Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for biocatalytic applications

Article abstract of DOI:10.1016/j.molcatb.2016.10.018

Monolignol oxidoreductases from the berberine bridge enzyme-like (BBE-like) protein family (pfam 08031) catalyze the oxidation of monolignols to the corresponding aldehydes. In this report, we explore the potential of a monolignol oxidoreductase from Arabidopsis thaliana (AtBBE-like protein 15) as biocatalyst for oxidative reactions. For this study we employed a variant with enhanced reactivity towards oxygen, which was obtained by a single amino acid exchange (L182V). The pH and temperature optima of the purified AtBBE-like protein 15 L182V were determined as well as the tolerance toward organic co-solvents; furthermore the substrate scope was characterized. The enzyme has a temperature optimum of 50 °C and retains more than 50% activity between pH 5 and pH 10 within 5 min. The enzyme shows increased activity in the presence of various co-solvents (10–50% v/v), including acetonitrile, 2-propanol, 1,4-dioxane, and dimethyl sulfoxide. Primary benzylic and primary or secondary allylic alcohols were accepted as substrates. The enantioselectivity E in the oxidation of secondary alcohols was good to excellent (E>34 to?>200).

Full text of DOI:10.1016/j.molcatb.2016.10.018

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Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Characterization of the monolignol oxidoreductase AtBBE-like protein
15 L182V for biocatalytic applications
Sabine Pilsa, Kordula Schnabla, Silvia Wallnera, Marko Kljajicb, Nina Kupresaninb,
Rolf Breinbauerb, Michael Fuchsc, Raquel Rochac, Joerg H. Schrittwieserc,
Wolfgang Kroutilc, Bastian Daniela,∗, Peter Macherouxa
a Graz University of Technology, Institute of Biochemistry, Graz, Austria
b Graz University of Technology, Institute of Organic Chemistry, Graz, Austria
c University of Graz, Department of Chemistry, Organic and Bioorganic Chemistry, NAWI Graz, Graz, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2016
Received in revised form 31 October 2016
Accepted 31 October 2016
Available online xxx
Monolignol oxidoreductases from the berberine bridge enzyme-like (BBE-like) protein family (pfam
08031) catalyze the oxidation of monolignols to the corresponding aldehydes. In this report, we explore
the potential of a monolignol oxidoreductase from Arabidopsis thaliana (AtBBE-like protein 15) as bio-
catalyst for oxidative reactions. For this study we employed a variant with enhanced reactivity towards
oxygen, which was obtained by a single amino acid exchange (L182V). The pH and temperature optima
of the purified AtBBE-like protein 15 L182V were determined as well as the tolerance toward organic
co-solvents; furthermore the substrate scope was characterized. The enzyme has a temperature opti-
mum of 50 ◦C and retains more than 50% activity between pH 5 and pH 10 within 5 min. The enzyme
shows increased activity in the presence of various co-solvents (10–50% v/v), including acetonitrile, 2-
propanol, 1,4-dioxane, and dimethyl sulfoxide. Primary benzylic and primary or secondary allylic alcohols
were accepted as substrates. The enantioselectivity E in the oxidation of secondary alcohols was good to
excellent (E>34 to >200).
Keywords:
Biocatalysis
Oxidoreductase
Enzyme
Flavin
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
also stable radical species (semiquinone) can be formed. A com-
prehensive overview of the function and mechanism of different
Flavoproteins are a diverse protein class employing either flavin
mononucleotide (FMN) or the flavin adenine dinucleotide (FAD)
for catalysis [1,2]. Among them, the BBE-likes (pfam 08031) can
be distinguished due to their bicovalent cofactor tethering [3]. The
namesake of this protein family is the berberine bridge enzyme
(BBE) from California poppy (Eschscholzia californica), which cat-
alyzes the oxidative ring closure from (S)-reticulin to (S)-scoulerine
during isoquinoline alkaloid biosynthesis [4,5].
The majority of flavoproteins, including the BBE-likes, catalyze
redox reactions, and they are involved in a plethora of biological
processes [6]. The catalytically active moiety of the flavin cofactor is
the isoalloxazine ring, whose properties are modulated by the pro-
tein environment in which it is embedded. The isoalloxazine ring
is capable of one or two electron exchange reactions: In addition
to the fully reduced (hydroquinone) or oxidized (quinone) state,
flavoproteins has been provided by Fagan et al. [6].
The catalytic cycle of a flavoprotein can be divided into two half
reactions: In the resting state the flavin cofactor is oxidized. In the
reductive half reaction, the flavin is reduced by a given substrate.
In the oxidative half reaction, the flavin reacts with an appropriate
electron acceptor, thus it becomes reoxidized and is subsequently
able to enter another catalytic cycle. The nature of the final electron
acceptor is a crucial attribute of flavoproteins. Enzymes that pro-
mote the reaction of the flavin cofactor with oxygen are considered
to function as oxidases, while enzymes that inhibit the reaction
of the reduced flavin with oxygen are defined as dehydrogenases.
For BBE-likes it has been shown that a single gatekeeper residue
controls the oxygen reactivity of the enzyme [7] (compare Fig. 1
panel A). The rationale behind the engineering of the AtBBE-like 15
L182V variant is described in [7], creation of the AtBBE-like vari-
ant is published in [8]. We have recently identified two BBE-likes
from Arabidopsis thaliana as monolignol oxidoreductases (AtBBE-
like protein 13 and AtBBE-like protein 15) [8]. These enzymes were
heterologously expressed in Komagataella phaffii (formerly Pichia
∗
Corresponding author.
E-mail address: bastian.daniel@tugraz.at (B. Daniel).
http://dx.doi.org/10.1016/j.molcatb.2016.10.018
1381-1177/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.
0/).
Please cite this article in press as: S. Pils, et al., Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for
biocatalytic applications, J. Mol. Catal. B: Enzym. (2016), http://dx.doi.org/10.1016/j.molcatb.2016.10.018

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Fig. 1. A: Oxygen reactivity motif of AtBBE-like protein 15; gate keeper residue Leu182 was changed to valine to turn the enzyme from a dehydrogenase to an oxidase; B:
Proposed reaction mechanism; C: Active site and substrate binding pocket colored by hydrophobicity (red: hydrophobic, blue: hydrophilic). Green residues determine the
shape of the active site, orange residues also act as a catalytic base. D: Substrate binding pocket; the green residues are responsible for the hydrophobic properties of the
substrate binding pocket.
pastoris), and a biochemical and structural characterization has
been conducted in our laboratory. Both enzymes were found to oxi-
dize monolignols (p-coumaryl 1a, coniferyl 1b and sinapyl alcohol
1c) to the corresponding aldehydes while reacting only sluggishly
with molecular oxygen and were hence considered as monolignol
dehydrogenases. The physiological electron acceptor for AtBBE-
like proteins 15 and 13 is not known yet; therefore, the sluggish
oxidative rate hampers the potential application of AtBBE-likes
as biocatalysts as the turnover rate is determined by the slowest
reaction rate. This means that it is virtually impossible to achieve
significant turnover rates with a dehydrogenase in the absence of an
appropriate electron acceptor. Moreover, while AtBBE-like 15 was
found to be stable and expressed with good yields, expression of
AtBBE-like 13 was very cumbersome and the enzyme was found to
be unstable. To overcome these limitations, the oxygen gatekeeper
residue in AtBBE-like protein 15 was changed from leucine to valine
to turn the enzyme into an oxidase. The resulting enzyme vari-
ant AtBBE-like protein 15 L182V reacts more than 400 times faster
with oxygen compared to the wild type enzyme [8]. This enables
the usage of this enzyme for steady state kinetics and the puta-
tive application of the enzyme as biocatalyst in oxidative reactions.
The structure of the oxygen reactivity motif harboring the varia-
tion L182V is shown in Fig. 1A, the proposed reaction mechanism
of AtBBE-like protein 15 is represented in panel B. A representa-
tion of an imprint of the active site and the substrate binding site
is shown in panel C and D. Figure one is created using the crystal
structure of the wild type enzyme (pdb entry: 4UD8).
pocket that is analogous to the oxyanion hole of serine proteases. In
this pocket oxygen is complexed by two backbone amides (compare
Fig. 1 panel A: Leu178 and Cys179). From this position the oxygen
reacts with the reduced flavin to form a hydroperoxy adduct at the
C4a position that will subsequently eliminate hydrogen peroxide
to yield oxidized flavin. The accessibility of this pocket is sterically
controlled by a single gate keeper residue corresponding to Leu182
in AtBBE-like 15 (compare Fig. 1, panel A). In order to promote the
reaction of the reduced flavin with oxygen oxidases feature a valine
in this position, which will allow oxygen to bind in the pocket. In
contrast, dehydrogenases sterically block access to the pocket with
a larger amino acid side chain and thus prevent the reoxidation
of the reduced flavin cofactor [7,9]. The isoalloxazine ring and the
active site are located at the interface of both domains. The active
site forming residues are shown in Fig. 1C. Tyr 193 is proposed
to act as catalytic base. It is stabilized in the deprotonated form
by Tyr479 and is positioned by Lys436 via a cation-␲ interaction
(compare Fig. 1B). Tyr193 deprotonates the allylic alcohol to facili-
tate the hydride transfer to the flavin. The enzyme possesses a wide
surface accessible cavity harboring the substrate binding site and
the active site [8]. This funnel-like cavity was visualized and ana-
lyzed using the CASoX tool (Fig. 1C and D) [10,11]. Residues with a
major contribution to the binding site are shown in green in Fig. 1D,
also the flavin cofactor shown in yellow contributes to the binding
site. In the vicinity of the binding site is the active site. In Fig. 1C in
dark green the residues that contribute to the active site are shown.
In dark green Tyr117, Gln438, Ile409, Arg292, Val178 and Cys179
are contracting the cavity and thereby form the entrance to the
active site. The catalytic base motif (orange residues Fig. 1 panel
C), formed by Tyr193, Lys436 and Tyr479, is located opposite to
The structure of the enzyme can be divided in a FAD-binding
domain and a substrate binding domain [8]. The oxygen reactivity
motif is located in the FAD-binding domain and creates an oxygen
Please cite this article in press as: S. Pils, et al., Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for
biocatalytic applications, J. Mol. Catal. B: Enzym. (2016), http://dx.doi.org/10.1016/j.molcatb.2016.10.018

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this entrance [8]. Asn411 also contributes to the properties of the
active site. On the one hand it is involved in the complexation of a
water molecule together with Gln438, on the other hand it is a puta-
tive binding partner to anchor the monolignols prior to oxidation.
His115 and Cys179 are responsible for the bicovalent attachment
of the FAD, which confers a very tight steric control of the position
of the cofactor to the enzyme (Fig. 1C) [3].
The application of flavin-containing enzymes as oxidative cata-
lysts is highly desirable as they combine the advantage of using
molecular oxygen as the most environmentally benign oxidant
with the substrate-, regio-, and enantioselectivity of an enzyme
[12]. Therefore we initiated a feasibility study to determine the
potential of AtBBE-like protein 15 L182V as a biocatalyst. In partic-
ular, the substrate scope, the pH and temperature optima, solvent
tolerance, and enantioselectivity were in the focus of our study.
2.1.2. Melting point of AtBBE-like protein 15 at different pH
values
Thermofluor experiments were performed using a Biorad® CFX
Connect Real time PCR system (BioRad, Hercules, CA, USA). The
experiments were performed using Sypro® Orange as fluorescent
dye in 50 mM MES buffer pH 7.0. The total volume in each well
was 25 ␮L with a protein concentration of 0.4 mg/mL. The starting
temperature of 20 ◦C was kept for 5 min and then the temperature
was increased at a rate of 0.5 ◦C/min to 95 ◦C. Melting temperatures
were determined using the program Biorad CFX Manager 3.0.
2.2. Influence of temperature
2.2.1. Temperature optimum
The temperature optimum of AtBBE-like 15 L182V was deter-
mined spectrophotometrically by measuring the formation of
coniferyl aldehyde (2b) from coniferyl alcohol (1b). The reaction
was performed in 1.5 mL plastic reaction tubes in a thermomixer
(Eppendorf, Hamburg, Germany) in a temperature range of 20 ◦C to
70 ◦C. 500 ␮L 50 mM MES buffer pH 7 containing 0.2 ␮M AtBBE-like
15 L182V was preheated for 10 min at 500 rpm. The reaction was
started by the addition of 100 ␮L coniferyl alcohol (1b) solution to
reach a final concentration of 3.33 mM. The reaction was followed
over 10 min, whereby every 60 s a sample was taken. To this end,
20 ␮L of the reaction solution was mixed with 10 ␮L 1 M TRIS-buffer
pH 11. 2 ␮L of this solution was immediately used to determine the
product formation with a NanoDrop 2000 spectrometer (Thermo
Fisher Scientific, Waltham, USA). The coniferyl aldehyde (2b) con-
centration was measured at 402 nm, the extinction coefficient of
37120 M−1 cm−1 was determined with an authentic standard under
the given conditions.
2. Experimental
All chemicals were from Sigma Aldrich or Acros Organics,
respectively and were used as received. All solvents were from
Roth. NMR spectra were recorded with a Bruker NMR unit at 300
(1H) and 75 (13C) MHz, shifts are given in ppm and coupling con-
stants (J) are given in Hz. The AtBBE-like L182 V was expressed and
purified as described previously [8]; one Unit (U) is defined as the
amount of enzyme that forms one ␮mol substrate per minute. The
molecular mass of the catalyst is 60 kDa. Catalase from Micrococcus
lysodeikticus (CAS 9001-05-2) was from Sigma Aldrich.
All GC–MS measurements were carried out with an Agilent
7890A GC system, equipped with an Agilent 5975C mass-selective
detector (electron impact, 70 eV), Helium was used as carrier gas
at a flow rate of 0.55 mL/min. HPLC analysis was performed with
a Shimadzu HPLC system using the chiral stationary phase as indi-
cated. Optical rotation values were measured with a Perkin Elmer
Polarimeter 341.
Analytical thin layer chromatography (TLC) was carried out on
Merck TLC silica gel 60 F254 aluminium sheets and spots were
visualized by UV light (␭ = 254 and/or 366 nm) and/or by stain-
ing with potassium permanganate (0.3 g KMnO4, 20 g K2CO3, 5 mL
5 % aqueous NaOH in 300 mL H2O) which were ultimately heated
for development of the stains. For preparative product purification
a 50 to 100 fold excess of silica gel was used with respect to the
amount of dry crude product, depending on the separation prob-
lem. The dimensions of the column were selected in such a way
that the required amount of silica gel formed a pad between 10 cm
and 25 cm. The column was equilibrated first with the solvent or
solvent mixture, and the crude product diluted with the eluent was
applied onto the top of the silica pad.
2.2.2. Long-term stability of AtBBE-like 15 L182 V at different
temperatures
The enzyme was diluted in 50 mM TRIS/HCl buffer pH 8.0 to a
final concentration of 0.125 ␮M. 950 ␮L was incubated in plastic
reaction tubes in a thermomixer (Eppendorf, Hamburg, Germany)
at 20 ◦C, 30 ◦C, 40 ◦C, 50 ◦C, 60 ◦C, and 70 ◦C. After 24 h the solutions
were cooled to 20 ◦C and residual enzyme activity was determined
employing a Specord 200 Plus spectrophotometer (Analytik Jena,
Jena, Germany). The enzyme solution was transferred to a Hellma
SUPRASIL® cuvette (Hellma GmbH & Co. KG, Müllheim, Germany)
and the reaction was started by the addition of coniferyl alcohol
(1b) to a final concentration of 0.5 mM. Product formation was
followed for 5 min at 20 ◦C, the extinction coefficient of coniferyl
aldehyde (2b) under reaction conditions of 21000 cm−1 M−1 was
determined by an authentic standards. The experiments were
performed with and without the addition of glucose to a final con-
centration of 5 mM.
2.1. Influence of pH
2.1.1. Activity of AtBBE-like 15 L182V at different pH values
The pH optimum was determined with an optical oxygen meter
FireSting O2 (Pyro Science GmbH, Aachen, Germany) equipped
with a retractable needle-type oxygen sensor (Pyro Science GmbH,
Aachen, Germany). The reaction was performed in a measuring
cell with an integrated magnetic stirrer in triplicate at 25 ◦C and
500 rpm. 575 ␮L 50 mM MES buffer pH 7 containing 1.05 ␮M AtBBE-
like 15 L182 V was stirred until a stable oxygen level was reached.
The reaction was started by the addition of 25 ␮L sinapyl alcohol
(1c) solution to reach a final substrate concentration of 0.2 mM. The
reaction was recorded for at least 5 min. After the initial consump-
tion of 30 ␮M of oxygen data was not taken in account any more.
The consumption of oxygen reflects the conversion of the substrate
during the reaction.
2.3. Influence of co-solvents
2.3.1. Activity of AtBBE-like 15 L182V in the presence of 10%
co-solvents
The measurements were conducted as described in Section 2.2.1
in the presence of 10% (v/v) of the respective co-solvents. Methanol,
ethanol, 2-propanol, 1-butanol, acetone, acetonitrile, tetrahydro-
furan (THF), N,N-dimethylformamide (DMF), dimethyl sulfoxide
(DMSO) and 1,4-dioxane were employed. The activity determined
in pure buffer was set to 100%. The presence of co-solvents was
not found to influence the extinction coefficient of the coniferyl
aldehyde.
Please cite this article in press as: S. Pils, et al., Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for
biocatalytic applications, J. Mol. Catal. B: Enzym. (2016), http://dx.doi.org/10.1016/j.molcatb.2016.10.018

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Table 1
GS-MS analytics: Retention times employing a non-chiral stationary phase.
Substance
Method
Retention time starting
material [min]
Retention time product
[min]
Retention time aldehyde
(authentic standard) [min]
Cinnamyl alcohol (1d)
Benzyl alcohol (1g)
Piperonyl alcohol (1h)
trans-2-Hexen-1-ol (1j)
cis-2-Hexen-1-ol (1k)
Crotyl alcohol (1i)
3-Phenyl-2-propyn-1-ol
(1l)
M2
M1
M2
M2
M2
M3
M1
13.4
3.4
14.4
6.3
6.3
3.2
12.9
7.55
13.8
6.0
6.0
3.0
12.9
8.0
13.9
6.0
3.0
5.1
6.2
5.1
(Z)-2-Fluoro-3-phenyl-
prop-2-en-1-ol
M1
6.2
5.7
(1e)
1-Phenyletanol (1p)
3-Phenyl-1-propanol (1q)
Geranylamine (3a)
M1
M1
M2
3.6
12.3
12.3
3.6
12.3
12.3
11.3
12.8
2.3.2. Activity in the presence of 10–50% (v/v) of organic
co-solvents
2.4.1.3. Method M3. Inlet temperature: 250 ◦C; split ratio: 90:1;
injection volume: 1 ␮L; column flow rate: 0.7 mL/min; oven pro-
gram: 40 ◦C for 5 min, 5 ◦C/min to 100 ◦C, 25 ◦C/min to 200 ◦C, 200 ◦C
for 1 min; MS transfer line temperature: 300 ◦C, MS source temper-
ature: 230 ◦C, MS quadrupole temperature: 150 ◦C; MS scan range:
m/z = 33–400.
The products were identified by their retention times deter-
mined with authentic standards, which are summarized in Table 1.
If no standard was available the products were identified by the
MS-spectrum.
The measurements were conducted as described in Section 2.3.1
in the presence of 10–50% (v/v) co-solvent. Only these solvents
were investigated which were found to increase the activity of
the enzyme in the experiments described in Section 2.2.1 (ethanol,
2-propanol, 1-butanol, acetonitrile, tetrahydrofuran, dimethyl sul-
foxide, and 1,4-dioxane).
2.3.3. Long-term stability in the presence of 30% (v/v) of organic
co-solvents
The measurements were conducted as described in section 2.3.1
in the presence of 30% co-solvents. The initial enzyme activity and
the residual activity were determined after 24 h incubation at 25 ◦C
and 500 rpm in a thermomixer (Eppendorf, Hamburg, Germany).
2.4.2. Substrate-specific activity of AtBBE-like 15 L182V
Substrate solutions (2 mM) were provided in MES buffer pH 7.00
in a measuring cell with a magnetic stirrer at 1000 rpm and 25 ◦C.
The reaction was started by the addition of 25 ␮L enzyme solution.
The substrates and the final enzyme concentrations are summa-
rized in Table 2. The course of the reaction was followed by the
oxygen consumption determined with an optical oxygen meter
FireSting O2 (Pyro Science GmbH, Aachen, Germany) equipped a
retractable needle type oxygen probe (OXR50-UHS, PyroScience,
Germany). The probe was calibrated with the oxygen saturated
buffer 50 mM MES pH 7,00 and a saturated Na2SO3-solution.
2.4. Substrate screening
Reactions were carried out in 50 mM MES buffer pH 7.0 in the
presence of 20% (v/v) DMSO. A total volume of 2 mL reaction mix-
ture was incubated in 11 mL glass test tube with screw cap (Pyrex,
Darmstadt, Germany) at 30 ◦C in an orbital shaker at 110 rpm. The
reaction was started by the addition of 10 ␮L enzyme to achieve
a final enzyme concentration of 1.2 ␮M. After 24 h the reaction
mixture was extracted with ethyl acetate (2 × 1 mL), the combined
organic phase was dried with Na2SO4 and subjected to GC analysis
to identify putative products.
2.5. Stereo specificity
Kinetic resolutions were carried out with substrates 1l, 1 m 1n
and 1o as described in Section 2.4. The products were identified by
coinjection with authentic samples on GC–FID.
2.4.1. GC–MS
GC–MS analyses were carried out on an Agilent 7890A GC
system equipped with an Agilent J&W HP–5 ms capillary col-
2.5.1. GC–FID (chiral stationary phase)
GC–FID analyses for the optical purity of 3-penten-2-ol (1n), 3-
octen-2-ol (1m), and cyclohex-2-en-1-ol (1o) were carried out on
an Agilent 7890A GC system equipped with a Varian CP-Chirasil
Dex-CB capillary column (25 m × 0.32 mm × 0.25 ␮m; stationary
phase: ␤-cyclodextrin bonded to dimethylpolysiloxane) (Agilent
Technologies, Santa Clara, USA) using hydrogen as carrier gas.
Method M5 (used for 1n): Inlet temperature: 220 ◦C; split
ratio: 90:1; injection volume: 1 ␮L; column flow rate: 1 mL/min;
oven program: 60 ◦C for 1 min, 20 ◦C/min to 70 ◦C, 70 ◦C for 5 min,
20 ◦C/min to 180 ◦C, 180 ◦C for 1 min; detector temperature: 250 ◦C.
Method M6 (used for 1o and 1m): Inlet temperature: 220 ◦C;
split ratio: 90:1; injection volume: 1 ␮L; column flow rate:
1 mL/min; oven program: 60 ◦C for 1 min, 20 ◦C/min to 95 ◦C, 95 ◦C
for 9 min, 20 ◦C/min to 180 ◦C, 180 ◦C for 1 min; detector tempera-
ture: 250 ◦C.
umn (30 m × 0.25 mm × 0.25 ␮m; stationary phase: bonded
&
cross-linked 5%-phenyl-methylpolysiloxane) (Agilent Technolo-
gies, Santa Clara, USA) and coupled to an Agilent 5975C mass-
selective detector (electron impact ionisation, 70 eV; quadrupole
mass selection) using helium as carrier gas.
2.4.1.1. Method M1. Inlet temperature: 250 ◦C; split ratio: 90:1;
injection volume: 1 ␮L; column flow rate: 0.7 mL/min; oven
program: 100 ◦C for 0.5 min, 10 ◦C/min to 300 ◦C; MS transfer
line temperature: 300 ◦C, MS source temperature: 230 ◦C, MS
quadrupole temperature: 150 ◦C; MS scan range: m/z = 33–400.
2.4.1.2. Method M2. Inlet temperature: 250 ◦C; split ratio: 90:1;
injection volume: 1 ␮L; column flow rate: 0.7 mL/min; oven pro-
gram: 40 ◦C for 2 min, 10 ◦C/min to 180 ◦C, 180 ◦C for 1 min; MS
transfer line temperature: 300 ◦C, MS source temperature: 230 ◦C,
MS quadrupole temperature: 150 ◦C; MS scan range: m/z = 33–400.
4-Phenyl-3-buten-2-ol (1l) was converted into the corre-
sponding acetate derivative prior to chiral-phase GC analysis
by reaction with acetic anhydride (20 ␮L/mL sample) and
Please cite this article in press as: S. Pils, et al., Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for
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13C NMR (100 MHz, CDCl3): 136.8, 133.7, 129.5, 128.7, 127.8, 126.6,
69.1, 23.5; MS (EI): m/z: 148 (54), 130 (47), 115 (66), 105 (100), 91
(60), 77 (45);
Table 2
Reaction mixtures.
Substrate
Enzyme concentration [␮M]
HPLC analysis on chiral stationary phase {Daicel Chiralcel OD-H,
n-heptane/2-propanol 90/10, 0.7 mL/min, 25 ◦C, tret(enantiomer
1) = 11.5 min, tret(enantiomer 2) = 16.8 min}: tret(major iso-
mer) = 11.5 min, ee = 94%; the physical data is in consistency with
literature [12].
Sinapyl alcohol (1c)
0.125
0.125
0.125
0.625
2.5
Coniferyl alcohol (1b)
p-Coumaryl alcohol (1a)
Cinnamyl alcohol (1d)
Piperonyl alcohol (1h)
4-Phenylbut-3-en-2-ol (1l)
Crotyl alcohol (1i)
2.5
2.5
2l: 1H NMR (300 MHz, CDCl3): 7.56-7.49 (m, 3H), 7.42-7.39 (m,
3H), 6.72 (d, J = 16.3, 1H), 2.39 (s, 3H); 13C NMR (100 MHz, CDCl3):
198.4, 143.4, 134.4, 130.5, 129.0, 128.3, 127.2, 27.6; MS (EI): m/z:
146 (60), 145 (67), 131 (71), 103 (100), 77 (50); the physical data is
in consistency with a commercial sample.
Table 3
GC-FID analytics: Retention times employing a chiral stationary phase.
Substance
Method
Retention
Retention
Retention time
time alc. 1 time alc. 2 ketone [min]
[min]
21.7
[min]
22.0
2.6. Synthesis of (Z)-3-fluoro-3-phenyl-prop-2-en-1-ol (1e)
4-Phenylbut-3-en-2-ol M7
19.3
(acetyl derivative) (1l)
2.6.1. Synthesis of ethyl (Z)-3-fluoro-3-phenylacrylate
3-Octen-2-ol (1m)
3-Penten-2-ol (1n)
Cyclohex-2-en-1-ol
(1o)
M6
M5
M6
10.2
5.4
9.8
10.5
5.6
10.2
9.0
4.1
7.6
The synthesis of ethyl (Z)-3-fluoro-3-phenylacrylate was per-
formed according to the work of Li et al. [14]. In a 5 mL round
bottom flask AgF (140 mg, 1.10 mmol, 1.9 eq.) was added to a solu-
tion of 2 mL acetonitrile and 0.1 mL H2O. Ethyl 3-phenylpropiolate
(94.5 ␮L, 0.57 mmol, 1.0 eq.) was then added to the brownish sus-
pension and the reaction was heated to 90 ◦C for 22 h. The reaction
mixture was then cooled to room temperature and the solvents
were removed under reduced pressure. The crude product was
taken up in 10 mL H2O and washed with Et2O (3 × 10 mL). The
combined organic layers were dried over Na2SO4, filtered and con-
centrated under reduced pressure to give the crude product which
was purified via silica column chromatography with cyclohex-
ane/ethyl acetate 50:1 to 20:1 as eluent. The yield was 133 mg
(0.685 mmol, 60%)colorlessoil (Rf = 0.24, cyclohexane/ethylacetate
18:1).
4-dimethylaminopyridine (DMAP; 1 mg/mL sample) at room tem-
perature for 3 h.
Chiral GC–FID analyses of the resulting 4-phenyl-3-buten-
2-yl acetate (1l acetate) were carried out on an Agilent
7890A GC system equipped with a Restek Rt® -␤DEXse capillary
column (25 m × 0.32 mm × 0.25 ␮m; stationary phase: 2,3-di-O-
ethyl-6-O-tert-butyldimethylsilyl-␤-cyclodextrin added into 14%-
cyanopropylphenyl/86%-dimethylpolysiloxane) using hydrogen as
carrier gas.
Method M7 (used for 1l): Inlet temperature: 220 ◦C; split ratio:
50:1; injection volume: 1 ␮L; column flow rate: 2 mL/min; oven
program: 60 ◦C for 1 min, 5 ◦C/min to 180 ◦C; detector temperature:
250 ◦C.
The retention times of the observed in these measurements are
summarized in Table 3.
1H NMR (300 MHz, CDCl3): ı 7.65 (d, 3JH,H = 6.7 Hz, 2H, H4 + H6),
3
7.52–7.37 (m, 3H, H1-3), 5.90 (d, JH,F = 33.3 Hz, 1H, H8), 4.26 (q,
3JH,H = 7.1 Hz, 2H, H10), 1.33 (t, 3JH,H = 7.1 Hz, 3H, H11).
13C NMR (76 MHz, CDCl3): ı 166.40 (Cq, d, JC,F = 277.6 Hz, C7),
164.18 (Cq, d, JC,F = 2.2 Hz, C9), 131.60 (CH, C1 + C3), 130.81 (Cq, d,
JC,F = 26.2 Hz, C5), 128.98 (CH, d, JC,F = 1.9 Hz, C4 + C6), 125.78 (CH,
d, JC,F = 7.9 Hz, C2), 97.35 (CH, d, JC,F = 6.9 Hz, C8), 60.55 (CH2, C10),
14.41 (CH3, C11).
2.5.2. HPLC–MS (chiral stationary phase)
HPLC analysis was performed with a Shimadzu HPLC system
using equipped with a CHIRALCEL OD-H column (Chiral Technolo-
gies Inc., West Chester, United States). Heptane/2-PrOH = 90/10
was employed as mobile phase at flow rate of 0.7 mL/min
and 25 ◦C. For the respective retention times were observed;
2.6.2. Reduction of ethyl (Z)-3-fluoro-3-phenylacrylate to
(Z)-3-fluoro-3-phenylprop-2-en-1-ol (1e)
t
ret[(R)-enantiomer] = 11.9 min,
tret[(S)-enantiomer] = 17.6 min,
In a 10 mL Schlenk-tube ethyl (Z)-3-fluoro-3-phenylacrylate
(100 mg, 0.515 mmol, 1.0 eq.) was dissolved in 3 mL CH2Cl2 and
cooled to −78 ◦C via an acetone/dry ice bath. 1.2 mL (1.192 mmol,
2.3 eq.) DIBAL-H (1.0 M in toluene) were slowly added to the col-
orless solution at −78 ◦C. The reaction mixture was then warmed
to 0 ◦C over a period of 2 h. Subsequently, the reaction was trans-
ferred into an 80 mL Schlenk-tube, diluted with CH2Cl2 (15 mL) and
quenched by the addition of H2O (1 mL). Rochelle-salt (sat.) (20 mL)
was added and the two phases were stirred vigorously for 19 h.
The aqueous phase was then washed with CH2Cl2 (3 × 20 mL) and
the combined organic layers were washed with 1 M HCl (1 × 50 mL)
and brine (1 × 50 mL), dried over Na2SO4, filtered and concentrated
under reduced pressure. The crude product was purified via column
chromatography with cyclohexane/ethyl acetate 5:1 as eluent. The
yield was 71 mg (0.466 mmol, 91%) colorless oil (Rf = 0.18, cyclo-
hexane/ethyl acetate 5:1).
and tret(ketone) = 10.5 min.
2.5.3. Scale up kinetic resolution
The substrate 1l (100 mg, 0.68 mmol) was solubilized in DMSO
(14 mL), mixed with MES buffer (50 mM, pH = 7.0, 56 mL) and incu-
bated for 3 h at 30 ◦C in a shaker at 120 rpm. The oxidase AtBBE-like
15 (350 ␮L, 140 ␮M) and the catalase from Micrococcus lysodeik-
ticus (1050 ␮L, 170000 U/mL) were added and the reaction was
incubated for 20 h at 30 ◦C and 120 rpm. The reaction mixture was
extracted with diethyl ether (4 × 40 mL), the combined organic
phase was dried over with Na2SO4, filtered and concentrated in
vacuo. The crude product was purified by flash chromatography
(hexane/diethyl ether 5:1). The fractions were collected and con-
centrated in vacuo to give compounds 1l (white solid, 35 mg,
0.24 mmol, 35%) and 2l (yellow oil, 41 mg, 0.28 mmol, 41%) with
following physical properties:
1H NMR (300 MHz, CDCl3): ı 7.58–7.30 (m, 5H, H1-6), 5.66 (dt,
3JH,H,F = 36.6, 7.1 Hz, 1H, H8), 4.45 (dd, 3JH,H,F = 7.1, 1.9 Hz, 1H, H9),
1.82 (s, 1H, OH).
(R)-1l: [␣]D20 = +14.7 (c = 1.00, MeOH); lit. :[1] [␣]D20 = −17.8
[c = 0.32, MeOH, (S)-enantiomer]; 1H NMR (300 MHz, CDCl3): 7.40-
7.21 (m, 5H), 6.57 (d, J = 15.9, 1H), 6.26 (dd, J1 = 15.9, J2 = 6.4, 1H),
4.49 (dquint, J1 = 6.4, J2 = 1.0, 1H), 1.70 (bs, 1H), 1.37 (d, J = 6.4, 3H);
13C NMR (76 MHz, CDCl3): ı 158.22 (Cq, d, JC,F = 251.2 Hz, C7),
131.87 (Cq, d, JC,F = 28.6 Hz, C5), 129.44 (CH, C1 + C3), 128.66 (CH, d,
Please cite this article in press as: S. Pils, et al., Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for
biocatalytic applications, J. Mol. Catal. B: Enzym. (2016), http://dx.doi.org/10.1016/j.molcatb.2016.10.018

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0.20
JC,F = 2.0 Hz, C4 + C6), 124.55 (CH, d, JC,F = 7.2 Hz, C2), 104.92 (CH, d,
JC,F = 15.3 Hz, C8), 56.23 (CH2, d, JC,F = 7.7 Hz, C9).
A
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
3. Results and discussion
AtBBE-like protein 15 L182V was found to oxidize allylic and
benzylic alcohols to the corresponding aldehydes. In Scheme 1 the
reaction equation and the structure of substances that were used
for enzymatic assays are shown.
3
4
5
6
7
pH
8
9
10
11
3.1. pH dependency of the activity of AtBBE-like protein 15 L182V
The pH profile of the enzyme was investigated in a range of 4
to pH 10 (Fig. 2a). Thermofluor experiments were conducted to
determine the melting point on the enzyme in a pH-range from 5
to 10 (Fig. 2b).
The enzyme is active over a broad pH range, with highest activity
at pH 7, and between pH 5 and pH 10 the enzyme retains more than
50% activity (5 min). The highest melting point of the enzyme was
found to be in HEPES buffer pH 7.0 (60 ◦C).
60
59
58
57
56
55
54
53
52
51
50
B
3.2. Temperature dependent performance of AtBBE-like 15 L182V
The initial enzyme activity was measured in a temperature
range of 20 to 70 ◦C (Fig. 3A). The residual activity of the enzyme
was determined after 24 h incubation at temperatures ranging from
20 ◦C to 70 ◦C in the presence and absence of glucose (Fig. 3B).
The highest activity was found at 50 ◦C. The findings are in good
agreement with the melting point of the wild type enzyme of 60 ◦C.
Fig. 2. A: pH optimum of AtBBE-like 15 L182V. Plotted is the observed consumption
of ␮mol oxygen per mg enzyme per minute. B: Melting point of AtBBE-like protein
15 at different pH values.
Fig. 3. A: Temperature optimum of AtBBE-like 15 L182V. B: Residual enzyme activity after incubation at different temperatures for 24 h in the presence (grey bars) and
absence (striped bars) of 5 mM glucose. The initial activity determined prior incubation without glucose was set to 100%.
Please cite this article in press as: S. Pils, et al., Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for
biocatalytic applications, J. Mol. Catal. B: Enzym. (2016), http://dx.doi.org/10.1016/j.molcatb.2016.10.018

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Scheme 1. Reaction equation and substrates used for enzymatic assays.
No loss of activity after 24 h can be observed at 30 ◦C. At 40 and 50 ◦C
a residual activity of 77% and 38% are found, respectively. Glucose
was shown to increase the melting temperature of the enzyme by
15 ◦C; in the long term experiment, a stabilizing effect of glucose at
40 ◦C can be observed, while at 50 ◦C the residual enzyme activity
is decreased by glucose [8].
250
200
150
100
50
3.3. Activity in the presence of 10% (v/v) of organic co-solvents
0
While an aqueous system is the natural environment of enzymes
the solubility of nonpolar substrates is limited under purely aque-
ous conditions. This limitation can be overcome by the addition
of co-solvents. A high tolerance of the enzyme towards organic
solvents is also required if a thermomorphic solvent system is sup-
posed to be applied for enzyme recycling [15]. As oxygen is the final
electron acceptor of AtBBE-like protein 15 L182V, also the oxygen
solubility is an important factor for the overall performance of the
catalyst. The addition of co-solvent is not only beneficial for sub-
strate solubility but can also enhance the oxygen solubility [16].
To determine the effect of organic solvents on the enzyme stabil-
ity, measurements in the presence of 10% (v/v) of various organic
solvents were conducted. The course of the reaction was followed
spectrophotometrically. The enzyme activity in buffer was set to
100%. In Fig. 4 the activities found in the presence of organic sol-
vents are shown.
Fig. 4. Activity of AtBBE-like protein 15 L182V with 10% (v/v) of organic co-solvents.
3.4. Activity in the presence of 10–50% (v/v) of organic co-solvents
The high solvent tolerance of the enzyme encouraged us to fur-
ther increase the co-solvent concentration to probe the enzyme
stability. Experiments were conducted with all solvents that were
found to increase the enzyme activity. The enzyme activity was
determined with acetonitrile, 2-propanol, 1,4-dioxane, tetrahydro-
furan, 1-butanol, dimethyl sulfoxide, and ethanol at concentrations
of 10%, 20%, 30%, 40% and 50% (v/v). The measured activities are
shown in Fig. 5.
An increased activity of the enzyme was found in acetonitrile,
2-propanol, 1,4-dioxane, tetrahydrofuran, 1-butanol, dimethyl sul-
foxide and ethanol. The highest degree of activation was found for
ethanol with 188% of the activity observed in buffer. A decreased
activity was determined in the presence of methanol, dimethyl-
formamide and acetone, with the lowest activity found with of
methanol (29%).
An increasing solvent concentration led to an increased enzyme
activity in all cases except for tetrahydrofuran, which was not tol-
erated at higher concentrations. The highest acitivity was found
in 40% ethanol with 346% of the activity measured in buffer.
The enzyme was shown not only to tolerate high solvent con-
centrations, also the activity can be enhanced. Still, high solvent
concentrations can lead to the denaturation and thereby deacti-
Please cite this article in press as: S. Pils, et al., Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for
biocatalytic applications, J. Mol. Catal. B: Enzym. (2016), http://dx.doi.org/10.1016/j.molcatb.2016.10.018

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400
350
300
250
200
150
100
50
0
Acetonitrile 2-Propanol 1,4-Dioxane
THF
1-Butanol
DMSO
Ethanol
Fig. 5. Effect on the enzyme activity of different solvents at concentrations between 10% and 50% (v/v).
Fig. 6. Long-term activity of AtBBE-like protein 15 L182V with 30% (v/v) of organic co-solvents with t1 = 0 h, t2 = 24 h.
Table 4
vation of the enzyme. A good long-term stability of the enzyme
under the given reaction conditions is essential to achieve a high
space/time yield.
Substrate conversion determined by GC.
Substance
Conversion [%]
Cinnamyl alcohol (1d)
(Z)-3-Fluoro-3-phenyl-prop-2-en-1-ol (1e)
3-Phenyl-2-propyn-1-ol (1f)
Benzyl alcohol (1g)
Piperonyl alcohol (1h)
Crotyl alcohol (1i)
97
>99
19
>99
49
15
3.5. Long-term stability in the presence of 30% (v/v) of organic
co-solvents
trans-2-Hexen-1-ol (1j)
cis-2-Hexen-1-ol (1k)
1-Phenyl-ethanol (1p)
3-Phenyl-propanol (1q)
Geranylamine (3a)
47
28
n.d.
n.d.
n.d.
The presence of various organic solvents was found to enhance
the activity of the enzyme. To test the long-term stability of the
enzyme, it was incubated with 30% (v/v) of the respective solvents
for 24 h (t2) before the reaction was started. The activity in buffer
was set to 100%, the starting and residual activities are depicted in
Fig 6.
the allylic ketone formed in situ to reach a theoretical yield of 100%
While no deactivation of the enzyme in buffer was detectable,
the presence of organic solvents led to enhanced activity but also
a decreased life-time of the enzyme. The highest activity after 24 h
incubation was determined in 30% DMSO. The enzyme activity in
DMSO was 177% initially and dropped to 122% within 24 h. Also
for other solvents a lower but still acceptable retention of the ini-
tial activity was determined. The initial activity dropped in ethanol
from 218% to 38%, in 2-propanol from 194% to 47% and in 1-butanol
from 201% to 33%. Especially the long-term stability in 2-propanol
creates interesting options with regard to coupled reactions with
NADH dependent alcohol dehydrogenases [17]. 2-Propanol is fre-
quently used to regenerate NADH from NAD+. In a one-pot process,
AtBBE-like 15 L182V could be employed for kinetic resolutions of
secondary allylic alcohols, while an alcohol dehydrogenase with
NADH regeneration by 2-propanol could be employed to reduce
of the desired enantiopure alcohol.
3.6. Substrate screening
In order to elucidate the substrate scope of AtBBE-like 15 L182V,
the enzyme was screened for activity with various alcohols. Exper-
iments were conducted in HEPES-buffer pH 7.0 containing 20%
DMSO with a substrate concentration of 10 mM.
The substances 1a-o were converted to the corresponding alde-
hydes, as confirmed by GC–MS analysis. The formation of carboxylic
acids, which has been reported as a follow-up reaction in the oxi-
dation of primary alcohols by other flavin-dependent oxidases, has
never been observed in our experiments. The conversions observed
by GC–MS are summarized in Table 4.
Please cite this article in press as: S. Pils, et al., Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for
biocatalytic applications, J. Mol. Catal. B: Enzym. (2016), http://dx.doi.org/10.1016/j.molcatb.2016.10.018

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0.6
0.5
0.4
0.3
0.2
0.1
0
allylic alcohol was found to be oriented towards the N5 position of
the flavin, while the pro-R proton was not [8].
4. Conclusions
In the present study we have probed the applicability of a mono-
lignol oxidoreductase for biocatalytic transformations. We have
employed the AtBBE-like 15 L182V variant that was engineered
previously toward higher oxygen reactivity. We have elucidated
the pH- and temperature optima of the enzyme, the substrate scope
and tolerance towards organic co-solvents. AtBBE-like protein 15
L182V retained more than 50% of activity in a broad pH range
between 5 and 10 for 5 min. The highest activity was reached at
50 ◦C, no deactivation of the enzyme was found after incubation
at 30 ◦C for 24 h. The enzyme exhibits an enhanced activity in the
presence of organic co-solvents. The most suitable co-solvent was
found to be DMSO: in 30% DMSO the enzyme Exhibits 177% of
the activity determined for an aqueous system; after 24 h at 20 ◦C
122% of enzyme activity is remained. Various allylic and benzylic
alcohols were accepted as substrates and converted to the corre-
sponding aldehydes. The enzyme preferably converts S-alcohols
and was successfully applied in a kinetic resolution in preparative
scale. Aliphatic alcohols were not converted by the enzyme. There-
fore AtBBE-like protein 15 L182V could be useful as a chemo- and
enantioselective enzyme for future biocatalytic applications.
1a
1b
1c
1d
1h
1i
1l
Fig. 7. Specific activity of AtBBE-like protein 15 L182V with p-coumaryl alcohol (1a),
coniferyl alcohol (1b), sinapyl alcohol (1c), cinnamyl alcohol (1d), piperonly alcohol
(1h), crotyl alcohohl (1i) and 4-Phenylbut-3-en-2-ol (1l).
Table 5
Kinetic resolutions with AtBBE-like protein 15 L182V.
Substance
Ketone [%]
Sum alcohols
[%]
e.e. [%]
>99 (R)
E
4-Phenylbut-3-en-2-ol 58
(acetyl derivative) (1l)
3-Octen-2-ol (1m)
3-Penten-2-ol (1n)
Cyclohex-2-en-1-ol
(1o)
42
>34
55
12
35
45
88
65
>99 (R)
20 n.d.
53 (R)
>47
>200
>200
Acknowledgments
This work was supported by grants from the Austrian Science
Foundation (FWF) (Doctoral program “Molecular Enzymology”
W901) to PM, RB and WK, and P28678 to PM.
All allylic and primary benzylic alcohols that were tested
were accepted as substrate. No conversion was detected (n.d.) for
aliphatic and secondary benzylic alcohols (1p-q) as well as genany-
lamine 3a.
References
3.6.1. Substrate specific activity of AtBBE-like 15 L182V
To evaluate the activity toward the different substance cate-
gories that were found to be converted, the specific activity of the
enzyme was assayed employing cinnamyl alcohol derivatives (1a-
d, 1l), a benzylic alcohol (piperinyl alcohol 1 h), and an allyl alcohol
(crotyl alcohol 1i). The determined activities are summarized in
Fig. 7.
The activities displayed in Fig. 7 are in good agreement with the
conversions summarized in Table 4. The highest activity is found
employing cinnamyl alcohol derivatives, ranging from 0.47 U/mg
for sinapyl alcohol (1c) to 0.13 U/mg for cinnamyl alcohol. The activ-
ity towards a benzylic alcohol (piperonyl alcohol 1 h, 0.008 U/mg)
and towards crotyl alchohol (1i, 0.002 U/mg) is one and two orders
of magnitude lower, respectively.
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[2] D. Leys, N.S. Scrutton, Curr. Opin. Struct. Biol. 41 (2016) 19–26.
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3.7. Kinetic resolutions
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[13] X. Chen, H. Zhou, K. Zhang, J. Li, H. Huang, Org. Lett. 16 (2014) 3912–3915.
[14] Y. Li, X. Liu, D. Ma, B. Liu, H. Jiang, Adv. Synth. Catal. 354 (2012) 2683–2688.
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[18] J.L.L. Rakels, A.J.J. Straathof, J.J. Heijnen, Enzyme Microb. Technol. 15 (1993)
1051–1056.
As secondary allylic alcohols were found to be converted by
the enzyme, this substance class was chosen for testing the enan-
tioselectivity of the enzyme. Kinetic resolutions were conducted
starting from racemic alcohols. Enantiomeric excess (e.e.) of the
remaining substrate as well as conversion were determined by GC
analysis on chiral phases, and the enantioselectivity (E) was calcu-
lated according to Rakels et al. [18]. The results are summarized in
Table 5.
The absolute configuration of the remaining alcohols 1l, 1 m and
1o was determined according to [13], [19] and [20], respectively.
The results indicate that the enzyme shows a high enantios-
electivity in the oxidation of secondary allylic alcohols. While
the (S)-enantiomer is converted, the (R)-enantiomer remains
untouched. This is in good agreement with a productive docking
mode we have published previously, in that the pro-S proton of the
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[20] H.J. Gais, T. Jagusch, N. Spalthoff, F. Gerhards, M. Frank, G. Raabe, Chem.—A
Eur. J. 9 (2003) 4202–4221.
Please cite this article in press as: S. Pils, et al., Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for
biocatalytic applications, J. Mol. Catal. B: Enzym. (2016), http://dx.doi.org/10.1016/j.molcatb.2016.10.018