Altering the substrate specificity and enantioselectivity of phenylacetone monooxygenase by structure-inspired enzyme redesign

Article abstract of DOI:10.1002/adsc.200700045

Of all presently available Baeyer-Villiger monooxygenases, phenylacetone monooxygenase (PAMO) is the only representative for which a structure has been determined. While it is an attractive biocatalyst because of its thermostability, it is only active wit

Full text of DOI:10.1002/adsc.200700045

FULL PAPERS
DOI: 10.1002/adsc.200700045
Altering the Substrate Specificity and Enantioselectivity of
Phenylacetone Monooxygenase by Structure-Inspired Enzyme
Redesign
a
b
b
Daniel E. Torres PazmiÇo, Radka Snajdrova, Daniela V. Rial,
b,
a,
Marko D. Mihovilovic, * and Marco W. Fraaije *
University of Groningen, Department of Chemistry, Biotechnology Group, Nijenborgh 4, 9747 AG Groningen,
a
The Netherlands
Fax : (+31)-50-363-4165; e-mail: m.w.fraaije@rug.nl
Vienna University of Technology, Institute of Applied Synthetic Chemistry, Getreidemarkt 9/163-OC, 1060 Vienna,
b
Austria
Fax : (+43)-1-58801-15499; e-mail: mmihovil@pop.tuwien.ac.at
Received: January 23, 2007
Abstract: Of all presently available Baeyer–Villiger type PAMO shows no activity. An interesting finding
monooxygenases, phenylacetone monooxygenase was that the mutant is able to convert indole into
(
PAMO) is the only representative for which a struc- indigo blue: a reaction that has never been reported
ture has been determined. While it is an attractive before for a Baeyer–Villiger monooxygenase. In ad-
biocatalyst because of its thermostability, it is only dition to an altered substrate specificity, the enantio-
active with a limited number of substrates. By means selectivity towards several sulfides was dramatically
of a comparison of the PAMO structure and a mod- improved. This newly designed Baeyer–Villiger
eled structure of the sequence-related cyclopenta- monooxygenase extends the scope of oxidation reac-
none monooxygenase, several active-site residues tions feasible with these atypical monooxygenases.
were selected for a mutagenesis study in order to
alter the substrate specificity. The M446G PAMO Keywords: asymmetric catalysis; Baeyer–Villiger
mutant was found to be active with a number of aro- monooxygenase; biotransformations; enzyme rede-
matic ketones, amines and sulfides for which wild- sign; indigo blue; sulfoxidation
Introduction
biooxidations are of great value in organic synthesis
and the pharmaceutical chemistry. While only a few
[
2]
The number of processes in which biocatalysts are in- BVMOs were known and have been explored for bio-
volved is steadily growing. Biocatalysts are frequently catalytic purposes since the beginning of the 1970s, in
exploited for their exquisite regio- or enantioselectivi- recent years several other BVMOs have been report-
[
3]
ty for the synthesis of pharmaceutical building blocks ed in literature.
Biocatalytic studies on these
while they are also increasingly being explored for ap- BVMOs have revealed that these biocatalysts do not
plication in the synthesis of low-value chemicals. only catalyze Baeyer–Villiger oxidations but also are
Alongside with this increase in biocatalyst implemen- able to oxidize sulfides and other heteroatom-contain-
tation, more and more biocatalysts have become ing compounds. Except for this promiscuity in reactiv-
available. Due to the efforts of biocatalyst discovery ity, BVMOs are often very enantio-, regio- and/or
and redesign in the last decade, a huge number of chemoselective while accepting a broad range of sub-
novel enzymes have emerged on the market. How- strates. Recently we have obtained a novel BVMO,
ever, while the arsenal of hydrolytic enzymes is quite phenylacetone
monooxygenase
(PAMO,
EC
extensive nowadays, the number of applicable oxida- 1.14.13.92), which offers several unique and attractive
tive biocatalysts is lagging behind. To satisfy the grow- features: (1) it is thermostable and tolerant towards
[
4]
ing demand for novel oxidative biocatalysts, we focus organic solvents, (2) it has been shown to catalyze
on the discovery and redesign of such enzymes. enantioselective Baeyer–Villiger oxidations and sul-
An interesting class of oxidative biocatalysts is rep- foxidations, and (3) it represents the only BVMO
[
5]
[
6]
resented by the Baeyer–Villiger monooxygenases for which a crystal structure is available. While a
[1]
(
BVMOs). Products obtained by BVMO-mediated number of substrates, mainly aromatic, have been
Adv. Synth. Catal. 2007, 349, 1361 – 1368
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1361

Daniel E. Torres PazmiÇo et al.
FULL PAPERS
identified for phenylacetone monooxygenase from specificity, the active-site redesign results in improved
Thermobifida fusca, it shows poor or no activity with enantioselective behavior. Furthermore, we applied a
aliphatic compounds. This contrasts with the relaxed parallel screening methodology recently developed
substrate acceptance profiles of cyclohexanone mono- for the evaluation of whole cells biocatalyst perfor-
oxygenase (CHMO, EC 1.14.13.22) from Acinetobact- mance and stereoselectivity, demonstrating that wild-
[
7]
er sp. NCIMB 9871 and cyclopentanone monooxy- type and M446G PAMO can be exploited using
[
12]
genase (CPMO, EC 1.14.13.16) from Comamonas sp. whole cells.
[
8]
strain NCIMB 9872, which are the most extensively
[9]
studied BVMOs. These enzymes efficiently convert
a wide range of aliphatic ketones and are complemen- Results and Discussion
tary in respect to their enantioselectivity towards sev-
eral of these ketones. Unfortunately, CHMO and Cloning, Purification and Characterization of PAMO
CPMO display a poor stability in comparison to Mutants
[10]
PAMO.
While they display widely differing substrate specif- Based on the structural differences of the (predicted)
icities, CPMO and PAMO are closely related as they active sites of CPMO and PAMO, we prepared a
share 41% sequence identity. This level of sequence single, a double and a triple mutant of PAMO by
homology has allowed us to build a structural model means of site-directed mutagenesis: (i) M446G, (ii)
of CPMO. Inspection of the active site of CPMO re- Q152F/L153G and (iii) Q152F/L153G/M446G. Over-
vealed that most residues located at the re face of the expression and purification of these mutants (see Ex-
bound FAD cofactor (e.g., PAMO residues N58, D66, perimental Section) resulted in three soluble mutant
S196, Q200, K336, R337, F389, Y495 and W501), proteins containing a tightly bound FAD cofactor. All
forming the substrate binding pocket, are identical in mutants were tested for activity with several potential
both enzymes. Conservation of R337 is not surprising substrates (phenylacetone, cyclopentanone, cyclohexa-
as it is the key active site residue which assists in cat- none) by following the rate of NADPH consumption
[6]
alysis. Only three residues were strikingly different: in time at 340 nm.
Q152, L153 and M446 in PAMO align with F156,
The double and the triple PAMO mutants were
G157 and G453 in CPMO (Figure 1). The F156 and found to be inactive with these ketones as only a slow
G157 residues of CPMO have recently been con- conversion of NADPH was observed with concomi-
firmed as hotspots in tuning enantioselectivity of tant production of hydrogen peroxide. This indicates
[11]
CPMO.
that these mutants are still able to bind NADPH and
In this paper we report the change of PAMO sub- reduce the flavin cofactor, while they have lost the
strate specificity by replacing M446: the M446G ability to perform oxygenation reactions. As a result
PAMO mutant. In addition to an altered substrate they merely act as NADPH oxidase with rates of
ꢀ
1
ꢀ1
0
.06 s for the Q152F/L153G mutant and 0.6 s for
the M446G/Q152F/L153G mutant. A similar rate
ꢀ
1
(
0.05 s ) of uncoupled NADPH consumption has
been observed for wild-type PAMO when no sub-
[
3d]
strate is present. Although the modeled active site
of CPMO appears quite similar to that of PAMO,
these results indicate that the Q/L!F/G substitutions
in PAMO result in an active site architecture that is
not effective in proper binding and/or positioning of
substrates. Improper positioning of substrates with res-
pect to the flavin cofactor will hinder an effective nu-
cleophilic attack of the peroxyflavin that is required
for oxygenation of the substrate. A recent study has
shown that the corresponding residues in CPMO can
be varied to some extent thereby influencing the
enantioselectivity. However, it should be noted that
all reported conversions using these CPMO mutants
resulted in relatively poor conversions when com-
[
11,13]
pared with wild type CPMO.
This again hints to
the fact that these residues are crucial for proper cat-
alysis.
Figure 1. Representation of the active-sites of PAMO
The M446G PAMO mutant showed significant ac-
tivity towards phenylacetone (1) while no activity was
(
green) and CPMO (purple).
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Adv. Synth. Catal. 2007, 349, 1361 – 1368

Substrate Specificity and Enantioselectivity of Phenylacetone Monooxygenase
FULL PAPERS
observed with cyclopentanone and cyclohexanone. BVMOs are not able to perform hydroxylations and
[
16]
The mutant displayed an identical uncoupling rate are very poor epoxidation catalysts.
We also con-
when compared to wild-type PAMO. In the absence firmed that wild-type PAMO and M446G PAMO are
of a suitable substrate the mutant enzyme oxidized unable to hydroxylate or epoxidize the indole-related
ꢀ
1
NADPH at a rate of 0.1 s . A hint for an altered sub- compounds styrene, indene and cinnamyl alcohol. We
strate specificity for the M446G mutant was already concluded that indigo formation from indole is most
obtained during growth of E. coli cells expressing the likely the result of N-oxidation of indole, as has been
respective enzyme. During growth of M446G PAMO- previously suggested for a distantly related flavin-con-
[
14d]
expressing cells, the culture medium turned blue sug- taining monooxygenase.
The formed indole N-
gesting that indigo blue was formed during cultiva- oxide is expected to rearrange into indoxyl, which
tion. Indigo production has been observed in E. coli would yield indigo blue upon spontaneous dimeriza-
before when expressing other types of mono- or dioxy- tion. Such a rearrangement has also been observed
[14]
[17]
genases.
This observation suggests that the endo- for related N-oxides. To our knowledge such a syn-
genous indole (14), generated from tryptophan by thetic route towards indigo blue has not been de-
[
15]
tryptophanase in E. coli, is oxidized by this specific scribed before and it may offer interesting alternative
PAMO mutant and subsequently undergoes spontane- routes for synthesis of indigo derivatives. In addition
ous dimerization to yield indigo blue. Thus far, no to indole, also another secondary amine, N-methyl-
other BVMO has been found which is able to form benzylamine, was accepted by M446G PAMO while
indigo blue from indole.
this amine is not a substrate for wild-type PAMO. In
contrast, it was previously demonstrated that wild-
type PAMO oxidizes the tertiary amine, N,N-dime-
thylbenzylamine, while this amine is not accepted by
M446G PAMO. This indicates that the engineered
mutant readily accepts secondary amines. These re-
[
5]
Substrate Specificity and Steady-State Kinetics of
M446G PAMO
Using isolated enzyme, the substrate acceptance pro- sults show that replacing M446G has dramatic effects
file of M446G PAMO was explored in comparison on the substrate acceptance profile.
with the wild-type enzyme. This has revealed that, be-
sides phenylacetone (1) and indole (14), M446G
PAMO accepts a number of other organic substrates, Enantioselective Sulfoxidations by Purified M446G
including ketones/aldehydes, sulfides and amines. The PAMO
steady-state kinetic parameters for these substrates
are shown in Table 1. Compared to wild-type PAMO, While it has been observed in previous studies that
the apparent affinity (K ) of the mutant enzyme to- wild-type PAMO efficiently oxidizes a wide range of
M
[
5]
wards the physiological substrate, phenylacetone (1), aromatic sulfides (Table 1), the enzyme mediated
decreased 10-fold. The activity of the mutant enzyme oxidation was often not very enantioselective. As can
was found to be identical to that of wild-type PAMO: be seen in Table 1, the M446G PAMO also readily
ꢀ
1
k =3.0 s . Using phenylacetone as substrate the converts a number of aromatic sulfides. In fact, one
cat
thermostability of the M446G mutant was tested at sulfide was discovered that is not accepted by wild-
5
08C. A half-life time of 55ꢁ6 h was found which is type enzyme while it is oxidized by the mutant. To de-
[3d]
comparable to that of wild-type PAMO. This shows termine the enantioselectivity of this oxidation, 2 mL
that the mutation does not affect the thermostability. reactions were performed using purified mutant
The catalytic efficiency towards several known enzyme in the presence of glucose 6-phosphate dehy-
PAMO substrates 2, 5, and 9 was also not affected by drogenase for regeneration of NADPH. Analysis of
the mutation. Interestingly, while some substrates ac- these conversions by chiral GC showed that the M!
cepted by wild-type PAMO were not accepted by G mutation resulted in a substantial increase in enan-
M446G PAMO (4-hydroxyacetophenone and N,N-di- tioselectivity of several product sulfoxides (Table 2).
methylbenzylamine), the PAMO mutant showed ac- The enantioselectivity with thioanisole (7) increased
tivity towards several aromatic substrates (3, 11, 13 from a moderate (ee =41%) to an excellent enantio-
wt
and 14) that are not converted by wild-type PAMO.
selectivity (eeM446G =93%). Even more impressive re-
We were also able to confirm that purified M446G sults were obtained for sulfides 8 and 9 for which the
PAMO is capable of forming indigo blue from indole wild-type enzyme displayed almost no enantioselec-
as substrate. The ability of this mutant to convert tivity (ee =6%) while the mutant enzyme shows an
wt
indole was unexpected as the BVMO-catalyzed pro- excellent enantioselectivity (eeM446G =92 and 95%, re-
duction of indigo blue from indole has not been ob- spectively). With all three sulfides, the corresponding
served before. Previous studies have shown that mono- (R)-sulfoxides were predominantly formed. In con-
oxygenases are able to produce indigo upon epoxida- trast, conversion of benzyl methyl sulfide (10) yields
[
14a–c]
tion or hydroxylation of indole.
However, mainly the (S)-sulfoxide. The enantioselectivity of
Adv. Synth. Catal. 2007, 349, 1361 – 1368
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Daniel E. Torres PazmiÇo et al.
FULL PAPERS
Table 1. Steady-state kinetic parameters of wild-type and M446G PAMO.
[
a,b]
[a]
Substrate
Structure
Wild-type PAMO
KM [mM]
M446G PAMO
KM [mM]
ꢀ
1
ꢀ1 ꢀ1
ꢀ1
ꢀ1 ꢀ1
No.
kcat [s ]
k /K [M s ]
cat
kcat [s ]
k /K [M s ]
cat M
M
Ketones/Aldehydes
1
3.0
2.3
0.08
0.1
37500
23000
3.0
3.1
1.0
0.2
3000
2
3
16000
1800
-
-
<1
2.9
-
1.6
-
4
5
0.34
2.2
150
<1
1.4
6.9
200
55
>0.30
>0.02
>2.5
>5.0
130
4
6
>0.06
>1.1
Sulfides
7
>0.12
2.1
>2.5
0.86
1.5
1.6
-
47
>1.3
>0.8
>0.35
0.53
>2.5
>2.0
>2.0
2.0
500
400
180
270
55
8
9
2400
170
1100
<1
0.25
1.8
1
0
1
1
-
>0.11
>1.7
Amines
1
2
3
0.03
-
1.0
-
32
-
-
<1
1
<1
>0.30
>20
16
14
-
-
<1
0.30
2.4
130
[
[
a]
Due to solubility limitations, lower limits for kcat and KM are given in some cases.
Some of the kinetic parameters of wild-type PAMO were adapted from previous studies.
b]
[3d,5]
.
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Substrate Specificity and Enantioselectivity of Phenylacetone Monooxygenase
FULL PAPERS
Table 2. Enantioselective sulfoxidations by wild-type and The fact that replacement of M446 can already result
M446G PAMO.
in dramatic changes in substrate specificity and enan-
tioselectivity suggests that specific interaction of one
or two methanol molecules in the active site may ex-
plain the observed solvent effect.
Substrate
No. Structure
Wild-type PAMO
M446G PAMO
Conversion ee
[a]
[a]
Conversion ee
%]
[
[%] [%]
[%]
4
1
93
(R)
7
8
9
94
>99
>99
>99
>99
Whole Cell Baeyer–Villiger Oxidation of Cyclic
Ketones
(R)
6
92
(R)
69
To probe whether wild-type and M446G PAMO can
be exploited as whole cell biocatalysts, the biooxida-
tion of several prochiral cycloketones was evaluated.
Screening experiments were performed in multi-well
plastic dishes and conversion and enantioselectivity of
microbial reactions were evaluated by chiral GC anal-
ysis. It was found that both PAMO variants were ef-
fective as whole cell biocatalysts. In general, wild-type
PAMO showed higher conversion of ketones but
lower enantioselectivity. On the other hand, M446G
PAMO oxidized ketones to the corresponding lac-
tones with higher enantioselectivity, but conversions
(R)
6
95
(R)
94
(S)
9
8
59
(S)
10
>99
(S)
[
a]
The predominantly formed enantiomer in parentheses.
mutant M446G towards this sulfide was found to be were poor to moderate in all cases. Data included in
lower than that of wild-type PAMO as the ee de- Table 3 represent a selection of results with striking
creased from 98% to 59%. This demonstrates that variations in the biocatalytic performance of wild-
the M446G mutant nicely complements the catalytic type PAMO and the M446G mutant. Interestingly,
repertoire of the wild-type enzyme. Interestingly, the the M446G mutant was able to oxidize 15 while wild-
M446G replacement mimics to some extent the effect type PAMO performed best with ketone 17 (Table 3).
of addition of methanol to wild-type PAMO. Recently This demonstrates the subtle changes in substrate spe-
it was shown that usage of up to 30% methanol is tol- cificity as a result of replacing M by G in position 446
[
4b]
erated by PAMO and affects the enantioselectivity.
of PAMO.
Table 3. Conversions of prochiral cycloketones using wild-type and M446G PAMO expressing cells.
Substrate
Structure
Wild-type PAMO
Conversion ratio lactones
M446G PAMO
Conversion ratio lactones
[a]
[b]
[a]
[b]
No.
ee [%]
ee [%]
15
-
-
4%
28 (ꢀ)
1
6
6
23%
35%
5 (+)
traces
12%
10 (ꢀ)
65 (ꢀ)
11 (ꢀ)
1
7
8
65%;
63%;
50:50
90:10
92/50
71/96
traces;
3%;
70:30
87:13
>99/45
1
97/48
[
[
a]
Ratio of “normal”/“abnormal” lactones.
Sign of specific rotation in parentheses, while ee is given for normal/abnormal lactone.
b]
Adv. Synth. Catal. 2007, 349, 1361 – 1368
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Daniel E. Torres PazmiÇo et al.
FULL PAPERS
Conclusions
In recent years it has become popular to employ
random mutagenesis approaches (directed evolution)
for enzyme redesign. However, such an approach is
[
18]
often not very efficient. In fact, it appears that for
effectively altering the substrate specificity of a bio-
catalyst one should preferentially target active site or
[
18,19]
ꢁfirst shellꢂ residues.
However, such a structure-in-
spired approach is only feasible when a structure of
the targeted biocatalyst is available. For the current
study, we exploited the recently determined crystal
structure of PAMO. A comparison of the PAMO
structure and a homology-built model of CPMO re-
vealed that both active sites are remarkably similar.
Only three residues could be identified as significantly
different: Q152, L153 and M446. It was postulated
that these residues determine to some extent the mo-
lecular basis for the two widely different substrate
specificities of PAMO and CPMO. While the Q152F/
L153G and Q152F/L153G/M446G PAMO mutants
were found to be inactive, the M446G mutant shows
interesting novel catalytic features while retaining its
thermostability. Several new compounds were identi-
fied as substrate for this PAMO variant (e.g., indole
and benzaldehyde). An altered substrate binding
pocket may explain the substantial changes in sub-
strate specificity and enantioselectivity towards sul-
fides and ketones.
Figure 2. Structure of PAMO in which the FAD cofactor is
shown in yellow sticks. The crucial active-site R337 is shown
in blue sticks. Observed mutations that effect enantioselec-
tivity of BVMOs are shown by spheres (M446, Q152 and
The exact binding mode of substrates in PAMO is
uncertain due to the lack of a crystal structure con-
taining a bound substrate. However, several random
[11,20]
L153 are indicated).
Residues that are within 15 Š from
and directed mutagenesis studies have been per- the isoalloxazine moiety of the FAD cofactor are in red,
formed with PAMO, CHMO and CPMO, providing more distant residues are in yellow.
clues about the residues that form the substrate bind-
[11,13,20]
ing pocket.
An inventory of all mutations that
affect enantioselectivity shows that many targeted res-
idues, including M446, align a pocket at the re-face of
the flavin cofactor (Figure 2). Future mutagenesis
studies can exploit the localization of these hotspots
that determine the plasticity of the substrate binding
pocket of BVMOs. By this, BVMOs can be created
that extend the catalytic potential of the presently
available BVMOs and may ultimately combine sub-
strate promiscuity with thermal stability.
has been built using the CPH models server (www.cbs.dtu.
dk/services/CPHmodels).
Site-Directed Mutagenesis
For mutagenesis, the previously constructed plasmid
[3d]
pPAMO was used as first template. The plasmid contains
the pamo gene (accession number YP 289549) which is con-
trolled by the PBAD promoter. For preparation of the triple
mutant, the single mutated plasmid pPAMO (M446G) was
used as template. Mutants were constructed using the
Experimental Section
ꢃ
QuickChange Site-Directed Mutagenesis Kit of Stratagene
with pPAMO as template following the recommendations of
the manufacturer. M446 was mutated to G with primer 5’-
GCGCTCAGCAACGGCCTGGTCTCTATC-3’ and its
complementary primer. Q152 and L153 were mutated to F
Materials
All chemicals or enzymes were obtained from ACROS Or-
ganics, Jülich Fine Chemicals, Roche Applied Sciences and
Sigma–Aldrich. Oligonucleotide primers were obtained
from Sigma Genosys. DNA sequencing was done at GATC
or
G with primer 5’-ATGGCCAGCGGCTTTGGCTC-
CGTCCCGCAG-3’ and its complementary primer. The mu-
tated nucleotides are shown in bold. The resulting plasmids
were transformed into E. coli TOP10.
(
Konstanz, Germany). The figures have been prepared using
the PyMol software (www.pymol.org). The CPMO model
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Adv. Synth. Catal. 2007, 349, 1361 – 1368

Substrate Specificity and Enantioselectivity of Phenylacetone Monooxygenase
FULL PAPERS
Overexpression and Purification of Wild-Type
PAMO and its Mutants
column). The absolute configuration of the lactones was de-
termined by comparison with the currently available litera-
ture for CHMO from Acinetobacter sp. NCIMB 9871 and
CPMO from Comamonas sp. strain NCIMB 9872.
Wild-type PAMO and the mutants were overexpressed in
ꢀ
1
TB medium containing 50 mgmL ampicillin and 0.2% l-
arabinose at a temperature of 378C. Purification of the
PAMO mutants was performed as previously described for
[3d]
wild-type PAMO.
Acknowledgements
Steady-State Kinetics
CERC3 and COST D25/0005/03 are gratefully acknowledged
for funding and support.
Enzyme concentrations were measured photometrically by
monitoring the absorption of the FAD cofactor at 441 nm
ꢀ
1
ꢀ1
(
e441nm =12.4 mM cm ). The activities of the purified en-
zymes were determined spectrophotometrically by monitor-
ing the decrease of NADPH in time at 340 nm (e
=
40nm
References
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ꢀ
1
ꢀ1
6
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ments were performed on a Perkin–Elmer Lambda Bio40
spectrophotometer at a temperature of 258C. The obtained
data were fitted to the Michaelis–Menten equation by non-
linear regression analysis (SigmaPlot version 10.0 for Win-
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Enantioselective Oxidations using Purified Enzyme
Enzymatic conversion of substrates 7–10 by wild-type
PAMO and mutant M446G were performed at 308C in a
2
1
mL reaction mixture containing 50 mM Tris-HCl, pH 9.0,
00 mM NADPH, 1% (v/v) DMSO, 5 mM glucose 6-phos-
phate, 5 Uglucose 6-phosphate dehydrogenase, 2.5 mM
enzyme and 2.5 mM of the substrate of interest. After
90 min the reaction was stopped by extraction with ethylace-
tate, dried over MgSO and analyzed by gas chromatogra-
4
phy. Chiral and achiral GC analyses were performed on a
Shimadzu GC17 instrument equipped with an FID and a
Chiraldex G-TA column (Alltech, 30 m”0.25 mm”
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.125 mm) or an HP1 column (Agilent, 30 m”0.25 mm”
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ꢀ
1
medium containing 200 mgmL ampicillin and inoculated
with 1% v/v of an overnight preculture of TOP10 cells bear-
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on an orbital shaker. When the appropriate OD590 was
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0.1% w/v) together with the substrates 6, 15–18 (1 mg/12-
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cultivation, samples were taken, extracted with ethylacetate
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