In situ formation of H2O2 for P450 peroxygenases

Article abstract of DOI:10.1016/j.bmc.2014.05.074

An in situ H₂O₂ generation approach to promote P450 peroxygenases catalysis was developed through the use of the nicotinamide cofactor analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) and flavin mononucleotide (FMN). Final productivity could be enhanced due to higher enzyme stability at low H₂O₂ concentrations. The H₂O₂ generation represented the rate-limiting step, however it could be easily controlled by varying both FMN and BNAH concentrations. Further characterization can result in an optimized ratio of FMN/BNAH/O₂/biocatalyst enabling high reaction rates while minimizing H₂O₂-related inactivation of the enzyme.

Full text of DOI:10.1016/j.bmc.2014.05.074

Bioorganic & Medicinal Chemistry 22 (2014) 5692–5696
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
In situ formation of H2O2 for P450 peroxygenases
Caroline E. Paul a, , Ekaterina Churakova a, , Elmer Maurits a, Marco Girhard b, Vlada B. Urlacher b,
,
⇑
Frank Hollmann a,
⇑
a Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands
b Institute of Biochemistry, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
An in situ H2O2 generation approach to promote P450 peroxygenases catalysis was developed through
the use of the nicotinamide cofactor analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) and flavin
mononucleotide (FMN). Final productivity could be enhanced due to higher enzyme stability at low
H2O2 concentrations. The H2O2 generation represented the rate-limiting step, however it could be easily
controlled by varying both FMN and BNAH concentrations. Further characterization can result in an opti-
mized ratio of FMN/BNAH/O2/biocatalyst enabling high reaction rates while minimizing H2O2-related
inactivation of the enzyme.
Received 18 April 2014
Revised 23 May 2014
Accepted 28 May 2014
Available online 11 June 2014
Keywords:
P450 peroxygenases
mNADHs
Ó 2014 Elsevier Ltd. All rights reserved.
In situ H2O2 generation
Hydrogen peroxide shunt
Oxyfunctionalization
1. Introduction
chemical oxidation/oxyfunctionalisation catalysts. Furthermore,
both enzymes were demonstrated to exhibit significant activity in
Synthetic nicotinamide cofactor analogues (mNADHs) such as
1-benzyl-1,4-dihydronicotinamide (BNAH, Scheme 1) were previ-
ously shown to be functional mimics of their natural, more complex
counterparts.1 For example, they have been demonstrated to pro-
mote alcohol dehydrogenase-catalyzed reduction reactions,2–4
monooxygenase-catalyzed oxyfunctionalizations,5,6 and, more
recently, to be excellent substitutes for NAD(P)H in ene-reductase-
mediated C@C-bond reductions.7
In the course of our ongoingefforts to exploit the syntheticpoten-
tial of mNADHs for biocatalysis, we realized that the reactivity of, for
example, BNAH towards free flavins exceeds the reactivity of their
native counterparts by orders of magnitude. Combined with the
high reactivity of reduced flavins towards molecular oxygen (yield-
ing oxidized flavins and hydrogen peroxide),8 this might be
exploited as a convenient system for the catalytic in situ generation
of H2O2 to promote peroxidase reactions (Scheme 2).
the so-called hydrogen peroxide shunt pathway,9–11 as does also
P450Sp from Sphingomonas paucimobilis—the first characterized
a
enzyme of the CYP152 subfamily.12 That is, these enzymes are capa-
ble of forming the catalytically active ferric hydro-peroxy species
directly from the resting state of the enzyme using H2O2 as oxidant.
From a preparative point of view, this reactivity is highly interesting
as it circumvents the need of an expensive nicotinamide cofactor
together with a regeneration system, as well as the (likewise com-
plicated) electron transport chain.13–15 Unfortunately, heme-
dependent enzymes are prone to fast inactivation in the presence
of elevated H2O2 concentrations due to oxidative modification of
the heme prosthetic group.16 Therefore, stoichiometric addition of
H2O2 has to be avoided. In recent years, several in situ H2O2 gener-
ation methods based on the reductive activation of molecular oxy-
gen have been reported, amongst them, enzymatic,17 chemical,18
electrochemical 19–22 and photochemical systems.11,23–25
To test our hypothesis, we chose the cytochrome P450 peroxy-
The aim of the present study was to evaluate the suitability of
the proposed chemical, flavin-catalyzed in situ H2O2-generation
genases P450Bsb (CYP152A1) from Bacillus subtilis9 and P450Cl
a
(CYP152A2) from Clostridium acetobutylicum.10 P450Bsb and
system to promote P450Cl and P450Bsb-catalyzed oxyfunctionali-
a
P450Cl catalyze the
a
- and b-hydroxylation of fatty acids thereby
sation reactions.
a
exhibiting an interesting chemical reactivity not found amongst
2. Results and discussion
2.1. In situ generation of H2O2
⇑
Corresponding authors. Tel.: +49 211 8113889 (V.B.U.), +31 15 2781957 (F.H.).
E-mail addresses: vlada.urlacher@hhu.de (V.B. Urlacher), f.hollmann@tudelft.nl
In a first set of experiments, we evaluated the parameters influ-
encing the H2O2-generation rate of the system BNAH/FMN/O2.
(F. Hollmann).
Both authors contributed equally.
http://dx.doi.org/10.1016/j.bmc.2014.05.074
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

C. E. Paul et al. / Bioorg. Med. Chem. 22 (2014) 5692–5696
5693
NH2
O
NH2
O
P
O
P
O
N
O
O
N+
N
O
O
O
OH OH
N+
NH2
OH
HO
N
BNA+
HO
OX
NAD+, X = H
NADP+, X = PO3H2
N
2H+ + 2 e-
NH2
O
NH2
O
P
O
P
O
H
H
O
O
N
O
O
O
N
N
H
H
OH OH
NH2
N
OH
HO
N
HO
OX
NADH,
N
BNAH
X = H
NADPH, X = PO3H2
Scheme 1. Comparison of the structures and basic electrochemical features of natural nicotinamide cofactors and synthetic analogues such as BNA.
For this, we varied independently the concentrations of BNAH
and FMN (Fig. 1). It is worth mentioning here that the reaction rate
can be determined by either following the characteristic UV-
absorption of BNAH (at 360 nm), or by following the concentration
of dissolved oxygen, or by quantifying the amount of H2O2 formed.
Within experimental error all methods gave the same results.
As shown in Figure 1, the initial reaction rate linearly depended
on both, the concentration of BNAH and FMN. A significant devia-
tion from linearity was observed only in the presence of very high
concentrations of either component. Considering the rather high
O2-reduction rates, we assume that this deviation from linearity
may be due to the poor solubility of O2 in aqueous media (ca.
0.25 mM) and O2-diffusion from the gas phase to the reaction mix-
ture becoming overall rate-limiting.
Overall, we concluded that the BNAH/FMN/O2 system allows
for highly predictable and reproducible in situ generation of
H2O2 necessary to promote P450-peroxygenase catalyzed
oxyfunctionalizations.
Figure 1. Initial depletion rate of BNAH with varying BNAH (
D) concentration
([FMN] = 120 M), and FMN (j) concentration ([BNAH] = 85 M).
l
l
2.2. Enzymatic hydroxylation
and 3, respectively (Scheme 1). The first experiments were per-
formed under arbitrarily chosen conditions with cell-free extracts
for 20 min. Pleasingly, the enzyme-catalyzed hydroxylation pro-
Once the generation of H2O2 with BNAH and FMN was
established, the process was applied for the hydroxylation of myris-
tic acid 1 by the heme-containing peroxygenases P450Cl and
a
ceeded smoothly, leading to 75% and 95% conversion for P450Cl
P450Bsb to obtain the 2- and/or 3-hydroxymyristic acid products 2
a
O
O
R
H
α
HO
NH2
N
N
O
9
2
OH
O
O2 H2
+
N
N
NH
N
H
O
O
O
H
O
HO
β
9
P450
3
O
R
N
NH2
N
O
O
O2
H2
NH
HO
N
9
1
Scheme 2. Proposed chemoenzymatic system for the hydroxylation of fatty acids. BNAH is used as stoichiometric reductant reducing FMN (flavin adenine mononucleotide).
The reduced FMN quickly reacts with molecular oxygen to yield H2O2 (hence FMN acts as transfer hydrogenation catalyst between BNAH and O2). Finally, H2O2 regenerates
the catalytically active ferric hydro-peroxy species in P450 peroxygenases (such as P450Bsb and P450Cl ) to perform selective hydroxylation of, for example, myristic acid.
a

5694
C. E. Paul et al. / Bioorg. Med. Chem. 22 (2014) 5692–5696
Figure 2. Hydroxylation time course of myristic acid with in situ H2O2 generation; P450Cl (N), P450Bsb (h). Left: with cell-free extracts; right: with purified enzymes. Tris–
a
HCl buffer (50 mM, pH 7.5), [myristic acid] = 200 lM, [BNAH] = 600 lM, [FMN] = 60 lM, [P450] = 1 lM, 30 °C.
and P450Bsb, respectively (Fig. 2, left). The product distribution (a:b
ratio of the hydroxylated products 2 and 3) was 24:1 for P450Cl and
a
1:1.7 for P450Bsb, respectively. These results are in good accordance
with the product distributions observed previously and therefore
appears to reflect the intrinsic selectivity of these enzymes.9À11,26
It should be mentioned here that no other (by)products than the
ones mentioned have been observed in all reactions.
Performing the same reactions with purified enzyme led to even
higher product concentrations, achieving full conversion after only
5 min (Fig. 2, right). We attribute this apparent higher activity to the
presence of endogenous catalases in the crude Escherichia coli cell
extracts competing with P450Cl or P450Bsb for the H2O2 generated.
a
In the case of the purified enzymes, both exhibited identical
activities (turnover frequency of 40 minÀ1), which may indicate that
the chemocatalytic H2O2 generation reaction is overall rate-
limiting. In fact, with 40 minÀ1 the catalytic activities significantly
fall back behind the rates observed with stoichiometric amounts
of H2O2 being 200 and 1200 minÀ1 for P450Cl or P450Bsb, respec-
a
tively.9,10 Under these conditions, however, rapid inactivation of
the enzymes was also observed resulting in maximally 40% conver-
sion of the starting material. Therefore, we conclude that the lower
activity in our case is over-compensated by the higher robustness of
the reaction scheme leading to overall higher productivities. Similar
observations were made using photochemical in situ generation of
H2O2.11
Figure 4. Product 2 and 3 formation with varying FMN concentrations; P450Cl (N),
a
P450Bsb (h). Tris–HCl buffer (50 mM, pH 7.5), [myristic acid] = 200
[BNAH] = 600 M, [P450] = 1 M (purified enzyme), 30 °C, 5 min.
lM,
l
l
BNAH and FMN while keeping all other parameters constant (Figs. 3
and 4). Especially increasing the BNAH concentration had a signifi-
cant influenceon the overall reaction rate (Fig. 3). However, it should
be mentioned here that these experiments were performed using
crude cell extracts putatively containing endogenous E. coli cata-
lases, resulting in an unproductive consumption of H2O2. Increasing
2.3. BNAH and FMN concentrations
Hoping to optimize the enzymes’ performance by increasing the
H2O2 generation rate, we systematically varied the concentrationsof
the FMN concentration above 25
lM did not result in any significant
increase of the overall reaction rate (Fig. 4). Interestingly enough, in
both series a maximal activity of both, P450Cl and P450Bsb of ca.
a
35 minÀ1 was observed, corresponding to a product accumulation
rate of ca. 2.1 mM hÀ1. This corresponds well to the value under
which in previous studies O2 diffusion limitation has been suspected
to become overall rate-limiting.23
We therefore expect that further studies optimizing the aera-
tion of the reaction mixtures (or reducing the biocatalysts’ concen-
trations) will yield higher activities for P450Cl and P450Bsb
.
a
2.4. Semi-preparative scale
Encouraged by the promising results of the process, a semi-
preparative scale of the P450Cl -catalyzed hydroxylation was
a
carried out with 10 mM of myristic acid 1. Despite the low solubility
of both BNAH and myristic acid 1, we avoided using co-solvents
and performed the reaction as a suspension containing the aque-
ous mixture of the insoluble substrates. However, the reaction
Figure 3. Turnover frequency (TF) with varying BNAH concentrations; P450Cl (N),
a
P450Bsb (h). Tris–HCl buffer (50 mM, pH 7.5), [myristic acid] = 200 lM,
[FMN] = 60 lM, [P450] = 1 lM (cell-free extracts), 30 °C, 5 min.

C. E. Paul et al. / Bioorg. Med. Chem. 22 (2014) 5692–5696
5695
yielded only 4% of the hydroxylated products 2 and 3 over a reac-
tion period of 70 h. Even though this corresponds to a doubling of
the overall product concentration compared to the experiments
described above, the overall yield was disappointingly low. Moder-
ate shaking was applied to maintain the suspension; hence, the
enzyme may have undergone mechanical stress caused by the sus-
pension. Or the surfactant properties of the reagents may have
impaired the enzyme stability. Also, inhibitory effects of the ele-
vated reagent concentrations cannot be excluded at present. Fur-
ther studies are necessary to clarify the aspects of P450
peroxygenases stability and putative inactivation at elevated
(co)-substrates concentrations and to establish a practical system
for fatty acid hydroxylation.
H2O2-related inactivation of the enzyme. Especially optimized aer-
ation procedures are expected to fully exploit the preparative
potential of this new approach.
So far, we have used BNAH in stoichiometric amounts. Envisag-
ing preparative scale application of the in situ H2O2 generation
method presented here we are going to evaluate recycling of the
cofactor, as well as its in situ regeneration. For the latter approach,
especially organometallic catalysts such as [Cp⁄Rh(bpy)H]+ cou-
pling the reduction of BNA+ to oxidation of formic acid, appear
most promising.4–6
We believe that the novel in situ H2O2 system has a great poten-
tial and its future application can be extended to more biocatalytic
systems.
2.5. Control experiments
4. Experimental
Several control experiments were performed to detect the pres-
ence of possible background reactions. Thus, with respect to the
standard enzyme-catalyzed reaction with full conversion (Fig. 5,
column 1), the same reaction was carried out leaving out either
BNAH or FMN or both (Fig. 5, columns 2–4). While the controls
in the absence of BNAH were essentially negative, a small but sig-
nificant product formation in the absence of FMN (as the H2O2-
generation catalyst) was observed.
4.1. General details
All commercial reagents and solvents were purchased at the
highest purity available and used as received. Cell-free extracts as
well as purified P450Cl and P450Bsb were obtained as previously
a
reported.10 P450 concentrations of the enzyme preparations
were determined by CO-difference spectral assay with
e
450–490 = 91 mMÀ1 cmÀ1 27
. Catalase (2100 U/mg) from bovine liver
Principally, spontaneous aerobic oxidation of BNAH yielding
H2O2 may account for this. However, control experiments on the
stability of BNAH under the reaction conditions revealed that the
rate of this autoxidation is too low to explain the background activ-
ity observed. At present, we also cannot fully exclude the presence
of trace amounts of flavins (or other redox-active dyes originating
from E. coli) to have remained in the enzyme preparations.
was purchased from Sigma–Aldrich. Myristic acid, lauric acid, 2
hydroxymyristic acid, FMN, diethyl ether (Et2O), hydrochloric acid
(HCl), tris(hydroxymethyl)-aminomethane (Tris), ethyl acetate
(EtOAc), dimethylsulfoxide, and N,O-bis(trimethylsilyl) trifluoroac-
etamide (BSTFA) containing trimethylchlorosilane (99:1) were
purchased from Sigma–Aldrich in analytical pure grade and used
as received. Melting points were taken on a Büchi melting point
B-450 and are uncorrected. UV assays were performed on a
Shimadzu UV–Vis spectrophotometer UV-2401 PC, with disposable
plastic cuvettes of 1.5 mL in volume. NMR spectra were recorded on
a Bruker Avance spectrometer at 300 (1H) and 75 (13C) MHz. GC-MS
analyses were carried out on a Shimadzu GC-2010 apparatus
coupled to a Shimadzu GCMS-QP2010S mass-selective detector
with an AOC-20i auto-injector.
3. Conclusions
Overall, we demonstrated that the novel in situ H2O2 generation
method presented here is a promising approach to promote P450
peroxygenases catalysis. Thus, the overall productivity could be
enhanced due to higher enzyme operational stability at tailored
H2O2 generation rates. Even though this work reports the proof-
of-concept only, the most important parameters determining the
efficiency to the overall system have been identified. The
generation of H2O2 was the rate-limiting step, however it could be
easily controlled via appropriate FMN and BNAH concentrations.
Further characterization will result in an optimized ratio of FMN/
BNAH/O2/biocatalyst enabling high reaction rates while minimizing
4.2. Synthesis
4.2.1. 1-Benzyl-3-carbamoylpyridinium bromide (BNA+)
Following
a
previous procedure.7 Nicotinamide (4.88 g,
40 mmol) was dissolved in acetonitrile (40 mL) and benzyl bromide
(4.8 mL, 40 mmol) was added. The reaction mixture was refluxed for
15 h, after which time a precipitate was observed. The solution was
cooled and Et2O (50 mL) was added to further precipitate the final
product. After filtering and washing with Et2O (3 Â 10 mL), the bro-
mide salt 1-benzyl-3-carbamoylpyridium bromide BNA+ was
obtained as a white solid powder (10.9 g, 93%). Mp: 214–215 °C;
the NMR data recorded corresponded to the previous procedure.7
4.2.2. 1-Benzyl-1,4-dihydronicotinamide (BNAH)
Following a previous procedure.7 Under nitrogen atmosphere, 1-
benzyl-3-carbamoylpyridinium bromide BNA+ (2.93 g, 10 mmol)
was dissolved in distilled H2O (60 mL) and NaHCO3 (4.20 g,
50 mmol) was added. Sodium dithionite Na2S2O4 (8.71 g, 50 mmol)
was then added in small portions and the reaction mixture was stir-
red at room temperature for 3 h in the dark, during which time the
solution turned from orange to yellow as the yellow product precip-
itated. The solid was filtered, washed with ice-cold H2O (3 Â 10 mL)
and dried over phosphorus pentoxide (P2O5) under reduced pres-
sure to afford product 1-benzyl-1,4-dihydronicotinamide BNAH as
a bright yellow powder (1.71 g, 80%). Mp: 111–114 °C; the NMR
data recorded corresponded to the previous procedure.7
Figure 5. Product 2 and 3 formation with control reactions; P450Cl (grey), P450Bsb
a
(white). Tris–HCl buffer (50 mM, pH 7.5), [myristic acid] = 200
lM, [FMN] = 60 lM,
[BNAH] = 600 M, [P450] = 1 M (purified enzyme), 30 °C, 20 min.
l
l

5696
C. E. Paul et al. / Bioorg. Med. Chem. 22 (2014) 5692–5696
4.3. Enzyme-catalyzed reaction conditions
4.6. Oxygen Clark electrode
The following buffers and stock solutions were used: Tris–HCl
buffer (50 mM, pH 7.5), FMN (1 mM aqueous solution), myristic
acid (20 mM in DMSO), lauric acid (20 mM in DMSO). Fresh stock
solution of BNAH (10–100 mM) was prepared in methanol before
each use.
The reactions were performed in microcentrifuge plastic tubes
(2 mL in volume) at 30 °C, under ambient atmosphere, unless
otherwise mentioned. The reaction mixture contained Tris–HCl
buffer (50 mM, pH 7.5; final volume of 1 mL), myristic acid
Oxygen consumption was measured on Strathkelvin 782
2-Channel oxygen system using Strathkelvin 1302 Clark-type
microcathode oxygen electrodes. The measurements were done
in a water-jacketed respiration chamber made from precision bore
glass covered by aluminium foil to prevent flavin illumination by
light; the water-jacket was connected to a thermally controlled
water system at 25 °C. The chamber was placed on a magnetic stir-
rer. The electrode was calibrated prior to measurements.
The following buffers and concentrations were used: phosphate
(200
l
M), FMN (40–250
lM), BNAH (200–600
l
M), P450Cl
buffer KPi (50 mM, pH 7.0), BNAH (100 lM), FMN (10–250 lM).
a
(1 M) or P450Bsb (1
tion assays were mixed at 1200 rpm for 5–20 min depending on
the experiment. The reaction was stopped at the indicated time
intervals through the addition of concentrated HCl (40
w/w) followed by addition of lauric acid (10 L) as an external
standard. The reaction mixtures were extracted twice with 1 mL
Et2O. After evaporation of the Et2O, the remaining reagents were
l
l
M), DMSO (1% v/v), MeOH (1% v/v). The reac-
The buffer was saturated with oxygen prior to usage. The reactions
were initiated by the addition of FMN and the electrode holder was
immersed into the chamber immediately. Oxygen consumption
was followed by the decrease in oxygen concentration using the
Strathkelvin software. All experiments were performed in tripli-
cates obtaining a standard deviation below 15%. The oxygen con-
sumption rate correlated with the BNAH depletion rate (Fig. 1).
lL, 37%
l
dissolved in BSTFA (200 lL) and incubated in a thermoshaker for
30 min at 80 °C and 1200 rpm. After cooling down to room temper-
ature the samples were transferred to the glass GC vials and ana-
lyzed by GC or GC-MS. All control experiment were performed
identically excluding one of the reaction components. To confirm
the peroxygenase activity standard reaction assay was performed
upon addition of catalase from bovine liver (2100 U/mg).
Acknowledgments
This work was supported by the Marie Curie IEF ‘BIOTRAINS’
and ‘BIOMIMIC’ financed by the European Union through the 7th
Framework People Programme (Grant agreement number 238531
and 327647). Maarten Gorseling (Delft University of Technology)
and Melanie Wachtmeister (Heinrich-Heine University Düsseldorf)
are gratefully acknowledged for their technical assistance.
The semi-preparative scale was performed with the following
reaction conditions: Tris–HCl buffer (50 mM, pH 7.5), [myristic
acid] = 10 mM, [FMN] = 60 lM, [P450Cl ] = 1 lM, [BNAH] = 50
a
mM, at 30 °C during 70 h.
References and notes
1. Paul, C. E.; Arends, I. W. C. E.; Hollmann, F. ACS Catal. 2014, 4, 788.
2. Sicsic, S.; Durand, P.; Langrene, S.; Goffic, F. L. E. Eur. J. Biochem. 1986, 155, 403.
3. Sicsic, S.; Durand, P.; Langrene, S.; Legoffic, F. FEBS Lett. 1984, 176, 321.
4. Lo, H. C.; Fish, R. H. Angew. Chem., Int. Ed. 2002, 41, 478.
5. Ryan, J. D.; Fish, R. H.; Clark, D. S. ChemBioChem 2008, 9, 2579.
6. Lutz, J.; Hollmann, F.; Ho, T. V.; Schnyder, A.; Fish, R. H.; Schmid, A. J.
Organomet. Chem. 2004, 689, 4783.
7. Paul, C. E.; Gargiulo, S.; Opperman, D. J.; Lavandera, I.; Gotor-Fernandez, V.;
Gotor, V.; Taglieber, A.; Arends, I. W. C. E.; Hollmann, F. Org. Lett. 2013, 15, 180.
8. Massey, V. J. Biol. Chem. 1994, 269, 22459.
9. Matsunaga, I.; Ueda, A.; Fujiwara, N.; Sumimoto, T.; Ichihara, K. Lipids 1999, 34,
841.
10. Girhard, M.; Schuster, S.; Dietrich, M.; Dürre, P.; Urlacher, V. B. Biochem.
Biophys. Res. Commun. 2007, 362, 114.
11. Girhard, M.; Kunigk, E.; Tihovsky, S.; Shumyantseva, V. V.; Urlacher, V. B.
Biotechnol. Appl. Biochem. 2013, 60, 111.
4.4. GC analysis
The progress of the reaction was followed using gas chromatog-
raphy (GC) and the final product concentrations were calculated
based on calibration curve equations using an external
standard (lauric acid). GC-MS analysis was performed with He as
carrier gas and
(25 m  0.25 mm  0.4
a
Varian FactorFour VF-1ms column
m). Column oven program: 50 °C, 60 °
l
C/min to 212 °C, 8 min, 60 °C/min to 325 °C, 5 min; linear velocity:
37 cm/s; split ratio: 5. Characteristic mass fragmentation patterns
were obtained from the EI-MS using ion source (200 °C); scan
mode with an m/z range 40–600 in 0.5 s.
12. Matsunaga, I.; Yamada, M.; Kusunose, E.; Nishiuchi, Y.; Yano, I.; Ichihara, K.
FEBS Lett. 1996, 386, 252.
Myristic acid (retention time tR 5.54 min), lauric acid (tR
4.25 min) and 2-hydroxymyristic acid (tR 7.45 min) were also
assigned using authentic standards. The peak of 3-hydroxymyristic
acid at 7.69 min was identified by the mass fragmentation pattern
13. Urlacher, V. B.; Girhard, M. Trends Biotechnol. 2012, 30, 26.
14. Urlacher, V. B.; Eiben, S. Trends Biotechnol. 2006, 24, 324.
15. Urlacher, V.; Schmid, R. D. Curr. Opin. Biotechnol. 2002, 13, 557.
16. Valderrama, B.; Ayala, M.; Vazquez-Duhalt, R. Chem. Biol. 2002, 9, 555.
17. van Deurzen, M. P. J.; van Rantwijk, F.; Sheldon, R. A. Tetrahedron 1997, 53,
13183.
in the GC-MS chromatogram. The regioselectivity for a:b hydroxyl-
ation were 1:1.7 for P450Bsb and 24:1 for P450Cl
.
a
18. Karmee, S. K.; Roosen, C.; Kohlmann, C.; Lütz, S.; Greiner, L.; Leitner, W. Green
Chem. 2009, 11, 1052.
19. Lutz, S.; Vuorilehto, K.; Liese, A. Biotechnol. Bioeng. 2007, 98, 525.
20. La Rotta Hernandez, C. E.; Lütz, S.; Liese, A.; Bon, E. P. S. Enzyme Microb. Technol.
2005, 37, 582.
21. Getrey, L.; Krieg, T.; Hollmann, F.; Schrader, J.; Holtmann, D. Green Chem. 2014,
16, 1104.
22. Krieg, T.; Huttmann, S.; Mangold, K.-M.; Schrader, J.; Holtmann, D. Green Chem.
2011, 13, 2686.
23. Churakova, E.; Arends, I. W. C. E.; Hollmann, F. ChemCatChem 2013, 5, 565.
24. Churakova, E.; Kluge, M.; Ullrich, R.; Arends, I.; Hofrichter, M.; Hollmann, F.
Angew. Chem., Int. Ed. 2011, 50, 10716.
25. Perez, D. I.; Mifsud Grau, M.; Arends, I. W. C. E.; Hollmann, F. Chem. Commun.
2009, 6848.
26. Honda Malca, S.; Girhard, M.; Schuster, S.; Dürre, P.; Urlacher, V. B. Biochimica
et Biophysica Acta (BBA)—Proteins Proteomics 2011, 1814, 257.
27. Omura, T.; Sato, R. J. Biol. Chem. 1964, 239, 2370.
4.5. UV spectrophotometry
The depletion rate of BNAH was determined with a UV–Vis
spectrophotometer following the decrease of the characteristic
UV absorbance of BNAH at 360 nm. The experiments were per-
formed in disposable plastic UV cuvettes (1.5 mL in volume) at
30 °C with Tris–HCl buffer (50 mM pH 7.5), on a Shimadzu UV-
2401 PC spectrophotometer with a Julabo F12 Refrigerated/Heat-
ing Circulator. The stock solution of BNAH was diluted in buffer
to final concentration of 10–400
bated for 5 min at 30 °C. The reactions were initiated by the addi-
tion of the FMN solution to a final concentration of 10–250 M. All
lM, and the samples were incu-
l
experiments were performed in triplicates obtaining a standard
deviation below 15%.