Enantioselective oxidation by a cyclohexanone monooxygenase from the xenobiotic-degrading Polaromonas sp. strain JS666

Article abstract of DOI:10.1016/j.molcatb.2012.03.002

A cyclohexanone monooxygenase (CHMO) from the xenobiotic-degrading Polaromonas sp. strain JS666 was heterologously expressed in Escherichia coli, and its ability to catalyze enantio- and regiodivergent oxidations of prochiral and racemic ketones was investigated. The expression system was also used to evaluate this enzyme's potential role in the oxidation of cis-1,2-dichloroethene (cDCE), a groundwater pollutant for which strain JS666 is the only known assimilator. The substrate enantiopreference and -selectivity of the strain JS666 CHMO is similar to that of other CHMO-type enzymes; of note is this enzyme's excellent stereodiscrimination of 2-substituted cyclic ketones. The expression system exhibits no activity with ethene or cDCE as substrates under the tested conditions. Phylogenetic analysis shows that sequence variability among cyclohexanone monooxygenases could be a rich source of new enzyme activities and attributes.

Full text of DOI:10.1016/j.molcatb.2012.03.002

Journal of Molecular Catalysis B: Enzymatic 78 (2012) 105–110
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enantioselective oxidation by a cyclohexanone monooxygenase from the
xenobiotic-degrading Polaromonas sp. strain JS666
Anne K. Alexandera, David Biedermannb, Michael J. Finkb, Marko D. Mihovilovicb, Timothy E. Mattesa,∗
a The University of Iowa, Department of Civil and Environmental Engineering, 4105 Seamans Center, Iowa City, IA 52242, USA
b Vienna University of Technology, Institute of Applied Synthetic Chemistry, Getreidemarkt 9/163-OC, A-1060 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 April 2011
Received in revised form 30 January 2012
Accepted 8 March 2012
Available online 17 March 2012
A cyclohexanone monooxygenase (CHMO) from the xenobiotic-degrading Polaromonas sp. strain JS666
was heterologously expressed in Escherichia coli, and its ability to catalyze enantio- and regiodivergent
oxidations of prochiral and racemic ketones was investigated. The expression system was also used to
evaluate this enzyme’s potential role in the oxidation of cis-1,2-dichloroethene (cDCE), a groundwater
pollutant for which strain JS666 is the only known assimilator. The substrate enantiopreference and
-selectivity of the strain JS666 CHMO is similar to that of other CHMO-type enzymes; of note is this
enzyme’s excellent stereodiscrimination of 2-substituted cyclic ketones. The expression system exhibits
no activity with ethene or cDCE as substrates under the tested conditions. Phylogenetic analysis shows
that sequence variability among cyclohexanone monooxygenases could be a rich source of new enzyme
activities and attributes.
Keywords:
Cyclohexanone monooxygenase
Enantioselective oxidation
Baeyer-Villiger oxidation
Chiral lactone
© 2012 Elsevier B.V. All rights reserved.
Cis-1,2-dichloroethene
1. Introduction
Here we report the cloning and heterologous expression of a
CHMO from Polaromonas sp. strain JS666, a ␤-proteobacterium
Cyclohexanone monooxygenases (CHMOs) are a subset of the
family of enzymes known as Baeyer-Villiger monooxygenases
(BVMOs; E.C. 1.4.13.22), so named because they perform the clas-
sical Baeyer-Villiger oxidation reaction. Enzymatic Baeyer-Villiger
oxidations have several advantages over their traditional chemi-
cal counterparts; namely, they can achieve greater chemo-, regio-
and enantioselectivity while avoiding the use of potentially haz-
ardous organic peracids and organometallic catalysts [1,2]. They
have been a popular target of study over the last few decades
because of their versatility and enantiopure production of chiral
lactones, which are valuable building blocks in the pharmaceutical
industry.
capable of degrading a wide variety of xenobiotic compounds,
including the recalcitrant groundwater contaminant cis-1,2-
dichloroethene (cDCE) [7]. Differences in potentially important
amino acid residues are noted among the consortium of heterol-
ogously expressed CHMOs, and the phylogenetic relationship of
CHMOJS666 to these other enzymes is illustrated. The acceptance
and enantioselective oxidation of 32 ketone substrates is evaluated
and compared to reference biotransformations. The new heterolo-
gous expression system is also used to test the previously published
hypothesis that CHMOJS666 is involved in cDCE and ethene oxidation
[8].
The CHMO found in Acinetobacter sp. NCIMB 9871 is the
most extensively researched enzyme in this group, and has
exhibited activity on more than 100 ketone substrates [3,4].
Novel CHMOs have been discovered and characterized more
recently through genome mining and heterologous expression;
this increased enzyme availability has provided opportunities to
discover diverse activities and selectivities and to explore the
effects of changes in particular regions of the protein sequence
[5,6].
2. Experimental
2.1. Creation of heterologous expression system
The target CHMO gene (1620 bp, GenBank Accession No.
YP 552312) was amplified as a blunt-end PCR product with native
start- and stop-codons, using the proofreading Pfx DNA polymerase
(Invitrogen). The amplified gene was ligated into the pET 101/D-
TOPO vector (Invitrogen) to create the expression vector pAA2,
and was then sequenced to check for accuracy against its native
338 kb plasmid sequence (GenBank Accession No. CP000318).
Escherichia coli BL21 Star (DE3) from Invitrogen was used as the
expression strain for all analyses.
∗
Corresponding author. Tel.: +1 319 335 5065; fax: +1 319 335 5660.
E-mail address: tim-mattes@uiowa.edu (T.E. Mattes).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2012.03.002

106
A.K. Alexander et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 105–110
Fig. 1. Phylogenetic relationships between CHMO protein sequences, where branch length is proportional to inferred evolutionary distance, and GenBank protein acces-
sion numbers are as follows: Acidovorax sp. JS42: YP 986353; Acinetobacter sp. NCIMB 9871: BAA86293; Acinetobacter SE19: AAG10021; Arthrobacter sp. BP2: AAN37479;
Arthrobacter sp. L661: ABQ10653; Brachymonas petroleovorans: AAR99068; Polaromonas sp. JS666: YP 552312; Rhodococcus sp. HI-31: BAH56677; Rhodococcus sp. Phi1:
AAN37494; Rhodococcus sp. Phi2: AAN37491; Rhodococcus sp. TK6: AAR27824; Xanthobacter flavus: CAD10801. Sequences were aligned over 512 positions using MEGA4 [9];
the JS666 CHMO has 539 predicted amino acid residues.
2.2. CHMO expression assays: ketone substrates
soluble and insoluble fractions was assessed for a variety of temper-
atures and IPTG concentrations to determine expression conditions
that would maximize the amount of soluble CHMO.
Baffled Erlenmeyer flasks were used for preculture and cultiva-
tion broth growing. A LBamp preculture (200 mg/L ampicillin) was
inoculated with a bacterial single colony from an agar plate and
incubated at 37 ◦C in an orbital shaker overnight. The cultivation
medium (LBamp) was then inoculated with 2% (v/v) of the preculture
and incubated for 1–2 h under the same conditions until an optical
density of 0.2–0.6 was reached. IPTG (0.2 mM) and ␤-cyclodextrin
(4 mM) were added; the mixture was thoroughly mixed and split
in 1.0 mL aliquots into multi-well plates. Substrates were added as
0.8 M solutions in 1,4-dioxane to a final concentration of 4 mM. The
plates were sealed with adhesive film and incubated at 24 ◦C in an
orbital shaker for 24 h.
At selected time points during ketone oxidation assays, 0.5 mL
of culture medium was extracted with ethyl acetate plus methyl
benzoate as internal standard. The organic phase was removed and
dried over anhydrous MgSO4, then analyzed by chiral GC with an
Agilent 6897N gas chromatographer fitted with a Cyclodextrin E
column (Solutions and Tools GmbH, 25 m × 0.25 mm × 0.125 ␮m),
FID, and H2 carrier gas. Normal lactone peaks (see Section 3.5) were
identified through meta-chloroperbenzoic acid (mCPBA) oxidation
of racemic ketones, using a biphasic reaction protocol adapted from
Mohan and Whalen [11]. Ketone substrates were also oxidized by
E. coli cultures expressing the Acinetobacter sp. NCIMB 9871 CHMO
gene from vector pMM4 [12], using the same transformation and
culture conditions as described for pAA2. As the enantioselectivity
of the Acinetobacter CHMO is known for these substrates, these oxi-
dations provided identification controls for R- and S-enantiomers in
the chiral GC analysis. Enantioselectivity (E) values were calculated
using the Selectivity software developed by Faber et al. [13].
Headspace samples (100 ␮L) were taken from the cDCE- and
ethene-containing cultures at selected time points, and ana-
lyzed by GC; cDCE on a J&W Scientific GSQ capillary column
(30 m × 0.25 mm × 0.53 ␮m), and ethene and epoxyethane on a
Supelco 1% SP-1000 60/80 Carbopack B packed column, both with
FID and N2 carrier gas.
2.3. CHMO expression assays: alkene substrates
A freshly transformed overnight E. coli BL21 Star (DE3) culture
carrying the expression vector pAA2 was inoculated at 1% (v/v) in
LB broth with 200 mg/L ampicillin to begin each expression assay.
Cultures were grown at 23 1 ◦C with shaking at 200 rpm until an
OD600 of 0.5–0.7 was reached, at which point they were induced
with 0.1 mM (final concentration) IPTG and transferred to 20 mL
glass serum bottles sealed with PTFE-coated rubber septa (4 mL
per bottle). Neat cDCE (97%, Sigma) was dissolved in ethanol and
added to a final concentration of 2.5 mM (1% ethanol (v/v)) 2 h post-
induction. Gaseous ethene (4.1 ␮mol per bottle) was added to the
appropriate cultures through 0.22 ␮m pore-size filtered syringes
2 h after induction with IPTG.
3. Results and discussion
3.1. Phylogenetic analysis
2.4. Analytical methods
Investigations into sequence relationships between CHMO-type
enzymes can reveal both conserved domains and regions of vari-
ation that may account for differences in substrate acceptance
and enantioselectivity, and it can be expected that in general,
closely related sequences will exhibit similar activities. The phy-
logenetic relationships of sequenced CHMOs whose activities have
been verified through heterologous expression are shown in Fig. 1.
CHMOJS666 is most similar to CHMO from Brachymonas petroleovo-
rans (66% amino acid identity), and its sequence also clusters
with the CHMOs from Xanthobacter flavus and Acidovorax sp. JS42.
The CHMOJS666 has 62% amino acid identity with the well-studied
Acinetobacter sp. NCIMB 9871 CHMO. These amino acid similarities
Sequence alignment of heterologously expressed CHMO gene
sequences and phylogenetic tree calculation were conducted in
MEGA4, using the neighbor-joining method [9].
An adapted protocol for the EPICENTRE EasyLyse Bacterial Pro-
tein Extraction Solution [10] was used to separate the soluble and
insoluble protein fractions in total E. coli cell extracts obtained
by French press 2 h after induction with IPTG. These fractions
were resuspended in SDS loading buffer (Bio-Rad), heated at 95 ◦C
for 1 min, and then resolved with SDS-PAGE using precast Crite-
rion 12.5% polyacrylamide gels (Bio-Rad) and a constant 200 V for
55 min. The relative intensity of the CHMO protein band in the

A.K. Alexander et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 105–110
107
Table 1
Desymmetrization of prochiral cycloketones (substrates 1a–15a).
Substrate
R, Rꢀ, X
Compound no.
JS666
Reference biotransformation
% Conv.a
% eeb
% Yield/% Conv.a
% eeb
Biocatalyst
Reference
Bn
1
2
+
+
78 (−)
96 (−)
+++
+++
72
+++
61
88 (−)
95 (−)
79 (+)
99 (−)
75 (+)
CHMOXantho
CHMOXantho
CHMOBrevi1
CHMOXantho
CHMOBrevi1
[19]
[19]
[20]
[19]
[20]
3,4,5-triMeO-Bn
3,4-(OCH2O)-Bn
+++
36 (+)
3
Ph
4
5
+
+
97 (−)
98 (−)
+++
+++
83
98 (−)
98 (−)
64 (+)
CHMOXantho
CHMOXantho
CPMOComa
[19]
[19]
[21]
CO2Et
61
45
n.a.
n.a.
n.a.
n.a.
CHMOAcineto
CPMOComa
n.a.
[22]
[23]
n.a.
n.a.
n.a.
Me, Me
(CH2)5
++
6
7
n.c.
X = CH2
R = Me
X = O
R = H
X = N-Bn
R = H
+++
++
+++
++
97 (−)
99 (+)
n.a.
n.a.
n.a.
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
n.a.
[19]
[19]
n.a.
+
>99 (−)
n.a.
8
++
n.c.
9
n.a.
n.a.
10
11
X = CH2
n.c.
n.c.
n.c.
n.a.
n.a.
n.a.
71
n.c.
53
>99 (−)
n.a.
95 (+)
n.a.
CHMOXantho
CHMOAcineto
CPMOComa
n.a.
[19]
[24]
X = O
12
13
X = N-CO2Me
n.c.
n.a.
14
15
n.c.
+
n.a.
n.a.
n.a.
+++
n.a.
n.a.
n.a.
n.a.
CHMOAcineto
[25]
n.a.: not available, n.c.: no conversion, rac.: racemic.
a
Relative conversion (Conv.) of starting material determined by chiral phase GC after 24 h: +++ >90%, ++ 50–90%, + <50%.
Enantiomeric excess values determined by chiral phase GC; sign of optical rotation or absolute configuration is given in parentheses and assigned on the basis of reference
b
biotransformations.
still leave substantial room for sequence variation, which is a likely
source of novel activities, selectivities, and variations in enzyme
stability [6,14], and a compelling reason to continue to expand and
investigate the collection of known CHMOs.
All of the heterologously expressed CHMOs share the consen-
sus NADP+-binding motif and conserved substrate binding pocket
residues identified by Mirza et al. [1], but there are differences in
the “mobile loop” sequence hypothesized to flexibly close around
the substrate in the binding pocket (Fig. S1). These steric differences
may affect substrate acceptance and enantioselectivity, so variation
in these residues is a logical target of future research into CHMO
substrate range and enzyme engineering.
Scheme 1. Desymmetrization of prochiral cycloketones with BVMOs.
23 ◦C to 37 ◦C and IPTG concentrations from 0.01 mM to1.0 mM
were assessed with both soluble/insoluble protein fraction analysis
and cyclohexanone oxidation assays to find improved conditions
for CHMO expression and activity. Out of the tested combinations,
the best conditions for strain JS666’s CHMO activity in this heterolo-
gous expression system were found to be 23 ◦C (room temperature)
and 0.1–0.2 mM IPTG. No CHMO activity in the E. coli strain
3.2. Improvement of the CHMO expression system
Initial expression assays with E. coli BL21 Star (DE3) and plasmid
pAA2 using the vector manufacturer’s recommended conditions
(37 ◦C, 1.0 mM IPTG) exhibited no oxidation of cyclohexanone, even
though SDS-PAGE with total cell extracts showed high produc-
tion of a protein of the CHMO’s expected size. Given that insoluble
protein aggregates (or inclusion bodies) are often responsible for
reductions in heterologous protein activity [15], soluble/insoluble
protein fraction analysis was performed as described above, and
it was discovered that essentially all of the heterologous protein
was present in the insoluble phase (Fig. S2). Temperatures from
Scheme 2. Regiodivergent Baeyer-Villiger oxidations of fused bicyclic cyclobu-
tanones.

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A.K. Alexander et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 105–110
Table 2
Regiodivergent bio-oxidations of cycloketones (substrates 16a–25a).
Substrate
Compound no.
JS666
Reference Biotransformation
% Conv.a/ratiob
% eec
% Conv.a/ratiob
% eec
Biocatalyst
CHMOBrevi1
Reference
[26]
++d
38:62
8 (−)
85
51:49
96 (−)
16
17
62 (−)
>99 (−)
70
50:50
97 (−)
98 (−)
n.c.
n.c.
n.a.
n.a.
CHMOAcineto
CPDMO
[27]
[28]
78
48:52
98 (+)
89 (−)
18
+++
97:3
12 (+)
88 (n.d.)
n.c.
n.a.
CHMOAcineto
[29]
19
20
d
+
>99 (−)
36 (−)
+++/50:50
+++/100:0
95 (−), 94 (−)
CHMOAcineto
CPMOComa
[30,31]
[28]
63:36
rac.
+++
100:0
Complex product
mixture
e
e
e
21 (+)-menthone
22 (−)-menthone
n.a.
n.a.
n.a.
CHMOAcineto
[32,33]
[28]
+++
100:0
n.c.
n.c.
CPDMO
23 (+)-trans-
dihydrocarvone
+++
0:100
CHMOAcineto
[32,33]
24 (−)-trans-
+++
100:0
Complex product
mixture
e
n.a.
CHMOAcineto
[32,33]
dihydrocarvone
+
+++
0:100
25 (+)-trans-
carvomenthone
e
CHMOXantho
[34]
0:100
n.a.: not available, n.c.: no conversion, rac.: racemic.
a
Relative conversion (Conv.) of starting material determined by chiral phase GC after 24 h: +++ > 90%, ++ 50–90%, + < 50%.
Ratio of regioisomers (normal:abnormal).
Enantiomeric excess values determined by chiral phase GC; sign of optical rotation or absolute configuration is given in parentheses and assigned on the basis of reference
b
c
biotransformations; eenormal in italics, eeabnormal in normal font.
d
Starting material was racemic within measurement error.
Starting material was optically pure; no epimerization observed.
e
carrying pAA2 was observed at 37 ◦C (the manufacturer’s recom-
mended temperature), and only minimal activity was detected at
30 ◦C. Finding the optimal activity at a low temperature is logical
for a heterologously expressed protein from strain JS666, which has
a growth optimum between 20 and 25 ◦C [16].
contaminant cDCE [16]. The CHMO gene in strain JS666 is encoded
on the smaller of two plasmids [7], and is upregulated during
growth on cDCE as a sole carbon source [17]. Strain JS666’s degra-
dation of cDCE is observed to be of two distinct types: one relatively
rapid and maintainable over successive batch cultures (growth-
coupled), and one slow and limited in duration (cometabolic). It has
been hypothesized that CHMOJS666 is responsible for cometabolic-
type cDCE degradation by catalyzing the production and eventual
accumulation of the toxic cDCE epoxide (2,3-dichlorooxirane) [8].
This CHMO alkene-oxidation explanation is consistent with the
3.3. Ethene and cDCE oxidation
Polaromonas sp. strain JS666 is unique in its ability to
couple growth to aerobic oxidation of the toxic groundwater

A.K. Alexander et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 105–110
109
Scheme 3. Regiodivergent oxidation of ␤-substituted cycloketones to proximal and
Scheme 4. Regiodivergent transformations of optically pure terpenones 28a–31a.
distal lactones.
observed increase in ethene epoxidation rate (and accumulation of
epoxyethane with no culture growth) in JS666 cultures following
exposure to cDCE [16] or cyclohexanol [unpublished results].
Under the conditions described (in which heterologously
expressed CHMOJS666 readily converted cyclohexanone to ␧-
caprolactone), no activity was observed with either ethene or
cDCE. Specifically, no degradation of cDCE or ethene or production
of epoxyethane (detection limit < 0.1 ␮mol/bottle) was measured
during the 24 h period following introduction of the substrate
to induced cultures. This result suggests that CHMOJS666 is not
involved in either growth-coupled or cometabolic cDCE or ethene
oxidation, and implies the need to reevaluate past hypotheses
about the cDCE degradation pathway(s) in strain JS666. One impor-
tant caveat is that the CHMO may behave differently in E. coli than
in its native host strain; in fact, post-translational modification of
this enzyme was proposed by Jennings et al. [17].
Scheme 5. Schematic view of a kinetic resolution of 2-substituted cyclic ketones
catalyzed by BVMOs.
class CHMOJS666 has the same enantiopreference and similar selec-
tivity as other CHMO-type enzymes.
3.5. Regiodivergent biooxidations of cycloketones
Substrates 16a–25a were tested to assess the biocatalyst’s abil-
ity to perform regiodivergent biooxidations (Table 2). In contrast
to chemical Baeyer-Villiger oxidations, several BVMOs catalyze
a parallel kinetic resolution: one enantiomer is converted to
the expected migration product (rearrangement of the more
nucleophilic residue to the normal lactone) while the other is trans-
formed to the regioisomeric product (abnormal lactone). In general,
starting material remains almost racemic during the course of the
reaction, as the difference in reaction rates is rather small. Regiodi-
vergent oxidations of fused bicyclic cyclobutanones, ␤-substituted
cycloketones, and optically pure terpenones are illustrated in
Schemes 2–4, respectively.
CHMOJS666 does not show a specific trend in these sub-
strate classes. Of three fused bicyclic cyclobutanones, only
16a is accepted and transformed with low enantioselectivity
(eenormal = 8%, eeabnormal = 62%). 3-Methylcyclohexanone 20a was
preferentially transformed to the normal lactone 20b with excel-
lent stereoselectivity (>99% ee), however at a slow rate (conversion
3.4. Desymmetrization of prochiral cycloketones
The Polaromonas strain JS666 CHMO shows moderate accep-
tance of symmetric cycloketones (substrates 1a–14a, Table 1,
Scheme 1). All three tested 3-substituted cyclobutanones were
converted, but only piperonyl compound 3a reached >90% conver-
sion within 24 h, however with poor enantioselectivity (36% ee).
4-Substituted cyclohexanones 4a and 5a were both oxidized to the
corresponding (−)-lactones with high optical purity (97 and 98%
ee), but again at a slow rate (<50% conversion). The same behav-
ior was observed for cis-3,5-dimethylcyclohexanone 8a. Bridged
bicyclic ketones 11a–14a were not accepted for Baeyer-Villiger oxi-
dation. A known side reaction, epoxidation of the double bond, was
also not detected. Only polycyclic substrate 15a, adamantanone,
was slowly converted to the lactone. Generally, in this substrate
Table 3
Kinetic resolutions of 2-substituted cycloketones (substrates 26a–31a).
Substrate
R
Compound no. JS666
% Conv.a
Reference Biotransformation
a
a
% eeS, % eeP
E
% Conv.a
% eeS, % eeP
E
Biocatalyst
Reference
18 (+)
70 (−)
>99 (R)
30 (S)
81 (S)
>99 (R)
71 (−)
>99 (+)
61 (S)
35 (+)
CHMOAcineto [35]
CHMOAcineto [36]
CHMOAcineto [36]
CHMOXantho [34]
CHMOAcineto [18]
Me
26
27
28
29
30
35
7
35
6
61 (−)
>98 (R)
95 (S)
>98 (S)
>98 (R)
76 (−)
>99 (+)
95 (S)
Et
92b
75b
41
>200
>200
>200
>200
79
>200
>200
>200
>200
CH2 CH CH2
59
40
Ph
CH2 CH2 CN
32
42
99 (R)
97 (R)
n.a.
n.a.
13
45
n.c.
n.a.
CHMOXantho [28]
Bn
31
20
3
n.a.: not available, n.c.: no conversion, rac.: racemic.
a
Relative conversion (Conv.) of starting material and enantiomeric excess values determined by chiral phase GC; sign of optical rotation or absolute configuration is given
in parentheses and assigned on the basis of reference biotransformations; eeS (substrate) in italics, eeP (product) in normal font.
b
Expression assays for these substrates were conducted with the conditions described for alkene substrates (Section 2.3). 27a and 28a were dissolved in ethanol and added
to a final concentration of 2.5 mM (1% ethanol (v/v)) 2 h post-induction.

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A.K. Alexander et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 105–110
<50% after 24 h). Biotransformations with CHMOJS666 of terpenones
21a–25a yielded mostly either untouched starting material (22a
and 23a) or complex product mixtures (21a and 24a). The compo-
sition of these mixtures and identity of byproducts was not further
examined within this study. Only (+)-trans-dihydrocarvone 25a
was cleanly converted to abnormal lactone 25c at a low rate.
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Acknowledgments
Funding for this work was provided by the University of Iowa’s
Center for Biocatalysis and Bioprocessing, the Iowa Biotechnology
Byproducts Consortium, and a National Science Foundation Grad-
uate Research Fellowship for A.K.A. D.B. gratefully acknowledges a
postdoctoral fellowship by the Austrian Exchange Service (OeAD)
within the Austria/Czech Republic scientific cooperation program.
M.J.F. is generously funded by EU-FP7 within the project OxyGreen
(Grant# 212281).
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2012.03.002.