TBADH activity in water-miscible organic solvents: Correlations between enzyme performance, enantioselectivity and protein structure through spectroscopic studies

Article abstract of DOI:10.1039/b418040b

The enantioselective reduction of 2-pentanone to (R)- and (S)-2-pentanol by Thermoanaerobacter (formerly Thermoanaerobium) brockii alcohol dehydrogenase (TBADH) in mixtures of water and water-miscible organic solvents was investigated. Significant enzymatic activity was retained in up to 87% methanol, ethanol and acetonitrile. The changes in enzyme activity as a function of organic solvent were correlated to structural alterations of TBADH with a series of spectroscopic studies (fluorescence, fluorescence quenching and circular dichroism (CD)). Interestingly, this study shows that the tetrameric form of TBADH is not critical for catalytic performance. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b418040b

View Article Online / Journal Homepage / Table of Contents for this issue
A R T I C L E
TBADH activity in water-miscible organic solvents: correlations
between enzyme performance, enantioselectivity and protein
structure through spectroscopic studies
Linus Olofsson,* Ian A. Nicholls and Susanne Wikman*
Bioorganic & Biophysical Chemistry Laboratory, Department of Chemistry & Biomedical
Sciences, University of Kalmar, SE-391 82, Kalmar, Sweden. E-mail: linus.olofsson@hik.se;
Fax: +46-480-446244; Tel: +46-480-446710, susanne.wikman@hik.se; Fax: +46-480-446244;
Tel: +46-480-446272
Received 30th November 2004, Accepted 13th January 2005
First published as an Advance Article on the web 4th February 2005
The enantioselective reduction of 2-pentanone to (R)- and (S)-2-pentanol by Thermoanaerobacter (formerly
Thermoanaerobium) brockii alcohol dehydrogenase (TBADH) in mixtures of water and water-miscible organic
solvents was investigated. Significant enzymatic activity was retained in up to 87% methanol, ethanol and
acetonitrile. The changes in enzyme activity as a function of organic solvent were correlated to structural alterations
of TBADH with a series of spectroscopic studies (fluorescence, fluorescence quenching and circular dichroism (CD)).
Interestingly, this study shows that the tetrameric form of TBADH is not critical for catalytic performance.
Introduction
The utility of enzymes for the catalysis of asymmetric reactions
1
in aqueous media is well established. Nonetheless, it can be
advantageous, or even necessary, to employ organic media,
e.g. for increased substrate/product solubility and stability,
2,3
substrate specificity, regiospecificity and enantiopurity.
The Thermoanaerobacter (formerly Thermoanaerobium)
brockii (T. brockii) alcohol dehydrogenase (TBADH) is an
oxidoreductase derived from T. brockii, a thermophilic bac-
terium isolated from the hot springs of Yellowstone National
Park (USA). TBADH has been shown to possess stability
5
,6
towards elevated temperatures and some organic solvents and
demonstrates a specificity for small aliphatic ketone substrates
and exhibits high product enantioselectivity.
Scheme 1 Reduction of 2-pentanone to (R)- and (S)-2-pentanol by
TBADH in a coupled substrate reaction in which the cosubstrate
2-propanol is oxidized to acetone to achieve regeneration of the cofactor
4,7,8
In its native
4
NADPH.
form, the enzyme is a homotetramer with a subunit molecular
weight of 37 652 Da, with each monomer consisting of a
Furthermore, each
monomer contains four tryptophan residues, rendering TBADH
amenable for fluorescence-based spectroscopic studies.
9
10,11
cofactor domain and a catalytic domain.
series of protic and aprotic water-miscible organic solvents on
◦
the enzymatic reduction of 2-pentanone by TBADH at 20 C
was examined, Fig. 1. 2-Pentanone was chosen as substrate
since it has previously been used in an extensive investigation of
Although the potential of TBADH for asymmetric synthesis
in organic media is implicit from many previous studies, no thor-
ough investigation has examined the potential and consequences
of the use of TBADH in syntheses involving high concentrations
of water-miscible organic solvents.
In this study, the influence of a range of water-miscible
protic and aprotic organic solvents [methanol, ethanol, 1-
propanol, acetonitrile (ACN) and tetrahydrofuran (THF)], on
the enantioselectivity and enzymatic performance of TBADH
in the reduction of 2-pentanone to (R)- and (S)-2-pentanol is
presented, Scheme 1. Furthermore, the influence of the organic
solvents on the observed enzyme activities has been correlated
to secondary, tertiary and quaternary structural features of the
protein, using a series of fluorescence and circular dichroism
4
factors influencing the TBADH-mediated reduction of ketones.
4
Assay procedures previously developed by Keinan et al., using
2-propanol (22%, v/v) as a cosubstrate for cofactor recycling
were used in conjunction with commercial TBADH (used as
received). Reactions performed under these conditions, i.e. 78%
(v/v) buffer, afforded the products (R)- and (S)-2-pentanol in
60% total yield as determined by GC-MS analysis using a chiral
capillary column.
(
CD) studies. The relative resilience of TBADH to these solvents
is demonstrated in part by the product enantioselectivity and
yield, together with the collective insights gained from the
fluorescence and CD studies.
Results and discussion
Fig. 1 Conversion of 2-pentanone to (R)- and (S)-2-pentanol by
◦
Enzymatic reduction of 2-pentanone
TBADH at 20 C in methanol ( ), ethanol (×), 1-propanol ( ), ACN (–)
and THF (᭺), all samples containing 22% 2-propanol as cosubstrate for
cofactor recycling. At no data point did the standard deviation exceed
4.5% and the average standard deviation was 1.2%.
In order to investigate the utility of this enzyme for use in
monophasic asymmetric organic synthesis, the influence of a
7
5 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 5 0 – 7 5 5
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5

View Article Online
Table 1 Characteristics of organic solvents
a
b
c
d
e
Solvent
C
50
DC
log P
e
I
Methanol
Ethanol
ACN
68
58
55
45
38
30.5
54.4
64.3
69.2
−0.74
−0.32
−0.34
0.34
32.7
24.6
35.9
20.4
7.6
5.1
4.3
5.8
3.9
4.0
1
-Propanol
THF
100
0.46
a
The solvent organic content in percentage at which enzyme activity
is 50% of the activity in buffer and cosubstrate mixture (22% total
b
13 c
organic content), see Fig. 1. Denaturation capacity.
Partitioning
13
coefficient of the solvent in the water–1-octanol biphasic system.
d
◦
14
e
15
Solvent dielectric constant at 25 C. Polarity index.
Fig. 2 SDS-PAGE analysis of TBADH purification steps showing
commercial TBADH (lane 1), fraction from Red Sepharose column
(lane 2), fraction from Superose column (lane 3) and SeeBlue plus2
protein standard (lane 4).
Increasing concentrations of methanol, up to 60% total
organic content, produced a decline in conversion efficiency,
to 70%, Fig. 1. Higher concentrations of methanol resulted in
a significant decline in activity, to 20% conversion at 87% total
organic solvent content. The use of ethanol and 1-propanol
presented similar trends, though the decrease in enzyme effi-
ciency was more pronounced, and appeared to be related to
the relative hydrophobicities of the three alcohols, Table 1.
Interestingly, despite the reduction in enzyme efficiency in media
of higher hydrophobicity, the enantiomeric excess (ee) of the
product (S)-alcohol remained relatively constant, 60 ± 4%, in
all organic solvent compositions and concentrations studied
environment. As seen in Fig. 3, the fluorescence emission, kmax
,
of TBADH in all organic solvents investigated behaved similarly,
and appeared to consist of three different regions; 0% to 30%
organic solvent (region 1), 30% to 60% organic solvent (region
2
) and 60% to 90% organic solvent (region 3). In region 1, kmax
is blue shifted (lowered) upon increasing the organic content
from 0% to 30%, this is followed by a red shift up until between
4
5% and 60% (region 2) and a blue shift at even higher organic
solvent contents (region 3).
(
not determined for 1-propanol, 87% total organic solvent
concentration, due to low yield). Improvement of ee could be
achieved by interruption of reaction at 50% conversion in order
4
to decrease time dependent racemization, and by optimizing
12
temperature and cosubstrate concentration.
In the cases of the aprotic solvents, ACN and THF, the former
resulted in a reactivity profile similar to that obtained with
the ethanol series, though enzyme reactivity was somewhat less
influenced by higher concentrations of this solvent. In THF, the
influence of this solvent on enzyme efficiency is already quite
apparent at lower concentrations, though again, and as also for
ACN, the product ee was stable at 60 ± 4% (ee determination
for THF at 71% and 87% not possible due to low yield).
From these data it is evident that TBADH is capable of
efficient function and product enantioselectivity at relatively
high concentrations of water-miscible organic solvents, though
elevated hydrophobicities result in successively lower conver-
sions. Several physical parameters have previously been used to
characterize enzyme catalysis in organic media, Table 1. While
the trends in Fig. 1 indicate a good correlation between TBADH
activity (C50), denaturation capacity (DC) and log P, at 87% total
organic content correlation with the dielectric constant (e) and
polarity index (I) of the solvent appear best.
◦
Fig. 3 Fluorescence kmax of TBADH at 20 C in methanol ( ), ethanol
(
×), 1-propanol ( ), ACN (–) and THF (᭺).
The blue shift in region 1 is interpreted as arising from a
decreased energy loss from excited Trp fluorophores to the
reorienting solvent dipoles of the increasingly more hydrophobic
surrounding media. One may well expect this trend to con-
tinue with increased hydrophobicity, but instead, a red shift
is observed (region 2). Evidently some of the Trp residues
suffer energy loss due to increased interaction with neighboring
molecules on account of increased environmental polarity. Thus
a dramatic change in Trp environment appears to take place in
the transition from region 1 to region 2.
Previous fluorescence studies of enzymes exposed to mixtures
of water and water-miscible organic solvents, using Trp as probes
for the environmental polarity, showed that an increase of Trp
exposure to the organic solvent–water mixture resulted in a
fluorescence emission red shift which was correlated to protein
However, based upon the solvent systems studied, all pa-
rameters support the general relationship between solvent
hydrophobicity and enzyme activity described above.
The robustness of this protein suggested studies on the
influence of these organic solvents on the structure of the
TBADH tetramer.
16–18
denaturation and concomitant activity loss.
In this study,
Purification of commercial TBADH for spectroscopic studies
TBADH fluorescence red shifts can be correlated neither to
protein denaturation nor to activity loss, as only a relatively
small, linear, decrease in enzyme activity was observed in the
organic solvents examined, see Fig. 1 and 3. However, the
observed changes in Trp environment might be explained by
disintegration of the TBADH tetramer.
X-Ray crystallographic studies show that the TBADH
tetramer may be represented as a dimer of two identical dimers
composed of subunits A and B (dimer AB) and subunits C and
D (dimer CD), Fig. 4. The largest areas of interaction in the
tetramer exist between subunit A and B of dimer AB and the
corresponding interface between subunits C and D; less inter-
Commercial TBADH was purified for use in the subsequent
spectroscopic studies. As seen in the SDS-PAGE gel, Fig. 2,
purified enzyme (lane 3) migrated as a single band with an
apparent molecular weight of ∼38 kDa per subunit as confirmed
by the SeeBlue protein standard band of alcohol dehydrogenase
at 38 kDa (lane 4).
10
Intrinsic tryptophan fluorescence of TBADH
Fluorescence studies were performed to investigate the pos-
sibility of correlating the organic solvent induced decrease
in TBADH activity to changes in TBADH tryptophan (Trp)
10,11,21
subunit contact is present between the two dimers.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 5 0 – 7 5 5
7 5 1

View Article Online
reached only 338 nm (though slightly suppressed by the organic
media), see Fig. 3, substantial TBADH structure should be
retained in all organic solvents and concentrations.
Quenching of TBADH fluorescence
To validate the basis for the observed changes in Trp flu-
orescence, fluorescence quenching studies were conducted to
establish the extent of Trp exposure. Dynamic quenching (Ksv
)
and static quenching (V) both arise from physical contact
between the quencher (acrylamide) and the fluorophore (the
excited indole ring of Trp). Dynamic quenching is a result of
collisional encounters between the fluorophore and the quencher
while static quenching occurs when the fluorophore is more
accessible to the quencher, resulting in complex formation
between the two. The presence of static quenching, which is seen
as upward curvature of a Stern–Volmer plot, Fig. 6, is quantified
by the term V, Table 2.
Fig. 4 TBADH tetramer side view (left) and from above (right). Trp’s
are coloured in blue and denoted with capital letters representing the
19,20
subunit. Illustrations were created with RasTop 2.0.3 software
the RSCB PDB molecular file 1BXZ.
using
10
The four tryptophans present in each TBADH monomer can
be divided into three categories, each with a different degree of
exposure to surrounding media: (a) Trp14 and Trp110, which are
partially exposed on the surface of both the monomer and in the
tetrameric structure, (Fig. 4), hence, their exposure to solvent
will not change upon tetramer dissociation. (b) Trp90, which
is almost completely solvent protected in the tetramer, being
buried in the subunit–subunit interface between subunit A and
D and between subunit B and C, (Fig. 4, right). Nevertheless,
Trp90 becomes freely exposed to the solvent upon separating the
tetramer into dimers, (Fig. 5), left. (c) Trp281, which is almost
completely buried in the dimer, but becomes freely accessible
to the solvent upon monomerisation, (Fig. 5, right). The local
subunit environments of Trp14 and Trp110 remain unaffected
despite tetramer disruption to dimers or monomers. Thus, the
fluorescence red shift in region 2 can be concluded to arise
from changes in the environments of Trp90 and/or Trp281 upon
transition from the more hydrophobic interior of the TBADH
tetramer to the less hydrophobic organic solvent–water mixture.
In the less polar solvents (methanol, ACN), intermediate red
shifts appear at 45% organic content, probably indicating the
disruption of tetramers into dimers, also exposing Trp90. At 60%
(
methanol, ACN) the red shifts are fully developed indicating
the disruption of dimers into monomers, a process leading to
the exposure of Trp281. The red shifts of kmax of the ethanol,
propanol, and THF series occur more abruptly, providing only a
narrow concentration range in which the dimeric form is present
before disrupting into monomers. The blue shift in region 3,
which is continuous, can thus be rationalized as a result from
the decreasing polarity of the medium, which influences all of
the Trp residues as all are now exposed to the solvent. The red
shifts (region 2) observed in ethanol, 1-propanol and THF, occur
at lower organic content (45%) than for the less hydrophobic
solvents, ACN and MeOH, (60%). Furthermore, the magnitude
of the red shift in THF is lower in THF due to its significantly
lower polarity, see Table 1.
Fig. 6 Stern–Volmer plots showing fluorescence quenching by acry-
lamide of Trp of TBADH in (a) MeOH, (b) ACN and (c) THF. Total
organic content is indicated by percentages in the figure. Trp fluorescence
in H
2
0
O (F ), divided by fluorescence at kmax for the respective organic
solvent concentration (F), is plotted against quencher concentration,
[
Q]. Note the difference in scale on the y-axis in (a, b) versus (c).
Fig. 6 shows the acrylamide quenching of Trp residues of
TBADH in (a) methanol, (b) ACN and (c) THF. The quenching
patterns of the homologous alcohol series represented by
methanol (a) are very similar, as judged by dynamic quenching
(
K
sv) and static quenching (V) in Table 2.
The Ksv for TBADH in water is 3.8 while V is 0, indicating
almost solely the presence of collisional quenching. Thus, the
indole rings of the partially exposed Trp110 and Trp14 on
the surface of the TBADH tetramer, appear to be sufficiently
protected from solvent to prevent complex formation with the
quencher. Upon exposure to 30% of the alcohols or ACN no
significant alteration in Ksv or V occurs, indicating that the Trp
residues are equally inaccessible to acrylamide in water or 30%
of these organic solvents.
Fig. 5 TBADH dimer (left) and monomer (right).
In order to achieve the fluorescence emission kmax of “totally
denatured” TBADH, treatment with GuHCl was performed
which resulted in a fluorescence emission kmax of 352 nm. Thus,
since the largest red shift of the fluorescence emission kmax
This is in agreement with the proposed tetrameric structure
in region 1, Fig. 3. The presence of static quenching, V, at 60%
of the alcohols or ACN suggests that the TBADH tetramer
dissolves into monomers causing exposure of the indole rings
7
5 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 5 0 – 7 5 5

View Article Online
Table 2 Quenching of Trp fluorescence by acrylamide
30% 60%
increased organic content (60% and 90%), which previously has
been attributed to the shielding of some chromophores when
present in large aggregates.
0
%
90%
22
Importantly, the CD spectrum of TBADH in 30% methanol
resembles the spectrum of native TBADH in water. In 60% and
90% methanol, the CD spectra suggest substantial structural
alterations despite the fact that the enzyme retained substantial
activity in 60% and 90% methanol. This further supports that
protein aggregation is responsible for these unexpectedly large
distortions. CD spectra of TBADH in the primary alcohols
methanol, ethanol and 1-propanol show the same trends (though
with accentuated effects for 1-propanol, results not shown). The
effect of aggregation is more pronounced in THF, Fig. 7(b)
already appearing at 30% THF, causing severe distortion of the
CD spectrum at 90% THF. Nevertheless, Fig. 7(c) shows that
the CD spectra of TBADH in water, 30% ACN or 60% ACN
are quite similar in shape; and that TBADH apparently retains
its structure and aggregation seems absent.
Thus, TBADH structure is well preserved in 60% ACN, and
since no major decrease in activity occurs in conjunction with in-
creasing the ACN concentration to 90%, its secondary structure
should remain intact, leaving aggregation as the cause for the
distorted CD spectrum at 90% ACN. Furthermore, since both
fluorescence, fluorescence quenching, and the activity behaviour
of TBADH in methanol, ethanol and ACN are very similar, it
is reasonable to expect that a native-like monomer structure is
present in the presence of various ranges of concentrations of
water-miscible organic solvents.
Solvent
K
SV
V
K
SV
V
K
SV
V
K
SV
V
Methanol
Ethanol
3.8
3.8
0
0
0
0
0
3.9
3.4
3.5
3.8
2.4
0
0
0
0
2.3
2.5
2.4
2.8
0.9 3.5 1.2
0.9 3.9 1.3
1.0 2.6 1.6
1.0 5.2 1.3
2.0 6.1 3.9
1
-Propanol 3.8
ACN
THF
3.8
3.8
0.7 3.1
Acrylamide (0–0.5 M final concentration) was added to TBADH pre-
incubated in mixtures of water and organic solvents. Fluorescence in
the absence of acrylamide/fluorescence in the presence of acrylamide
(
F /F), at kmax for each organic solvent concentration, was plotted
0
against acrylamide concentration (Stern–Volmer plot, see Experimental
section). Dynamic quenching constants KSV and static quenching
constants V were extracted from the plots using eqn. (1). In cases of
V < 0.5, the plots were considered essentially linear and V was set at
0
[
. KSV values were then determined using the simplified linear equation
eqn. (2)], which fits the tangents of the curves at 0 M acrylamide.
of Trp90 and Trp281 to the quencher, in agreement with the
interpretation of the fluorescence behaviour of TBADH in
region 2, Fig. 3. The slightly increased static quenching at 90%
organic content might indicate structural changes within the
monomers, causing further exposure of Trp14 and Trp110 to
the quencher. In terms of the influence of THF, static quenching
occurs at lower concentration (30%) and is more pronounced
at higher concentrations, which suggests an earlier induction of
tetramer disruption and more pronounced changes in monomer
structure resulting in increased exposure of the Trp indole rings.
The possibility of protein aggregation being responsible for the
reduced enzymatic activity upon increasing the organic content
is excluded since a) aggregation would lead to a decrease in
Trp exposure and thereby lower the static quenching (V), not
increasing it b) aggregation would keep Trp90 and Trp281 buried
within the aggregates and increasing the organic content would
only affect Trp14 and Trp110 on the surface of the aggregates,
thus producing a blue shift instead of a red shift.
General discussion
When enzymatic reactions are performed in neat organic
solvents the water deficiency prevents the protein from unfolding
3
since it is kinetically trapped in its native conformation.
However, the decreased protein flexibility also lowers enzyme
performance. In more polar solvents (methanol, ethanol, ACN),
the main factor reducing TBADH activity would probably be
water stripping, since polar organic solvents are capable of
solubilisation and removal of essential water from the enzyme
23,24
25
surface
and also affect the active site polarity. TBADH
Circular dichroism
denaturation appears to be relatively modest according to
CD measurements, but it is probably more pronounced for
the less polar solvents 1-propanol and THF (these solvents
being more effective in disrupting protein hydrogen bonding
Circular dichroism (CD) studies of TBADH secondary structure
were performed in order to examine the additional possibility
of structural changes within the monomers. In order to achieve
accurate CD signals a significantly higher TBADH concentra-
tion was used which in some cases induced protein aggregation
at higher concentrations of organic solvents. This is seen in the
methanol series, Fig. 7(a), by the presence of suppressed positive
ellipticity at 192 nm (absorptive flattening) in conjunction with
26
and hydrophobic interactions) according to their induction of
increased static quenching.
It has been argued that protein stability in TBADH
is enhanced by several factors, amongst them intersubunit
10,21
interactions.
However, upon exposure to the polar organic
Fig. 7 CD spectra of TBADH in (a) methanol, (b) THF, (c) ACN. TBADH in 30% organic solvent (---), 60% organic solvent (-·-), 90% organic
solvent (– –) and water (—). Measurements in THF only possible at 205–260 nm due to high absorbance at lower wavelengths.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 5 0 – 7 5 5
7 5 3

View Article Online
solvents employed in this study, disruption of the tetramer into
its subunits cannot be correlated to any decrease in enzyme
activity. That the enzyme is active in its monomeric form is
not particularly surprising since all proteins in a thermophilic
Purification of commercial TBADH for spectroscopic studies
Commercial TBADH was purified according to a procedure
adapted from Peretz et al. Samples were loaded onto a
Red Sepharose CL-6B XK 26/40 column (Pharmacia), pre-
equilibrated with buffer A (0.1 M NaCl, 0.1 mM EDTA,
27
10
organisms must be heat resistant, i.e. of high stability. This does
not contradict that oligomeric protein thermostability may be
enhanced by intersubunit interactions. Interestingly, the active
site-channel of each monomer is in contact with two amino
¨
0
.1 mM DTT, 25 mM Tris-HCl, pH 7.3), connected to an AKTA
FPLC system. TBADH was eluted using a linear gradient of
00 mL NaCl (0.1–0.8 M) and fractions containing TBADH
were pooled and run through a size exclusion Superose 12
6
10
acid residues from the neighbouring monomer, an interaction
which is lost upon monomerisation, though without causing any
obvious decline in enzyme activity.
−
1
1
0/30 column (Pharmacia), flow 0.1 mL min with buffer
A. TBADH fractions were concentrated by ultrafiltration with
a Vivaspin concentrator (Vivascience), 10 000 MW cut-off at
◦
Conclusion
2300 g, 4 C, 40 min. TBADH monomer size and purity
was confirmed by using NuPAGE 4–12% Bis-Tris pre-cast gel
kit with MES buffer, Coomassie staining and SeeBlue plus2
pre-stained protein standard (Invitrogen Life Technologies),
activity was confirmed using the assay described above, and
concentration was determined by Bio-Rad protein assay (Bio-
Rad Laboratories) with IgG as standard on Ultrospec 3000
Pharmacia Biotech (data not shown).
The influence of various water-miscible organic solvents on the
yield and enantioselectivity of the TBADH catalysed reaction of
2
-pentanone to (R)- and (S)-2-pentanol has been investigated.
Significant activity in up to 87% methanol, ethanol and ACN
was observed. The structural basis for the perturbation of
enzyme activity has been studied using a combination of
fluorescence and CD studies. Collectively, the spectroscopic
studies indicate that the enzyme is resilient to simple alcohols
and some aprotic media (ACN). Furthermore, this study has
shown that the oligomeric structure is not critical for catalytic
activity since the disruption of the tetramer into its subunits
cannot be correlated to any decrease in enzyme activity.
Intrinsic tryptophan fluorescence of TBADH
−
1
Purified TBADH (35 lg mL final concentration) was exposed
to mixtures of water and organic solvents (methanol, ethanol,
1-propanol, ACN, THF) in a series of organic solvent concen-
◦
trations (0–90%) for 30 min at 20 C. Tryptophan emission
◦
spectra were recorded (290 to 400 nm) at 20 C with a
Experimental
Hitachi F-2000 fluorescence spectrophotometer at an excitation
2
8
General
wavelength of 295 nm to minimise interference from tyrosine
29
and acrylamide. The excitation and emission bandpasses were
TBADH (EC 1.1.1.2, Lot 81H40692) and NADPH (Lot
−
1
set to 10 nm and the scan speed was set to 60 nm min .
Fluorescence emission maxima (kmax) were extracted from the
spectra and plotted against alcohol concentration.
4
1K7049), SIGMA (USA). All other solvents and reagents were
of analytical grade purchased from commercial suppliers and
used without further purification.
Quenching of fluorescence of TBADH
Enzymatic reduction of 2-pentanone
−
1
Purified TBADH (46 lg mL final concentration) was ex-
posed to mixtures of water and organic solvents (methanol,
ethanol, 1-propanol, ACN, THF) in a series of organic solvent
Reductions were performed in triplicate in a range of mixtures
of glycine buffer (20 mM, pH 7,8) and various organic solvents
(
methanol, ethanol, 1-propanol, ACN, THF). To a series of
◦
concentrations (0–90%) for 30 min at 20 C, followed by
Eppendorf tubes was added, in the following order: glycine
buffer (0, 300, 500, 800, 1250 lL, respectively), organic solvent
immediate addition of acrylamide (0–0.5 M final concentration).
Acrylamide was prepared as 8 M stock solutions in respective
organic solvents (0–90%). Tryptophan emission spectra were
recorded as for tryptophan fluorescence measurements above.
Fluorescence in absence of acrylamide at kmax for respective
(
(
1300, 950, 750, 450, 150, 0 lL, respectively), 2-pentanone
38.5 lL), 2-propanol (385 lL), 2-mercaptoethanol (25 lL,
2
34 mM in glycine buffer), NADPH (25 lL, 3.8 mM in glycine
−
1
buffer), commercial TBADH (200 lL, 10 U mL in glycine
alcohol concentration (F ), divided by the fluorescence in
0
buffer). The reaction mixtures were placed on a rocking table
presence of acrylamide (F), was plotted against acrylamide
concentration, [Q]. The following equation,
◦
for 20 h at 20 C, and reactions were interrupted by addition
29,30
of 0.5 g (NH
and supernatants were separated from pellets. To each pellet,
00 lL glycine buffer was added, then shaken and centrifuged
4
)
2
SO
4
. Samples were centrifuged at 4500g, 2 min
V
F
0
/F = (1 + KSV[Q])e /[Q]
(1)
2
was fitted to the plots to extract dynamic quenching constants
sv and static quenching constants V by a least-squares pro-
cedure using the KaleidaGraph for Windows software package
Synergy Software, Reading PA, USA). In cases of V being very
at 4500g, 5 min and the resulting supernatants were combined
with the corresponding supernatants from the previous step. The
K
combined supernatants were extracted with 4 × 1 mL CH
2
Cl
2
(
and phases separated by centrifugation at 1200g, 2 min. Finally,
low (<0.5), the plots were considered linear and the simplified
equation,
1
mL of each organic phase was placed in vials for GC-MS
◦
analysis. All procedures were performed at 20 C.
F
0
/F = (1 + KSV[Q])
was used to determine KSV from the linear regions of the plots
tangents at 0 M acrylamide).
(2)
GC-MS analysis of reduction products
(
Reduction substrate and products (1 lL) were analysed using a
CP-Chirasil-Dex CB 25 m chiral capillary column (Chrompack)
on a Shimatzu GC-17A equipped with a Shimatzu QP-5000
MS detector using the following analysis conditions: injector,
Circular dichroism
−
1
Purified TBADH (0.2 mg mL final concentration) was exposed
to mixtures of water and organic solvents (methanol, ethanol,
1-propanol, ACN, THF) in a series of organic solvent concentra-
◦
◦
2
7
4
50 C; interface, 200 C; column pressure, 45 psi; gas flow,
−
1
4 mL min ; split ratio, 60. Column oven temperature program:
◦
◦
◦
−1
◦
5
C (13 min) to 75 C, 10 C min . Baseline separation
tions (0–90%) for 30 min at 20 C. CD spectra were recorded on a
of 2-pentanone and (R)- and (S)-2-pentanol was achieved
and the corresponding peak areas were integrated to calculate
conversion and enantiomeric excess.
CD6 spectrodichrograph (Jobin-Yvon Instruments SA, France)
at 20 C with a path length of 0.5 mm. Data were collected in the
wavelength range 190–260 nm (205–260 nm in THF due to high
◦
7
5 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 5 0 – 7 5 5

View Article Online
absorbance) at 0.5 nm intervals with an integration time of 2 s.
Each spectrum, both sample and background, was the average of
11 Y. Korkhin, A. J. Kalb, M. Peretz, O. Bogin, Y. Burstein and F.
Frolow, J. Mol. Biol., 1998, 278, 967.
2 H. Yang, A. Jonsson, E. Wehtje, P. Adlercreutz and B. Mattiasson,
Biochim. Biophys. Acta, 1997, 1336, 51.
3 Y. L. Khmelnitsky, V. V. Mozhaev, A. B. Belova, M. V. Sergeeva and
K. Martinek, Eur. J. Biochem., 1991, 198, 31.
4 C. Reichardt, Solvents and Solvent Effects in Organic Chemistry, 3rd
edn., 2003, Wiley-VCH, Weinheim.
5 M. N. Gupta, R. Batra, R. Tyagi and A. Sharma, Biotechnol. Prog.,
1
1
1
1
5
scans, separately intercompared before summation to detect
anomalous alterations during the scanning period. Difference
spectra were generated by subtracting the background spectrum
from the corresponding sample spectrum.
Acknowledgements
1
997, 13, 284.
We thank Martin Karlsson and Professor Uno Carlsson, Uni-
versity of Link o¨ ping, Sweden, for help with CD spectroscopy
and Dr Per Ola Andersson (Kalmar) for many helpful discus-
sions. Financial support from the Swedish Research Council
16 T. Kijima, S. Yamamoto and H. Kise, Bull. Chem. Soc. Jpn., 1994,
7, 2819.
7 T. Kijima, S. Yamamoto and H. Kise, Enzyme Microb. Technol.,
6
1
1
1
996, 18, 2.
8 V. V. Mozhaev, Y. L. Khmelnitsky, M. V. Sergeeva, A. B. Belova,
(
VR), Carl Trygger’s Foundation, Graninge Foundation, KK
N. L. Klyachko, A. V. Levashov and K. Martinek, Eur. J. Biochem.,
Foundation and the University of Kalmar are gratefully ac-
knowledged.
1
989, 184, 597.
19 R. A. Sayle and E. J. Milner-White, Trends Biochem. Sci., 1995, 20,
3
74.
2
2
0 H. J. Bernstein, Trends Biochem. Sci., 2000, 25, 453.
1 Y. Korkhin, A. J. Kalb, M. Peretz, O. Bogin, Y. Burstein and E.
Frolow, Protein Sci., 1999, 8, 1241.
References
1
K. Drauz and H. Waldmann, eds. Enzyme Catalysis in Organic
Synthesis, 2nd edn, Vol. 1–3, 2002, Wiley-VCH, Weinheim.
G. Carrea and S. Riva, Angew. Chem., Int. Ed., 2000, 39, 2226.
A. M. Klibanov, Nature, 2001, 409, 241.
E. Keinan, E. K. Hafeli, K. K. Seth and R. Lamed, J. Am. Chem.
Soc., 1986, 108, 162.
22 K. R. Adina Gitter-Amir and Allan S. Schneider, Biochemistry, 1976,
15, 3131.
23 A. Zaks and A. M. Klibanov, J. Biol. Chem., 1988, 263, 3194.
24 L. A. S. Gorman and J. S. Dordick, Biotechnol. Bioeng., 1992, 39,
392.
2
3
4
5
6
R. J. Lamed and J. G. Zeikus, Biochem. J., 1981, 195, 183.
M. Miroliaei and M. Nemat-Gorgani, Int. J. Biochem. Cell Biol.,
25 H. Wehbi, J. Feng and M. F. Roberts, Biochim. Biophys. Acta, 2003,
1613, 15.
2
002, 34, 169.
26 J. Dordick, Enzyme Microb. Technol., 1989, 11, 194.
27 M. Peretz, O. Bogin, S. TelOr, A. Cohen, G. S. Li, J. S. Chen and Y.
Burstein, Anaerobe, 1997, 3, 259.
28 M. R. Eftink, Methods Biochem. Anal., 1991, 35, 127.
29 J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd edn.,
1999, Kluwer Academic/Plenum Publishers, New York.
30 L. M. Roberts and A. K. Dunker, Biochemistry, 1993, 32,
10479.
7
8
9
E. Keinan, K. K. Seth, R. Lamed, R. Ghirlando and S. P. Singh,
Biocatalysis, 1990, 3, 57.
K. Nakamura, R. Yamanaka, T. Matsuda and T. Harada, Tetrahe-
dron: Asymmetry, 2003, 14, 2659.
M. Peretz and Y. Burstein, Biochemistry, 1989, 28, 6549.
1
0 C. M. Li, J. Heatwole, S. Soelaiman and M. Shoham, Proteins:
Struct., Funct., Genet., 1999, 37, 619.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 7 5 0 – 7 5 5
7 5 5