
could be obtained, after 360 min reaction time. However,
the ee value culminated at a value lower than in the previ-
p
ous experiments, suggesting a slight modification of the
regio- and/or enantio-selectivity of the enzyme.
The plasmid containing the epoxide hydrolase gene from
S. tuberosum (pGEFII-StEH) was kindly provided by
Professor M. Arand (Institute of Pharmacology and Toxi-
cology, Z u¨ rich, Switzerland). Agarose 10BCL was
obtained from Iberagar S.A. and activated as described
These results clearly confirm the very high thermal stability
of the GA–StEH immobilizate, a very interesting property,
which opens the way to very long-running repeat batch
reactor experiments.
1
2
elsewhere.
Sepabeads epoxy-supports (Sepabead-EP)
were kindly donated by Resindion S.R.L. and derivatized

C. Mateo et al. / Tetrahedron: Asymmetry 18 (2007) 1233–1238
1237
as previously described.17 Enantiomeric excesses were
determined by chiral GC analysis carried out on a Shima-
dzu GC-20A equipped with FID and a fast Cyclosil-B col-
umn (Agilent, 13 m, 0.1 mm, 0.1 lm film, He) or a Chirasil-
DEX CB (Varian, 25 m, 0.25 mm, 0.25 lm film, He).
a sodium bicarbonate solution (2 mL, 0.2 M). The suspen-
sion was gently stirred for 1 h at 4 °C. After this period,
sodium bicarbonate (6 mL of 0.2 M, pH 10) containing
sodium borohydride (10 mg) was added and the suspension
was stirred for 30 min. Finally, the preparation was washed
with an excess of distilled water.
4
.2. Preparation of the enzymatic extract
4.5.3. Optimized procedure. GA support (2 g) was mixed
Escherichia coli BL21(DE3) was used as the host for the
cloning and functional expression of the EH gene from
S. tuberosum. E. coli competent cells were transformed with
the recombinant plasmid (pGEF-StEH). To produce
native EH, cells grown to an initial OD600 of 1.5 in a 1 L
fermentor were induced for 8 h with 0.4 mM IPTG
with an enzyme solution (20 mL of 1 mg/mL water), a
sodium bicarbonate solution at pH 10 (2 mL, 0.2 M) and
20% (w/v) of 164 kDa dextran. The suspension was stirred
gently for different time periods at 4 °C. In some cases, the
preparation was incubated at 25 °C for different time
periods. After this period, the enzyme preparation was
reduced and washed as described above to furnish GA
immobilizate.
(
20 °C, LB broth, 200 lg/mL ampicillin). Cells were
harvested by centrifugation, re-suspended in 20 mL of
0 mM carbonate ammonium buffer pH 7.6 and then
2
broken by a single pass through a French press. After
centrifugation at 20,000g for 30 min at 4 °C, the superna-
tant was freeze dried to produce about 0.5 g of crude enzy-
matic extract. This showed an activity of 1.4 U/mg and was
used as such in all experiments.
4.6. Enzyme stability against temperature
The different immobilizates were incubated at the indicated
temperature. At different time periods, samples were taken
from the preparation and the activity remaining on the
immobilizate measured. In each case, the initial activity
was considered to be the 100% value.
4
.3. Enzyme activity measurement
The enzymatic activity was measured against styrene oxide
at 25 °C and pH 7) as previously described by recording
the absorbance increment at 290 nm promoted by the benz-
4.7. Comparative selectivity study of the free StEH versus
the GA immobilizate
(
1
6
aldehyde formed. Thus, 2 mL of water, 20 lL of a
00 mM sodium periodate solution, and various amounts
The resolution process was started by the addition of rac-
styrene oxide (20 lL of a 1 M solution in DMF) to a solu-
tion of free StEH (0.62 mg of enzymatic extract in 2 mL of
phosphate buffer 10 mM, pH 7) or of immobilized StEH
onto optimized Glyoxyl-agarose supports (150 mg in
2 mL of phosphate buffer 10 mM, pH 7). This mixture
was maintained at 27 °C under gentle magnetic stirring.
At time intervals, about 150 lL of the reaction mixture
were withdrawn using a pipette tip equipped with a filter
in order to avoid removal of some support and extracted
with ethyl acetate (75 lL) under vortex agitation. The ee
of the residual epoxide and of the formed phenyl ethane-
diol was determined by injection (3 lL) of the ethyl acetate
phase on a fast chiral GC column (Cyclosil-B) using a tem-
perature programming (90 °C, 8 min; 60 °C/min; 150 °C,
15 min). The following retention times were obtained:
(S)-styrene oxide: 6.7 min and (R)-styrene oxide: 7.2 min;
(S)-phenyl ethanediol: 16.7 min and (R)-phenyl ethanediol:
16.9 min.
2
of a styrene oxide solution (from a stock solution 50 mM
in dimethyl formamide) were added to a spectroscopy cell.
Different enzyme preparations were added to this solution.
In parallel, a blank experiment was conducted using in the
absence of substrate.
4
.4. Immobilization on Sepabeads-EP supports
To 1 g of the different epoxy-supports (see Table 1) were
added 10 mL of an enzyme solution (1 mg/mL) in the
appropriate buffer (1 M sodium phosphate at pH 7 for
the hydrophobic commercial supports and 10 mM sodium
phosphate at pH 7 for the other supports). The suspension
was kept under mild stirring for 24 h at 25 °C and the enzy-
matic activity of both the suspension and the (filtered)
supernatant were determined. The suspension was filtered,
dried under vacuum, and a sodium hydrogen carbonate
(
10 mL of 0.1 M, pH 9) solution was added. Finally, the
residual epoxy groups were blocked by adding a glycine
solution (15 mL of 3 M, pH 8.5). The suspension was stir-
red during 24 h before washing and drying under vacuum.
4.8. Determination of the regioselectivity coefficients of
StEH immobilized on the GA support
2
7
Biohydrolyses were started by the addition of pure (R)- or
(S)-styrene oxide (2 lL) to a solution of immobilized StEH
onto GA support (150 mg in 2 mL of phosphate buffer
10 mM, pH 7) maintained at 27 °C under gentle magnetic
stirring. At three different time periods, the ee of the diols
formed was analyzed as described above.
4
.5. Immobilization on the Glyoxyl-agarose support
4
.5.1. Preparation of the Glyoxyl-agarose support (GA).
Glyoxyl-agarose 10 BCL gels (agarose-O-CH -CHO) were
prepared by etherification of agarose gels with glycidol
2
(
2,3-epoxypropanol) and further oxidation with sodium
1
2
periodate.
Complete biohydrolysis of meta-chlorostyrene oxide
(
10 mM) was carried out at 20 °C in 1 h using optimized
4
.5.2. Standard procedure. GA support (2 g) was mixed
Glyoxyl-agarose support (1 g in 2 mL of phosphate buffer
10 mM, pH 7). The ee of the formed diol was determined,
with an enzyme solution (20 mL of 1 mg/mL water) and

1238
C. Mateo et al. / Tetrahedron: Asymmetry 18 (2007) 1233–1238
after derivatization into the corresponding acetonide, by
injection on a chiral GC column (Chiralsil-DEX CB at
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. Gianfreda, L.; Scarfi, M. R. Mol. Cell. Biochem. 1991, 100,
1
35 °C; (R)-diol: 12.9 min and (S)-diol: 14.9 min).
8
9
7–128.
4.9. Study of the thermal stability of the GA–StEH
immobilizate using a repeated-batch reactor
9
. Manalov, R. J.; Kambourova, M. S.; Emanuilova, E. I.
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1
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The thermal stability of the StEH immobilized on GA sup-
port was investigated using a repeated-batch reactor. A
batch reactor similar to those described above was run at
11. Reddy, L. G.; Shankar, V. Crit. Rev. Biotechnol. 1983, 13,
55–273.
2
1
1
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2
7 °C until the enantiomeric excess of the residual styrene
oxide reached 99%. The residual epoxide and the formed
diol were then removed from the reactor by washing the
support four times with water (2 mL). The support was
either incubated in phosphate buffer (2 mL, 10 mM, pH
1
4. Mateo, C.; Abian, O.; Bernedo, M.; Cuenca, E.; Fuentes, M.;
Fernandez-Lorente, G.; Palomo, J. M.; Graz u´ , V.; Pessela, B.
C. C.; Giacomini, C.; Irazoqui, G.; Villarino, A.; Ovsejevi,
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7
) at 58 °C for several hours. The liquid phase was removed
by filtration under vacuum and the enantioselectivity of the
support was determined as described above before starting
a new resolution cycle.
15. Mateo, C.; Palomo, J. M.; Fuentes, M.; Betancor, L.; Grazu,
V.; Lopez-Gallego, F.; Pessela, B. C. C.; Hidalgo, A.;
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