Two-Step One-Pot Synthesis of Pinoresinol from Eugenol in an Enzymatic Cascade

Article abstract of DOI:10.1002/cctc.201500182

The phytoestrogen pinoresinol is a high-value compound that has a protective effect against diverse health disorders, and thus is of interest for the pharmaceutical industry. Isolation of pinoresinol from plants suffers from low yields, and its chemical synthesis involves several work-up steps. In this study we devised a novel two-step one-pot enzymatic cascade combining a vanillyl-alcohol oxidase and a laccase for the production of pinoresinol from eugenol via the intermediate coniferyl alcohol. Along with the well-characterized vanillyl-alcohol oxidase from Penicillium simplicissimum used to catalyze the oxidation of eugenol, enzyme screening revealed three bacterial laccases that were appropriate for the synthesis of pinoresinol from coniferyl alcohol. The cascade was optimized regarding enzyme ratios, pH value, and the presence of organic solvents. Under optimized conditions, pinoresinol concentration achieved 4.4 mM (1.6 gl^-1), and this compound was isolated and analyzed. Increasing value: The high-value compound pinoresinol is synthesized in a two-step one-pot process combining the vanillyl-alcohol oxidase from Penicillium simplicissimum (PsVAO) and a bacterial laccase starting from the inexpensive substrate eugenol. tBME=tert-butyl methyl ether.

Full text of DOI:10.1002/cctc.201500182

DOI: 10.1002/cctc.201500182
Full Papers
Two-Step One-Pot Synthesis of Pinoresinol from Eugenol
in an Enzymatic Cascade
[
a]
[a]
[a]
[b]
Esther Ricklefs, Marco Girhard, Katja Koschorreck, Martha S. Smit, and
[a]
Vlada B. Urlacher*
The phytoestrogen pinoresinol is a high-value compound that
has a protective effect against diverse health disorders, and
thus is of interest for the pharmaceutical industry. Isolation of
pinoresinol from plants suffers from low yields, and its chemi-
cal synthesis involves several work-up steps. In this study we
devised a novel two-step one-pot enzymatic cascade combin-
ing a vanillyl-alcohol oxidase and a laccase for the production
of pinoresinol from eugenol via the intermediate coniferyl alco-
hol. Along with the well-characterized vanillyl-alcohol oxidase
from Penicillium simplicissimum used to catalyze the oxidation
of eugenol, enzyme screening revealed three bacterial laccases
that were appropriate for the synthesis of pinoresinol from
coniferyl alcohol. The cascade was optimized regarding
enzyme ratios, pH value, and the presence of organic solvents.
Under optimized conditions, pinoresinol concentration ach-
À1
ieved 4.4 mm (1.6 gl ), and this compound was isolated and
analyzed.
Introduction
Recently, a focus in biocatalysis has been the development of
biocatalytic cascade reactions. This is reflected by the rising
tochrome P450 monooxygenases, enoate reductases and
Baeyer–Villiger monooxygenases have also become candidates
[
1–3]
[4]
[12]
number of published reviews
and books on this topic. The
for the implementation in synthetic cascade reactions. In ad-
simplest definition of the term “cascade reaction” is a combina-
tion of several (bio)synthetic reaction steps leading to a prod-
uct. Peculiarly attractive are so-called one-pot processes. Gen-
eral advantages of such one-pot systems are reduced time,
effort, and simplification of multistep synthetic reactions be-
cause no isolation and purification of intermediates (which
dition, the use of aldolases and hydroxynitrile lyases for CÀC
[13]
coupling reactions in cascades was reported. Laccases were
[14]
also used in cascade reactions for cofactor regeneration.
Very recently, a Trametes versicolor laccase/2,2,6,6-tetramethyl-
1-piperidinyloxylradical (TEMPO) system was applied in an one-
[15]
pot process for deracemisation of rac-2-phenyl-1-propanol.
[
2,5]
might be toxic or instable) are required. In addition, cascade
reactions often allow simultaneous regeneration of cofactors if
The implementation of a laccase into a linear enzymatic cas-
cade process for CÀC or CÀO coupling has, however, to the
best of our knowledge not been described in literature.
Herein, we describe the application of a novel enzymatic
[
3]
redox enzymes are applied.
One of the first cascades, reported in 1976, combined a bio-
transformation and a chemical step for the synthesis of enan-
tiomeric pure C -juvenile hormone from methyl 10,11-epoxy-
cascade combining
a vanillyl-alcohol oxidase (VAO) (EC
1.1.3.38) and a bacterial laccase (EC 1.10.3.2) for the production
of the complex and high-value compound pinoresinol from in-
expensive starting material. Besides its high market value, pi-
noresinol production is also interesting regarding possible ap-
plications in the medical field. Pinoresinol is one of the sim-
plest lignans belonging to the group of phytoestrogens. It is
a precursor of the mammalian lignans enterolactone and en-
terodiol, which are produced in the mammalian proximal
colon and for which health-supporting effects have been in-
16
[
6]
farnesate. In the same year an in vitro cascade reaction com-
bining six enzymes for the synthesis of threonine from aspartic
[
7]
acid was published.
Up to now most published enzymatic cascades involve
mainly dehydrogenases, ketoreductases, transaminases, and
amino acid dehydrogenases for the production of chiral alco-
[
8]
[9]
[10]
[11]
hols, amines, amides, or amino acids. More recently, cy-
[
16,17]
tensively discussed.
These lignans may reduce the risk of
[a] E. Ricklefs, Dr. M. Girhard, Dr. K. Koschorreck, Prof. Dr. V. B. Urlacher
[16,18]
hormone-dependent cancer in different organs, of osteo-
porosis, and of cardiovascular diseases owing to their anti-
Institute of Biochemistry
Heinrich Heine University
Universitätsstraße 1, 40225 Düsseldorf (Germany)
E-mail: vlada.urlacher@uni-duesseldorf.de
[19]
[20]
21]
[
oxidant and (anti)estrogenic properties. It has been shown
that pinoresinol inhibits proliferation and induces differentia-
[22]
[b] Prof. Dr. M. S. Smit
tion of human HL60 leukemia cells. Furthermore, the inhibi-
tory effect of pinoresinol on HIV-1 replication can be interest-
Department of Microbial, Biochemical and Food Biotechnology
University of the Free State
[23]
ing for chemotherapy. Pinoresinol is also a potential candi-
date as a hypoglycemic, antimelanogenic, and antifungal
205 Nelson Mandela Drive, Bloemfontein 9300 (South Africa)
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201500182.
[24]
agent.
ChemCatChem 2015, 7, 1857 – 1864
1857
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Pinoresinol can be isolated from seeds of flax and sesame,
or whole-grain cereals, legumes, fruits, and some vegetables.
However, the isolation from plants is not very effective and the
yields are quite low (e.g. 29 mg from 100 g fresh weight of
case is used in this process an additional acidification step is
mandatory.
For initial experiments the following bacterial laccases from
the institute’s enzyme collection were investigated: CgL1 from
[
25]
[34]
sesame seeds). The chemical synthesis of pinoresinol from
cheap materials is often quite complex, involving many individ-
Corynebacterium glutamicum,
CotA mutant K316N/D500G
(with improved expression in E. coli) from Bacillus licheniformis
[
26]
[35]
[36]
ual (and work-up) steps. Promising biocatalytic approaches
have been reported that employ laccases or peroxidases as
(CotA), Ssl1 from Streptomyces sviceus, and Tth from Ther-
[37]
mus thermophilus. Additionally, the fungal laccase Lcc from
Trametes versicolor, as well as HRP (both commercially avail-
able) were also tested because these enzymes have been
shown to catalyze the oxidation of 2 and, therefore, could be
used for comparison.
[
23,27–29]
catalysts and coniferyl alcohol as starting material.
A
general drawback of these reactions is that they suffer from
low selectivity and, therefore, low product concentrations (iso-
[
23,29]
lated yields of 4 or 10%).
Moreover, the starting com-
pound for this reaction, coniferyl alcohol, is quite expensive.
The novel enzymatic cascade described in this study utilizes
in the first step the inexpensive and abundant precursor euge-
nol 1, which is converted by VAO from Penicillium simplicissi-
mum (PsVAO) into the intermediate coniferyl alcohol 2. In
the second step 2 is oxidized by a bacterial laccase and under-
goes dimerization by means of CÀC and CÀO coupling, leading
to pinoresinol 3 (Scheme 1). Reaction parameters, such as
enzyme of choice, reaction time, pH value, enzyme ratios, and
addition of additives (e.g. organic solvents) were optimized at
an analytical scale to increase the product yield. Finally, pinore-
sinol 3 was produced and isolated on a semipreparative scale.
All enzymes were tested for their ability to produce 3 from
2 at different pH values (5.0, 7.5, and 9.0). The formation of 3
was observed with all bacterial laccases at pH 7.5 and pH 9.0,
but fungal Lcc showed no activity at these pH values. In con-
trast, at pH 5.0 3 was produced by fungal Lcc as well as by
bacterial CotA and Tth, but not by Ssl1 and CgL1. HRP was
active at all pH values tested and formation of 3 was observed.
In all cases racemic 3 was produced. Besides 3, two other
products were formed by all enzymes, owing to the catalytic
mechanism generating radicals (Figures S5 and S7B, Support-
[30]
[23,29]
ing Information), as also observed previously.
Development of the enzymatic cascade
[33]
Because PsVAO has its pH optimum at pH 10.0, and on the
basis of the screening results, the laccases CgL1, CotA, and
Ssl1, as well as HRP, were chosen first for investigation of the
one-pot cascade in combination with PsVAO (for further ex-
planation see the Supporting Information). Initial experiments
were performed at an analytical scale and comprised 1 mm eu-
Results and Discussion
Enzyme screening for conversion of coniferyl alcohol
[
31]
[27,29,31]
Earlier studies reported laccases from plants and fungi
that are capable of dimerizing coniferyl alcohol 2 to pinoresi-
nol 3. In addition, horseradish peroxidase (HRP) has been de-
À1
genol 1, 10 mUml PsVAO (volumetric activity was deter-
[
23]
À1
scribed to catalyze this reaction. To the best of our knowl-
edge no bacterial laccase has been reported for this reaction
to date, therefore, we decided to assess the potential of these
enzymes for the conversion of 2 into 3. Advantages of bacteri-
al laccases compared with their eukaryotic counterparts are
that the former can more easily be expressed in Escherichia
coli and are generally active at neutral to alkaline pH values,
mined with 1), and 10 mUml of either laccase or HRP (volu-
metric activity was determined with ABTS) at different pH
values (5.0, 7.5, and 9.0). As expected, no conversion of 1 was
observed at pH 5.0 because of the lack of PsVAO activity. In
contrast, at pH 7.5 and 9.0 conversion of 1 was completed
within 2.5 h and formation of the intermediate coniferyl alco-
hol 2 was observed. Enzyme-catalyzed conversion of 2 pro-
ceeded smoothly over time yielding the desired product pinor-
esinol 3 (Figure 1A). Depending on the enzyme used, the time
point at which complete conversion of the intermediate 2 was
achieved varied (Table S2 in the Supporting Information).
The highest yields of 3 were
[32]
whereas eukaryotic laccases often have acidic pH optima.
The latter point is of special importance for the development
of the projected enzymatic cascade, because the pH optimum
[
33]
reported for PsVAO is in the alkaline region. If a fungal lac-
obtained at pH 7.5, and were at
comparable levels for all laccas-
es, whereas HRP produced lower
amounts of 3 under the same
conditions (Figure 1B). The prod-
uct yields achieved at pH 9.0
were generally lower for all com-
binations and are summarized in
the Supporting Information (Fig-
ure S1).
Scheme 1. Formation of pinoresinol 3 in a linear one-pot cascade including PsVAO and a bacterial laccase. Al-
though PsVAO produces solely 2 from 1, it should be noted that for the conversion of 2 by laccases, other prod-
ucts as well as 3 were detected (Figure S5 and S7B, Supporting Information).
For reactions at pH 7.5 with
CotA 53 mm of 3 was detected
ChemCatChem 2015, 7, 1857 – 1864
www.chemcatchem.org
1858
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 2. Residual pinoresinol 3 after incubation with bacterial laccases for
À1
2
4 and 48 h. Reaction conditions: pH 7.5, 100 mm of 3, 10 mUml laccase.
Determined concentrations of 3 are normalized to the concentration at
t=0 h. Light grey: CotA, grey: Ssl1, dark grey: CgL1, black: control reaction
without enzyme.
these two CotA laccases of 63% enables the assumption that
their redox potentials are in the same range. Therefore, it
might be concluded that the conversion of 3 increases with in-
creasing redox potential of the laccase. To avoid depletion of 3
over time, we decided to focus on the laccases CgL1 and Ssl1
for further optimization of the cascade reaction.
Figure 1. Representative GC–MS chromatograms of the cascade reaction
and achieved pinoresinol 3 concentrations. Reaction conditions: pH 7.5,
Optimization of reaction conditions
À1
À1
1
mm of 1, 10 mUml PsVAO, 10 mUml of either a bacterial laccase or
Not only coniferyl alcohol 2, but also pinoresinol 3 are accept-
ed as substrates by the laccases, therefore, determination of
the optimal ratios of PsVAO and laccase is important regarding
fast conversion of 1 into 2, and also to ensure high concentra-
HRP. A) Reaction progress of the one-pot cascade combining PsVAO and
CotA. Peak numbers: [a]=eugenol, [b]=coniferyl alcohol, [c]=(Æ)-pinoresi-
nol, [IS]=internal standard. B) Concentrations of 3 determined for laccases
(CotA, Ssl1, CgL1) and HRP. Black: 2.5 h, dark grey: 5 h, grey: 7.5 h, light
grey: 24 h.
tions of 3. For reactions with Ssl1 a ratio of PsVAO/Ssl1 of 1:1
À1
(
10 mUml each) yielded the highest concentration of 3 (Fig-
after 2.5 h, for Ssl1 68 mm after 5 h, for CgL1 60 mm after 24 h,
and for HRP 40 mm after 24 h. Furthermore, it was observed for
Ssl1 and CotA that the amount of 3 decreased once the maxi-
mum concentration was achieved (Figure 1B). The control re-
action comprising only cell lysate of E. coli not expressing re-
combinant genes was negative (no formation of 2 and 3 was
observed), whereas for the control reaction comprising cell
lysate containing recombinant PsVAO low amounts of 3 were
detected (below 10 mm after 24 h). This background activity
can probably be attributed to the native laccase CueO from
ure 3A), whereas for reactions with CgL1 a ratio PsVAO/CgL1
À1
of 1:5 (10:50 mUml ) was found to be optimal (Figure 3B). Es-
pecially for Ssl1, it was noticed that when applied at higher
À1
concentrations (50–100 mUml ) the achieved concentrations
of 3 dropped to 20 mm.
To protect 3 from further oxidation by laccases, the addition
of organic solvents was investigated with the aim of in situ
product extraction. Moreover, previous studies have shown
that addition of organic solvents can affect phenol coupling re-
actions with regard to product ratios and the type of coupling
[
38]
[41]
E. coli.
(CÀC or CÀO coupling). The water-miscible organic solvents
The depletion of 3 that was observed during the reaction
can be explained by its further oxidation by the laccases. This
was confirmed by setting up control reactions containing 3 as
substrate (100 mm) and a laccase (CotA, Ssl1, or CgL1). All lac-
cases converted 3, although to different extents (Figure 2).
After 24 h conversions of 3 were 86 and 71% with CotA and
Ssl1, respectively. In contrast, CgL1 converted only 6% of 3
after 24 h. This might be explained by the lower redox poten-
methanol, acetonitrile, acetone, and ethanol, as well as the
water-immiscible organic solvents toluene, 3-methyl-1-butanol,
ethyl acetate, isooctane, and tert-butyl methyl ether (tBME)
were investigated.
Analysis of the effect of 20% (v/v) organic solvents on
PsVAO activity revealed only a minor impact of isooctane,
methanol, acetone, and ethanol on conversions of 1 (less than
5% deviation from control reaction without organic solvent).
In the presence of acetonitrile, toluene, and tBME, conversion
of 1 achieved 60–80% of that in the aqueous system, whereas
conversion of 1 was reduced to 30 or 20% after addition of 3-
methyl-1-butanol or ethyl acetate, respectively (Figure S2, Sup-
porting information).
[
34]
[36]
tial of CgL1 of 260 mV compared to that of Ssl1 (375 mV).
The redox potential of CotA from Bacillus licheniformis is not
known, but for CotA from Bacillus subtilis redox potentials of
[39]
[40]
5
25 and 455 mV have been reported (depending on the
analytical method used). The high sequence identity between
ChemCatChem 2015, 7, 1857 – 1864
www.chemcatchem.org
1859
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Figure 4. Concentrations of pinoresinol 3 in one-pot reactions with 20% (v/
v) of an organic solvent and 1 mm of 1. Reaction conditions: pH 7.5 in the
À1
À1
aqueous phase, 10 mUml PsVAO, 10 mUml Ssl1, 20% (v/v) of an organic
solvent. The reaction time for organic solvents resulting in conversions of
1
below 80% after 2.5 h (see Figure S2) was prolonged to 7.5 h. Grey: water-
miscible organic solvents, grey shaded: control reaction without organic sol-
vent, black: water-immiscible organic solvents.
Figure 3. Concentrations of pinoresinol 3 in the one-pot reaction containing
different ratios of PsVAO and a bacterial laccase. Reaction conditions: pH 7.5,
À1
À1
1
1
mm of 1, 10 mUml PsVAO, 1–100 mUml laccase. A) Ssl1, B) CgL1. :
&
À1
À1
À1
À1
À1
mUml , *: 5 mUml , ~: 10 mUml
, .
: 50 mUml , *: 100 mUml
&
Regarding production of 3 by Ssl1 the addition of acetone,
ethanol, and acetonitrile, as well as toluene and ethyl acetate
resulted in decreased yields. In the case of methanol and 3-
methyl-1-butanol the achieved product yields were in the
same range as in the aqueous system, whereas the addition of
isooctane and tBME resulted in a slight increase of 3 compared
to the aqueous system (Figure 4).
Figure 5. Concentrations of pinoresinol 3 in the one-pot cascade with 20%
(
v/v) of an organic solvent and 10 mm of 1. Reaction conditions: pH 7.5 in
À1
À1
the aqueous phase, 10 mUml PsVAO, 10 mUml Ssl1.
: 20% isooctane; ~: 20% tBME.
&
: aqueous system;
*
Based on these results, reactions with PsVAO and Ssl1 and
either tBME or isooctane were performed, and the concentra-
tion of 1 was increased to 10 mm. In the aqueous system the
concentration of 3 reached a maximum of 300 mm after 24 h
but the laccase CgL1 was applied instead of Ssl1, the yield of 3
was further increased to 1.25 mm after 96 h.
Considering that complete conversion at 10 mm of 1 was
achieved by PsVAO, we assumed that a substantial amount of
H O was produced that potentially could be harmful to the
(and then decreased), whereas in the systems with 20% isooc-
2
2
tane or tBME the concentration of 3 reached 340 and 660 mm
after 24 h and further increased up to 540 and 940 mm after
enzymes. Therefore, in an additional experiment catalase was
added to the optimized reaction cascade (20% tBME, 1:5 ratio
of PsVAO/CgL1, 10 mm of 1) for scavenging of H O . In this
72 h, respectively (Figure 5). Moreover, the depletion of 3 that
2
2
was observed in the aqueous system was not detected in the
biphasic systems.
case, however, the yield of 3 achieved was in the same range
as that without catalase (Figure S3, Supporting Information).
Another approach is the addition of peroxidases because
H O can be utilized as a cosubstrate. HRP was applied in the
These results are consistent with the determined partition
coefficients of 3: In the biphasic system with 20% tBME the
logP of 3 was 2.26, whereas its logP in the system with 20%
isooctane was À1.99 (Table S3, Supporting Information). Obvi-
ously, in situ extraction of 3 into the organic phase is more effi-
cient in the system with tBME. Thus, product 3 is no longer
available in the aqueous phase as a substrate for laccases,
leading to its accumulation at higher concentrations.
2
2
À1
one-pot process with PsVAO (20% tBME, 10 mU ml PsVAO,
À1
10 mUml HRP, 10 mm of 1). This process yielded 0.7 mm of 3
(Figure S4, Supporting information).
Finally, we also investigated a sequential mode of operation:
When 1 was almost completely converted by PsVAO to 2, lac-
case was added. When applying bacterial CgL1 under these
conditions, however, the concentration of 3 could not be fur-
ther increased (Figure S5, Supporting Information). Additional-
When the individually optimized reaction conditions (20%
tBME, 1:5 ratio of PsVAO/CgL1, 10 mm of 1) were combined,
ChemCatChem 2015, 7, 1857 – 1864
www.chemcatchem.org
1860
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
ly, the sequential application of fungal Lcc from T. versicolor
was tested; in this case, the laccase was added after adjust-
ment of the pH to 5.0. Complete conversion of 2 was achieved
Pfu-DNA-polymerase, and T4-ligase were from Thermo Scientific
(Schwerte, Germany). Oligonucleotides were from Eurofins MWG
Operon (Ebersberg, Germany).
7.5 h after addition of Lcc and at this time point the highest
concentration of 3 was achieved (850 mm). Thereafter, com-
plete depletion of 3 was observed within 16 h.
Bacterial strains and plasmids
Experiments on a semipreparative scale
Expression of the bacterial laccases and PsVAO was performed in
À
À
À
E. coli BL21 (DE3) (F ompT hsdS (r mB ) gal dcm (DE3)) (Novagen,
Encouraged by the above results, we performed further experi-
ments on the one-pot cascade with PsVAO and CgL1 at higher
eugenol 1 concentrations (25, 50, and 100 mm; Figure S6, Sup-
porting Information). When starting with 25 mm 1 the two-
B B
Darmstadt, Germany), except for Ssl1, which was produced in
À
À
À
r
E. coli BL21-Codon Plus (DE3)-RP (F ompT hsdS(rB mB ) dcm+ Tet
r
gal l(DE3) endA Hte [agrU proL Cam ] (Stratagene, Waldbronn, Ger-
many). Construction of the plasmids pET-16b_cgl1 (NC_003450
step cascade reached complete conversion within 120 h, and
[34]
region 3169415.3170896, GenBank: BAC00361.1),
pET22K316N/
GenBank:
À1
2
.7 mm of pinoresinol 3, corresponding to ꢀ1 gl , was deter-
D500G
(NCBI
RefSeq
and
Protein:
YP_077905.1,
(SSEG_02446;
[
35]
mined. At higher concentrations of 1 (50 and 100 mm) conver-
AAU22267.1),
pET22-ssl1
GenBank:
[42]
sions of 1 reached 88 and 53%, respectively, and in both cases
EDY55866.1) for expression of CgL1, CotA, and SsL1, respectively,
has been reported previously. The gene encoding PsVAO (Gen-
Bank: Y15627.1), obtained from Prof. Marco W. Fraaije (Groningen
Biomolecular Sciences and Biotechnology Institute, University of
Groningen, Netherlands), was amplified by polymerase chain reac-
tion (PCR) using the oligonucleotides
À1
4
.4 mm of 3, corresponding to ꢀ1.6 gl , was produced.
Finally, the reaction volume was increased (Figure S6, Sup-
porting Information) and the cascade was performed on a sem-
ipreparative scale under the optimized reaction conditions:
2
5 ml, 102.5 mg of 1 (25 mm), 20% (v/v) tBME, and PsVAO/
CgL1 in a ratio of 1:5. With this setup, 47% conversion of
was achieved after 120 h and 13 mg of 3 (corresponding to
5’-GCT AGC ATG TCC AAG ACA CAG GAA TTC AGG-3’ and
1
5’-GGA AGC TTT TAC AGT TTC CAA GTA ACA TGAC-3’
an isolated yield of 13%) could be isolated from the reaction
mixture by semipreparative HPLC (Figure S7, Supporting Infor-
mation) and analyzed by NMR spectroscopy (Figure S8, Sup-
porting Information).
and the amplicon was cloned into pET-28b(+) via restriction sites
NheI and HindIII to give plasmid pET-28b_psvao. The gene tth
(NCBI RefSeq Protein: YP_005339.1, GenBank: BAE16261.1) was am-
plified by PCR from the genomic DNA of T. thermophilus without
In a previous study reported by Pickel et al. 10% isolated
yield of 3 was reported for a biocatalytic reaction converting
coniferyl alcohol 2 by the commercially available laccase Lcc
the native TAT-secretion signal. The oligonucleotides
5’-GGA ATT CCA TAT GCT GGC GCG CAG GAG CTT-3’ and
[
29]
5’-CGG GAT CCT TAA CCC ACC TCG AGG ACT CCC-3’
from T. versicolor.
In another study, Mitsuhashi et al. per-
formed the same reaction with horseradish peroxidase and re-
were used. The PCR fragment was cloned into the plasmid pET-
22b(+) via NdeI and BamHI restriction sites resulting in the expres-
sion plasmid pET22b_tth. The gene sequences and their insertion
into the vectors were verified by automated DNA sequencing
[
23]
ported an isolated product yield of 4%. In comparison with
these studies our reaction employs an inexpensive and abun-
dant starting material (1 is 98% cheaper than 2), and the iso-
lated yield of 3 could be increased by 30 and 225%, respec-
tively.
(psvao—Inqaba Biotech, Pretoria, South Africa; tth—Eurofins MWG,
Ebersberg, Germany).
Conclusions
In conclusion, we have established a novel one-pot cascade
containing PsVAO and bacterial laccases for the synthesis of pi-
noresinol 3. To the best of our knowledge this is the first cas-
cade that combines such enzymes for the production of fine
chemicals. The reaction conditions have been optimized and
the application of the cascade on a semipreparative scale has
been demonstrated.
Heterologous gene expression in E. coli
Expression of the bacterial laccases and heat denaturation of E. coli
endogenous proteins (for laccase purification) was performed as
[34,35,42]
described elsewhere.
Detailed information on expression is
summarized in Table S1 (Supporting information). PsVAO was ex-
pressed in E. coli BL21 (DE3) cells carrying pET-28b_psvao in 50 ml
TB-medium supplemented with kanamycin (34 mgml ) at 378C,
À1
1
6
80 rpm. Protein expression was induced at an optical density at
00 nm (OD600) of 0.6 by addition of 0.5 mm isopropyl-b-d-thioga-
Experimental Section
lactopyranoside (IPTG). Thereafter, incubation conditions were
shifted to 308C, 140 rpm for 16 h. Cell harvesting was conducted
at 3200”g, 48C for 30 min and the pellet was resuspended in 5 ml
50 mm KPi (pH 7.5), supplemented with 0.1 mm phenylmethylsul-
fonyl fluoride (PMSF). Cells were lysed by sonification on ice and
cell debris was removed by centrifugation (48000”g, 30 min, 48C).
All enzyme preparations were employed as cleared cell lysates
without further purification.
Chemicals and enzymes
All chemical reagents were of analytical grade or higher and pur-
chased from Appli Chem (Darmstadt, Germany), Fluka (Buchs, Swit-
zerland), Sigma–Aldrich (Schnelldorf, Germany), or VWR (Darmstadt,
Germany). Laccase of Trametes versicolor (Lcc) and peroxidase from
horseradish (HRP) were from Sigma–Aldrich. Restriction enzymes,
ChemCatChem 2015, 7, 1857 – 1864
www.chemcatchem.org
1861
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Enzyme screening for conversion of coniferyl alcohol
nal standard, reaction mixtures were extracted twice with 1 ml
ethyl acetate and analyzed by GC–MS.
The volumetric activities of laccases and HRP were determined
with the substrate 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS) as described in the Supporting Information. Screening
for the production of 3 from 2 was carried out with 1 mm of 2
For volumetric scale-up 102.5 mg of 1 (corresponding to 25 mm) in
a reaction volume of 25 ml was used. Reactions were performed in
biphasic systems containing 50 mm KPi (pH 7.5), 20% (v/v) tBME,
À1
[
(
from a 50 mm stock solution dissolved in dimethyl sulfoxide
DMSO)] and 10 mUml of either laccase (CotA, SsL1, CgL1, Tth, or
PsVAO and CgL1 in a ratio of 1:5 (10:50 mUml ). After 120 h the
À1
reaction mixture was extracted four times with 12.5 ml ethyl ace-
tate, evaporated and resuspended in 8 ml of a methanol/0.1%
formic acid mixture in a ratio of 7:1. Pinoresinol 3 was isolated by
semipreparative HPLC (Figure S4, Supporting Information). For
Lcc) or HRP in 100 mm NaOAc (pH 5.0), 50 mm KPi (pH 7.5), or
5
3
0 mm Tris-HCl (pH 9.0). Reaction mixtures were incubated for
0 min at 258C, extracted twice with 300 ml ethyl acetate and ana-
lyzed by GC–MS.
product verification high-resolution mass spectrometry (HRMS)
1
(
Table S4, Supporting Information), H NMR (Figure S6A, Support-
13
ing Information), C NMR (Figure S6B, Supporting Information),
HMBC, COSY, and HSQC spectroscopy were performed and the
data were analyzed (further information is given in the Supporting
Information).
Cascade reaction
Development of the enzymatic cascade
1
4
4
3
The volumetric activities of laccases and HRP were determined
with ABTS as described above. The volumetric activity of PsVAO
was determined with 1 as substrate as described in the Supporting
Information. Reactions at analytical scale (1 ml reaction volume)
contained 1 mm of 1 (from a 50 mm stock solution dissolved in
(Æ)-Pinoresinol 3: H NMR (600 MHz, CD COCD ): d=6.98 (d,
J
J
J
3
3
3
(H ,H )=1.9 Hz, 2H; 2,2’-H), 6.83 (ddd, J (H5,5’,H6,6’)=8.1,
2,2’ 6,6’
4
(H2,2’,H6,6’)=2.0 Hz, J (H6,6’,H7,7’)=0.7 Hz, 2H; 6,6’-H), 6.78 (d,
3
(H5,5’,H6,6’)=8.1 Hz, 2H, 5,5’-H), 4.66 (d, J (H7,7’,H ’)=4.0 Hz, 2H;
7,7’-H), 4.21–4.18 (m, 2H; 9,9’-H), 3.83 (s, 6H; 3,3’-OCH ), 3.79 (dd,
8,8
3
À1
À1
3
4
DMSO), 10 mUml PsVAO and 10 mUml laccase or HRP in
00 mm NaOAc (pH 5.0), 50 mm KPi (pH 7.5), or 50 mm Tris-HCl
pH 9.0). Reactions were performed in an overhead shaker at 258C,
0 rpm. Samples were taken after 2.5, 5, 7.5, and 24 h. After addi-
J (H8,8’,H ’)=9.1, J (H ,H ’)=3.8 Hz, 2H, 9,9’-H), 3.11–3.04 ppm
9,9 7,7’ 9,9
13
1
(
2
(m, 2H, 8,8’-H); C NMR (151 MHz, CD OCD ): d=148.30 (C-3,3’),
3 3
146.82 (C-4,4’), 134.17 (C-1,1’), 119.61 (C-6,6’), 115.50 (C-5,5’), 110.60
(C-2,2’), 86.64 (C-7,7’), 72.21 (C-9,9’), 56.25 (3,3’-OCH ), 55.25 ppm
3
(C-8,8’); HRMS (ESI, neg.) calculated for C H O [MÀH]À m/z:
tion of an internal standard (see below), reaction mixtures were ex-
tracted twice with 300 ml ethyl acetate and analyzed by GC–MS.
20
21
6
357.1344, found: 357.1343
All reactions were performed in triplicate. Control reactions were
performed with cleared cell lysate of E. coli not containing heterol-
ogous proteins.
Determination of partition coefficients
For determination of partition coefficients (logP), 4 ml 50 mm KPi
pH 7.5) was supplemented with 2% (v/v) DMSO and 1 ml of an or-
(
Optimization of reaction conditions
ganic solvent (tBME or isooctane). 10 mm eugenol 1, 5 mm conifer-
yl alcohol 2 or 0.275 mm pinoresinol 3 were added, and incubated
in an overhead shaker at 258C for 2 h. 600 ml of the organic and
the aqueous phases were taken for analysis and 2 mm of the inter-
nal standard ferulic acid methyl ester (FSME) (from a 100 mm stock
solution in ethanol) was added. The aqueous sample was extracted
with 600 ml ethyl acetate. The samples from the organic and aque-
ous phase were analyzed by GC–MS. For calculation of the parti-
tion coefficient the peak areas (pa) of the substances were normal-
ized to the internal standard and the following mathematical for-
mula was used:
For determination of the optimal enzyme ratio 1 mm of 1 and
À1
1
0 mUml PsVAO were supplemented with a bacterial laccase
(
1
Ssl1 or CgL1) of different volumetric activities (1, 5, 10, 50, or
00 mUml ).
À1
To analyze the influence of organic solvents 20% (v/v) of an organ-
ic solvent was added to 50 mm KPi (pH 7.5) containing (i) 1 mm of
À1
À1
1
and 10 mUml PsVAO, or (ii) 1 mm or 10 mm of 1, 10 mUml
À1
PsVAO, and 10 mUml Ssl1.
Based on the determined optimum reaction parameters (10 mm of
, biphasic system with 20% tBME) the reaction was repeated with
1
log P ¼ logðpaorganicÞÀlogðpaaqueousÞ:
À1
À1
CgL1 (50 mUml ) or HRP (10 mUml ) instead of Ssl1
À1
(
10 mUml ). Optionally, the reaction with CgL1 was supplemented
À1
with 600 Uml catalase.
In the one-pot sequential reaction mode 10 mm of 1 in a biphasic
Product analysis and identification
À1
system was supplemented with 10 mUml PsVAO, and after a reac-
À1
Qualitative and quantitative GC–MS analysis
tion time of 72 h 10 mUml laccase was added. In the case of Lcc
pH adjustment was performed by dropwise addition of HCl.
Reaction mixtures were analyzed by GC–MS measurements on
a GC–MS 2010 (Shimadzu, Duisburg, Germany) equipped with an
FS-Supreme-5 column (30 m”0.25 mm”0.25 mm, Chromatogra-
phie Service GmbH, Langerwehe, Germany) and helium as carrier
gas. 0.5 ml of a sample was injected with a split of 5 and an injec-
tion temperature of 3008C. For separation of the compounds the
temperature program started at 1308C. The temperature was held
After addition of an internal standard, reaction mixtures were ex-
tracted twice with 300 ml ethyl acetate and analyzed by GC–MS.
Experiments on a semipreparative scale
À1
Concentration scale-up was performed in biphasic systems with
for 2 min, increased with a rate of 208Cmin to 3008C and held
5
0 mm KPi (pH 7.5), 20% (v/v) tBME, PsVAO and CgL1
for 8.5 min. Compounds were identified by their characteristic
mass-fragmentation patterns and retention times compared to au-
thentic reference compounds.
À1
(
10:50 mUml ), and different concentrations of 1 (25, 50, or
1
00 mm) in a reaction volume of 0.5 ml. After addition of an inter-
ChemCatChem 2015, 7, 1857 – 1864
www.chemcatchem.org
1862
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
For quantitative analysis of 3 a calibration curve was recorded uti-
lizing FSME as internal standard: (i) 100 mm from a 5 mm stock solu-
tion in DMSO in case of reactions with 1 mm or 10 mm of 1, or
[11] a) W. Hummel, H. Schütte, E. Schmidt, C. Wandrey, M.-R. Kula, Appl. Mi-
crobiol. Biotechnol. 1987, 26, 409–416; b) H.-S. Bae, S.-G. Lee, S.-P. Hong,
M.-S. Kwak, N. Esaki, K. Soda, M.-H. Sung, J. Mol. Catal. B 1999, 6, 241–
247; c) M. Fuchs, D. Koszelewski, K. Tauber, W. Kroutil, K. Faber, Chem.
(
ii) 2 mm from a 100 mm stock solution in case of higher concentra-
Commun. 2010, 46, 5500–5502; d) E.-S. Park, J.-Y. Dong, J.-S. Shin, Appl.
Microbiol. Biotechnol. 2014, 98, 651–660.
tions of 1. Two calibration curves were recorded: (i) with final con-
centrations of 3 of 20 to 100 mm and FSME in final concentration
of 100 mm; (ii) with final concentrations of 3 of 0.1 to 2.5 mm and
FSME in final concentration of 2 mm. The ratios of the peak area of
[
12] a) R. Agudo, M. T. Reetz, Chem. Commun. 2013, 49, 10914–10916;
b) C. A. Müller, B. Akkapurathu, T. Winkler, S. Staudt, W. Hummel, H.
Grçger, U. Schwaneberg, Adv. Synth. Catal. 2013, 355, 1787–1798; c) S.
Gargiulo, D. J. Opperman, U. Hanefeld, I. W. C. E. Arends, F. Hollmann,
Chem. Commun. 2012, 48, 6630–6632; d) T. Classen, M. Korpak, M.
Schçlzel, J. Pietruszka, ACS Catal. 2014, 4, 1321–1331; e) M. Gall, M.
Thomsen, C. Peters, I. V. Pavlidis, P. Jonczyk, P. P. Grünert, S. Beutel, T.
Scheper, E. Gross, M. Backes, T. Geißler, J. P. Ley, J.-M. Hilmer, G. Kramm-
er, G. J. Palm, W. Hinrichs, U. T. Bornscheuer, Angew. Chem. Int. Ed. 2014,
3
to that of the internal standard were plotted against the concen-
tration of 3 to give a straight-line calibration plot.
Pinoresinol purification by semipreparative HPLC
5
3, 1439–1442; Angew. Chem. 2014, 126, 1463–1466; f) N. Oberleitner,
Pinoresinol 3 was purified by HPLC equipped with an Eurospher II
C. Peters, J. Muschiol, M. Kadow, S. Saß, T. Bayer, P. Schaaf, N. Iqbal, F.
Rudroff, M. D. Mihovilovic, U. T. Bornscheuer, ChemCatChem 2013, 5,
1
00-10-C18 column (300”8 mm, pore size 100 Š, 10 mm particle
size, Bischoff, Leonberg, Germany). A solvent gradient of metha-
nol (B) and 0.1% formic acid (A) was applied as follows: Starting
from 20% B to 35% B in 10 min, hold for 10 min, increase to 70%
B within 28 min, then to 90% B within 1 sec, hold for 2 min.
3
524–3528; g) A. Pennec, F. Hollmann, M. S. Smit, D. J. Opperman,
ChemCatChem 2015, 7, 236–239; h) S. Schulz, M. Girhard, S. K. Gaßmey-
er, V. D. Jäger, D. Schwarze, A. Vogel, V. B. Urlacher, ChemCatChem 2015,
7, 601–604; i) S. Schmidt, C. Scherkus, J. Muschiol, U. Menyes, T. Winkler,
W. Hummel, H. Grçger, A. Liese, H.-G. Herz, U. T. Bornscheuer, Angew.
Chem. Int. Ed. 2015, 54, 2784–2787; Angew. Chem. 2015, 127, 2825–
2828.
Acknowledgements
[
13] a) M. Pe sˇ i c´ , C. López, J. López-Santín, G. Alvaro, Appl. Microbiol. Biotech-
nol. 2013, 97, 7173–7183; b) I. Sµnchez-Moreno, V. HØlaine, N. Poupard,
F. Charmantray, B. LØgeret, L. Hecquet, E. García-Junceda, R. Wohlge-
muth, C. GuØrard-HØlaine, M. Lemaire, Adv. Synth. Catal. 2012, 354,
Heinrich-Heine University is acknowledged for financial support.
We wish to thank Prof. Dr. Marco W. Fraaije (Laboratory of Bio-
chemistry, Groningen Biomolecular Sciences and Biotechnology
Institute, University of Groningen, Netherlands) for providing the
gene psvao.
1725–1730; c) S. Baum, F. van Rantwijk, A. Stolz, Adv. Synth. Catal. 2012,
3
54, 113–122; d) S. van Pelt, F. van Rantwijk, R. A. Sheldon, Adv. Synth.
Catal. 2009, 351, 397–404; e) T. Sehl, H. C. Hailes, J. M. Ward, R. Warden-
ga, E. von Lieres, H. Offermann, R. Westphal, M. Pohl, D. Rother, Angew.
Chem. Int. Ed. 2013, 52, 6772–6775; Angew. Chem. 2013, 125, 6904–
6908.
Keywords: bacterial laccase · biocatalysis · CÀC coupling ·
enzymes · natural products
[14] a) E. E. Ferrandi, D. Monti, I. Patel, R. Kittl, D. Haltrich, S. Riva, R. Ludwig,
Adv. Synth. Catal. 2012, 354, 2821–2828; b) S. Aksu, I. W. C. E. Arends, F.
Hollmann, Adv. Synth. Catal. 2009, 351, 1211–1216.
[
15] A. Díaz-Rodríguez, N. Ríos-Lombardía, J. H. Sattler, I. Lavandera, V.
Gotor-Fernµndez, W. Kroutil, V. Gotor, Catal. Sci. Technol. 2015, 5, 1443–
1446.
[
1] a) E. Ricca, B. Brucher, J. H. Schrittwieser, Adv. Synth. Catal. 2011, 353,
239–2262; b) R. C. Simon, N. Richter, E. Busto, W. Kroutil, ACS Catal.
014, 4, 129–143; c) J. Muschiol, C. Peters, N. Oberleitner, M. Mihovilov-
2
2
[16] H. Adlercreutz, Crit. Rev. Clin. Lab. Sci. 2007, 44, 483–525.
[17] a) A. M. Duncan, W. R. Phipps, M. S. Kurzer, Best Pract. Res. Clin. Endocri-
nol. Metab. 2003, 17, 253–271; b) R. A. Dixon, Annu. Rev. Plant Biol.
2004, 55, 225–261; c) T. Cornwell, W. Cohick, I. Raskin, Phytochemistry
2004, 65, 995–1016.
[18] a) A. Ayella, S. Lim, Y. Jiang, T. Iwamoto, D. Lin, J. Tomich, W. Wang, Nutr.
Res. 2010, 30, 762–769; b) S. Jafari, S. Saeidnia, M. Abdollahi, Curr.
Pharm. Biotechnol. 2014, 15, 409–421; c) M. Azrad, R. T. Vollmer, J.
Madden, M. Dewhirst, T. J. Polascik, D. C. Snyder, M. T. Ruffin, J. W. Moul,
D. E. Brenner, W. Demark-Wahnefried, J. Med. Food 2013, 16, 357–360.
[19] a) J. Feng, Z. Shi, Z. Ye, Biol. Pharm. Bull. 2008, 31, 1067–1070; b) S. M.
Sacco, L. U. Thompson, B. Ganss, W. E. Ward, J. Med. Food 2011, 14,
1208–1214.
ic, U. Bornscheuer, F. Rudroff, Chem. Commun. 2015, 51, 5798–5811.
2] A. Bruggink, R. Schoevaart, T. Kieboom, Org. Process Res. Dev. 2003, 7,
[
[
[
6
22–640.
3] S. Kara, J. H. Schrittwieser, F. Hollmann, M. B. Ansorge-Schumacher, Appl.
Microbiol. Biotechnol. 2014, 98, 1517–1529.
4] a) S. Riva, W.-D. Fessner, Cascade biocatalysis. Integrating stereoselective
and environmentally friendly reactions, Wiley-VCH, Weinheim, 2014; b) E.
Garcia-Junceda, Multi-step enzyme catalysis. Biotransformations and che-
moenzymatic synthesis, Wiley-VCH, Weinheim, 2008.
[
[
[
[
5] J. M. Woodley, Trends Biotechnol. 2008, 26, 321–327.
6] K. Imai, S. Marumo, T. Ohtaki, Tetrahedron Lett. 1976, 17, 1211–1214.
7] M. Szczesiul, D. E. Wampler, Biochemistry 1976, 15, 2236–2244.
8] a) C. V. Voss, C. C. Gruber, K. Faber, T. Knaus, P. Macheroux, W. Kroutil, J.
Am. Chem. Soc. 2008, 130, 13969–13972; b) S.-Y. Chen, C.-X. Yang, J.-P.
Wu, G. Xu, L.-R. Yang, Adv. Synth. Catal. 2013, 355, 3179–3190; c) V.
Gauchot, W. Kroutil, A. R. Schmitzer, Chem. Eur. J. 2010, 16, 6748–6751;
d) T. Sehl, H. C. Hailes, J. M. Ward, U. Menyes, M. Pohl, D. Rother, Green
Chem. 2014, 16, 3341–3348; e) J. H. Schrittwieser, F. Coccia, S. Kara, B.
Grischek, W. Kroutil, N. d’Alessandro, F. Hollmann, Green Chem. 2013,
[20] a) J. L. Adolphe, S. J. Whiting, B. H. Juurlink„ L. U. Thorpe, J. Alcorn, Br. J.
Nutr. 2010, 103, 929–938; b) J. Peterson, J. Dwyer, H. Adlercreutz, A.
Scalbert, P. Jacques, M. L. McCullough, Nutr. Rev. 2010, 68, 571–603.
[21] E. Aehle, U. Müller, P. C. Eklund, S. M. Willfçr, W. Sippl, B. Dräger, Phyto-
chemistry 2011, 72, 2396–2405.
[22] M. V. Sepporta, T. Mazza, G. Morozzi, R. Fabiani, Nutr. Cancer 2013, 65,
1208–1218.
1
5, 3318–3331; f) M. Schallmey, R. J. Floor, B. Hauer, M. Breuer, P. A.
[23] S. Mitsuhashi, T. Kishimoto, Y. Uraki, T. Okamoto, M. Ubukata, Bioorg.
Med. Chem. 2008, 16, 2645–2650.
Jekel, H. J. Wijma, B. W. Dijkstra, D. B. Janssen, ChemBioChem 2013, 14,
8
8
4
70–881; g) D. Kalaitzakis, I. Smonou, J. Org. Chem. 2010, 75, 8658–
661; h) C. V. Voss, C. C. Gruber, W. Kroutil, Angew. Chem. Int. Ed. 2008,
7, 741–745; Angew. Chem. 2008, 120, 753–757.
[24] a) A. Wikul, T. Damsud, K. Kataoka, P. Phuwapraisirisan, Bioorg. Med.
Chem. Lett. 2012, 22, 5215–5217; b) K. H. Kim, E. Moon, S. Y. Kim, K. R.
Lee, J. Agric. Food Chem. 2010, 58, 4779–4785; c) B. Hwang, J. Lee, Q.-
H. Liu, E.-R. Woo, D. G. Lee, Molecules 2010, 15, 3507–3516.
[25] I. E. Milder, I. C. Arts, B. van de Putte, D. P. Venema, P. C. Hollman, Br. J.
Nutr. 2005, 93, 393–402.
[
9] a) M. Schrewe, N. Ladkau, B. Bühler, A. Schmid, Adv. Synth. Catal. 2013,
55, 1693–1697; b) E. O’Reilly, C. Iglesias, D. Ghislieri, J. Hopwood, J. L.
Galman, R. C. Lloyd, N. J. Turner, Angew. Chem. Int. Ed. 2014, 53, 2447–
450; Angew. Chem. 2014, 126, 2479–2482.
10] F. van Rantwijk, A. Stolz, J. Mol. Catal. B 2015, 114, 25–30.
3
2
[26] a) S. C. Roy, K. K. Rana, C. Guin, J. Org. Chem. 2002, 67, 3242–3248; b) A.
Pelter, R. S. Ward, D. J. Watson, P. Collins, I. T. Kay, J. Chem. Soc. Perkin
[
ChemCatChem 2015, 7, 1857 – 1864
www.chemcatchem.org
1863
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Trans. 1 1982, 175–181; c) J. Maruyama, M. Kobayashi, M. Miyashita, I.
Kouno, H. Irie, Heterocycles 1994, 37, 839–845.
[36] M. Gunne, A. Hçppner, P.-L. Hagedoorn, V. B. Urlacher, FEBS J. 2014,
4307–4318.
[
[
[
27] M. Lahtinen, L. Viikari, P. Karhunen, J. Asikkala, K. Kruus, I. Kilpeläinen, J.
Mol. Catal. B 2013, 85, 169–177.
28] F. De Angelis, R. Nicoletti, N. Spreti, F. Verì, Angew. Chem. Int. Ed. 1999,
[37] K. Miyazaki, Extremophiles 2005, 9, 415–425.
[38] G. Grass, C. Rensing, Biochem. Biophys. Res. Commun. 2001, 286, 902–
908.
[39] P. Dur¼o, Z. Chen, C. S. Silva, C. M. Soares, M. M. Pereira, S. Todorovic, P.
Hildebrandt, I. Bento, P. F. Lindley, L. O. Martins, Biochem. J. 2008, 412,
339–346.
38, 1283–1285; Angew. Chem. 1999, 111, 1364–1367.
29] B. Pickel, M.-A. Constantin, J. Pfannstiel, J. Conrad, U. Beifuss, A. Schaller,
Angew. Chem. Int. Ed. 2010, 49, 202–204; Angew. Chem. 2010, 122,
2
07–209.
30] M. W. Fraaije, C. Veeger, W. J. van Berkel, Eur. J. Biochem. 1995, 234,
71–277.
31] T. Shiba, L. Xiao, T. Miyakoshi, C.-L. Chen, J. Mol. Catal. B 2000, 10, 605–
15.
[40] E. P. Melo, A. T. Fernandes, P. Dur¼o, L. O. Martins, Biochem. Soc. Trans.
2007, 35, 1579–1582.
[41] a) A. Intra, S. Nicotra, S. Riva, B. Danieli, Adv. Synth. Catal. 2005, 347,
973–977; b) S. Ncanana, L. Baratto, L. Roncaglia, S. Riva, S. G. Burton,
Adv. Synth. Catal. 2007, 349, 1507–1513.
[
[
2
6
[
[
32] P. Baldrian, FEMS Microbiol. Rev. 2006, 30, 215–242.
33] E. de Jong, W. J. van Berkel, R. P. van der Zwan, J. A. de Bont, Eur. J. Bio-
chem. 1992, 208, 651–657.
[42] M. Gunne, V. B. Urlacher, PLoS ONE 2012, 7, e52360.
[
34] E. Ricklefs, N. Winkler, K. Koschorreck, V. B. Urlacher, J. Biotechnol. 2014,
Received: February 24, 2015
Revised: April 7, 2015
1
91, 46–53.
[35] K. Koschorreck, R. D. Schmid, V. B. Urlacher, BMC Biotechnol. 2009, 9, 12.
Published online on June 3, 2015
ChemCatChem 2015, 7, 1857 – 1864
www.chemcatchem.org
1864
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim