Identification of an Esterase Isolated Using Metagenomic Technology which Displays an Unusual Substrate Scope and its Characterisation as an Enantioselective Biocatalyst

Article abstract of DOI:10.1002/adsc.201801691

Evaluation of an esterase annotated as 26D isolated from a marine metagenomic library is described. Esterase 26D was found to have a unique substrate scope, including synthetic transformations which could not be readily effected in a synthetically useful manner using commercially available enzymes. Esterase 26D was more selective towards substrates which had larger, more sterically demanding substituents (i. e. iso-propyl or tert-butyl groups) on the β-carbon, which is in contrast to previously tested commercially available enzymes which displayed a preference for substrates with sterically less demanding substituents (e.g. methyl group) at the β-carbon. (Figure presented.).

Full text of DOI:10.1002/adsc.201801691

Title: Identification of an Esterase Isolated Using Metagenomic
Technology which Displays an Unusual Substrate Scope and its
Characterisation as an Enantioselective Biocatalyst
Authors: Declan P Gavin, Edel J Murphy, Aoife M Foley, Ignacio Abreu
Castilla, F Jerry Reen, David F Woods, Stuart G Collins,
Fergal O'Gara, and Anita Maguire
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201801691
Link to VoR: http://dx.doi.org/10.1002/adsc.201801691

10.1002/adsc.201801691
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Identification of an Esterase Isolated Using Metagenomic Technology which Displays
an Unusual Substrate Scope and its Characterisation as an Enantioselective Biocatalyst
Declan P Gavin,a¥ Edel J Murphy,b¥ Aoife M Foley,a Ignacio Abreu Castilla,c F Jerry Reen,d
David F Woods,c Stuart G Collins,a Fergal O’Gara,c,e* Anita R Maguiref*
¥ These authors contributed equally to this publication
a School of Chemistry; Analytical and Biological Chemistry Research Facility; Synthesis and
Solid State Pharmaceutical Centre; University College Cork, T12 K8AF Cork, Ireland
b School of Chemistry; Analytical and Biological Chemistry Research Facility; University
College Cork, T12 K8AF Cork, Ireland
c
BIOMERIT Research Centre; School of Microbiology; University College Cork, T12
K8AF Cork, Ireland
d School of Microbiology, University College Cork, T12 K8AF Cork, Ireland
e
Human Microbiome Programme, School of Pharmacy and Biomedical Sciences, Curtin
Health Innovation Research Institute, Curtin University, Perth, WA 6102, Australia and
Telethon Kids Institute, Perth, WA 6008, Australia;
School of Chemistry, School of Pharmacy, Analytical and Biological Chemistry Research
f
Facility, Synthesis and Solid State Pharmaceutical Centre, University College Cork, T12
K8AF Cork, Ireland
* Correspondence: Anita Maguire a.maguire@ucc.ie; Fergal O’Gara f.ogara@ucc.ie
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201######
Abstract. Evaluation of an esterase annotated as 26D
isolated from a marine metagenomic library is
described. Esterase 26D was found to have a unique
substrate scope, including synthetic transformations
which could not be readily effected in a synthetically
useful manner using commercially available enzymes.
Esterase 26D was more selective towards substrates
which had larger, more sterically demanding
substituents (i.e. iso-propyl or tert-butyl groups) on the
β-carbon, which is in contrast to previously tested
commercially available enzymes which displayed a
preference for substrates with sterically less demanding
substituents (i.e. methyl group) at the β-carbon.
Keywords:
Esterase;
Metagenomic
Library;
Stereochemistry; Biocatalyst; Enantiopurity
Pharmaceutical Ingredients (APIs) in an efficient,
highly stereoselective manner has never been more
important.[1] Moreover, with increasing focus on
“green” synthetic routes, a requirement exists for
technologies which operate under milder conditions
and minimise waste generation. Biocatalysis has
emerged as a very favourable solution to these
challenges.[2]
Introduction
Due to the importance of stereochemistry in drug
discovery and the associated clinical and regulatory
implications,
the
capability
of
producing
enantiomerically pure synthons and Active
1
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10.1002/adsc.201801691
Advanced Synthesis & Catalysis
Biocatalysts are inherently stereoselective and many
can outperform alternative methods prevalent in
asymmetric synthesis, such as transition metal
catalysis or chiral auxiliary chemistry.[3] In addition
to the aforementioned advantages for enantioselective
synthesis, biocatalysts are also obtained from
renewable resources, can operate at neutral pH and
wide ranges of temperatures, and are safe for both
humans and the environment. Hence, there are
significant advantages to this enabling technology
when compared to “traditional” organic synthesis,
which often relies on harsh reaction conditions or
heavy metal catalysts.[2a, 4]
Hydrolytic biocatalysts are very important to
pharmaceutical, chemical and food industries.[5]
These enzymes can be used to effect achiral
transformations, however their primary use in
synthetic chemistry is in asymmetric synthesis, where
they can lead to enantioselective (dynamic) kinetic
resolutions of esters and related compounds.[6]
Esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) are
sub-groups of hydrolases. Esterases and lipases
catalyse the hydrolysis of short chain and long chain
glycerol esters, respectively.[6a, 7]
culture-independent techniques have now been
successfully employed. The construction and
functional screening of large metagenomic libraries
have successfully uncovered many novel biocatalysts.
[14] Furthermore the screening of these libraries can be
adapted to high-throughput (103 to 105 variants/day)
or ultrahigh-throughput (106 to 109 variants/day)
functional screening assays. [15]
Previously, our group [11] and others have had success
with enzyme mining in niche environments as a
biocatalyst source.[16] The marine environment, which
has bestowed upon us many pharmaceutically
interesting natural products, has proven to be a rich
source of protein diversity.[11, 17]
Exploring the potential of the novel biocatalysts in
enantioselective synthesis
Via the culture-independent sampling of the marine
environment, a number of enzymes were identified in
our laboratory as having hydrolytic activity through
use of a tributyrin screen. The hydrolase family of
enzymes make up approximately 55% of all enzymes
used in biocatalytic processes, with only 8% of these
being esterases.[18] Following a screen of target
substrates with these biocatalysts, esterase 26D was
identified as an active and selective enzyme when
tested, showing an altered substrate preference to
A common strategy employed for the discovery of
novel biocatalysts is through functional metagenomic
screening to discover proteins with novel
sequences.[8]
19]
There exists
a
requirement for new enzyme
previously tested commercial hydrolases.[1b,
discovery, not least from the point of view of
investigation of new activities and functionality,[9] as
well as from the perspective of novel substrate
specificity. The dereplication of functionally active
clones is a vital step in the identification of novel
biocatalysts. The DNA sequencing of the novel
biocatalysts can be searched against large sequence
databases such as NCBI, which currently contains
Esterase 26D shows greater selectivity when the
substituent on the β-carbon is a tert-butyl group
(sterically bulky) versus a methyl group.
Results and Discussion
[10]
over 125 million sequences of different proteins.
The novel esterase 26D was identified in a functional
screen of an Axinella dissimilis marine sponge
metagenomic library (n = 20,352 clones) constructed
in E. coli EPI300 cells using the fosmid vector
This step of bioinformatic dereplication of positive
clones at an early stage of the process, saves much
time and cost on downstream analysis. Furthermore,
these newly discovered biocatalysts can act as
templates for further evolution, starting from a point
at which the enzyme displays some activity for the
target transformation.
pCCFOS1.
sequence encoding the 26D esterase revealed
sequence identity (71%) with hydrolase
WP_108845108.1 from Phyllobacteriaceae
bacterium and hypothetical protein (70%)
Comparative analysis of the gene
a
a
OUW21162.1 from a Rhizobiales bacterium. The
clustering tree demonstrated that the 26D sequence
branched on the same node as the aforementioned
proteins, (WP_108845108.1 (■) and OUW21162.1
(●)) however importantly the 26D esterase was on a
distinct branching point to these (Figure 1). The
percentage identity of the sequences in the tree, range
from 44-71% compared to 26D. We theorized that
this novel sequence could give rise to a novel
functioning esterase with a unique substrate profile
when compared with existing esterase activities.
The marine environment is an unexploited resource
for the mining of hydrolases such as esterases and
[11]
lipases.
This niche environment harbours a wide
range of enzymes that tolerate varying reaction
conditions such as high salt concentration, high
pressure and pH tolerance, as well as functioning in
extreme temperatures, which enable their use in
diverse applications including industrial scale
[12]
transformation.
In the past when using traditional
microbiology techniques, only a minority of the
bacteria were culturable from a given environment
and therefore a vast array of important enzymes could
[13]
have been overlooked.
To combat this challenge,
2
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10.1002/adsc.201801691
Advanced Synthesis & Catalysis
Figure 1. Clustering Tree to define the uniqueness of the esterase positive clone 26D
Table 1. Commercial enzymes used in the kinetic resolution of 3-arylalkanoic acids.[19]
Entry
No. of enzymes which gave No. of enzymes which furnished
>10% conversion acid product in 90% ee
15
R
1
2
3
Me
Et
iPr
tBu
7
5
5
1[a]
1
2[b]
1
4
(a) Use of co-solvent required
(b) Heating to 40 C required
Previous work in our group investigated the kinetic
resolution of 3-arylalkanoic acids. Using a systematic
approach with commercial enzymes and by varying
the co-solvent, a range of compounds were resolved
with excellent enantiopurity (Table 1).[1b, 19] One of
the features of these compounds is that the chiral
3
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10.1002/adsc.201801691
Advanced Synthesis & Catalysis
centre is not adjacent to the reacting site (i.e. at the α-
carbon) but at the β-carbon, which brings about
significant challenges in terms of selectivity and
activity.[1b, 19-20]
Candida Antarctica Lipase B as the biocatalyst), the
optimum outcome achieved led to the acid product in
excellent enantiopurity (98% ee) with moderate
conversion (23%) in 72 hours at 40 °C (Scheme 1).
Even under the optimised conditions, the kinetic
resolution of 5 is of limited synthetic utility as the
enantiopurity of the recovered ester is, of necessity,
compromised by the limited extent of reaction.[19]
One possible explanation for this given by Müller et
al is that, given the paucity of natural products
featuring the t-butyl group, enzyme evolution in
nature to accommodate this sterically demanding
substituent is less likely.[21]
Notably, from this previously completed screen the
number of enzymes which processed the substrate
decreased as the steric bulk of the β-substituent
increased, Table 1.[19] Concurrently the number of
enzymes exhibiting enantiodiscrimination also
decreased. In particular, enzymatic hydrolysis of
substrate 5 bearing a t-butyl substituent was
extremely challenging, with only two of the
previously screened commercial hydrolases showing
activity. After considerable optimisation of the
conditions for the biotransformation (immobilised
Scheme 1: Previously screened hydrolysed-mediated kinetic resolution of (±)-ethyl 4,4-dimethyl-3-
phenylpentanoate (±)-5 at variable reaction times and temperature.[19]
In this work identifying an enzyme which would
hydrolyse substrate 5, the challenging substrate, in a
synthetically useful manner would be very attractive.
Accordingly, wild type esterase 26D was tested for
activity against the challenging ester substrate 5.
Gratifyingly, in the initial screens using whole cells
expressing the wild type esterase 26D both activity
and excellent enantioselectivity were seen with
substrate 5. Whole cells expressing the esterase
furnished the acid product in excellent enantiopurity
(98% ee) with 21% conversion (Ecalc) (Table 2, entry
5).
In light of this result, screening this novel enzyme
against the series of esters 1-4 was undertaken to
probe the substrate scope of the enzyme by varying
the alkyl substitution on the β-carbon (as summarised
in Table 2). Interestingly, esterase 26D, while active
across the series, displayed the opposite trend to all
other enzymes screened against this similar series,
with enantioselectivity increasing with increased
steric demand at the β-substituent. Hydrolysis of the
esters bearing ethyl (3) and isopropyl (4) substituents
proceeded with good enantioselectivity, while
decreased selectivity was seen with the smaller
methyl substituent, even though resolution of this
derivative was by far the most efficient in our earlier
study.[1b, 19]
Interestingly resolution of the trifluoromethyl
substituted ester 2 was more efficient than that of the
methyl derivative 1 under the same conditions.
While the hydrolysis of ester 5 was promising (Table
2, entry 5), to enhance its synthetic utility, process
optimisation was undertaken (Table 2, entry 6)
involving repeated additions of the biocatalyst at 0,
12 and 24 hours, which led to recovery of the ester in
73% ee albeit with a reduction in the enantiopurity of
the acid (91% ee) under these more forcing
conditions. This result demonstrates that with further
optimisation, esterase 26D can lead to a synthetically
feasible
kinetic
resolution,
whereby
both
enantiomeric series can be accessed. Use of
alternative co-solvents (1,4-dioxane, MTBE, EtOH),
variation of pH (6 to 7.8) and extended reaction (72
h) time were explored without significant impact.
4
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10.1002/adsc.201801691
Advanced Synthesis & Catalysis
Table 2. Kinetic resolution of 3-arylalkanoic acid ethyl esters 1-5 using esterase 26D
Conversion (%)
1H
% ee
Time
(h)
Entry
R1
E[22]
[b] [22]
Ecalc
Ester
Acid
NMR[a]
1
2
3
4
5
6
7
Me[c]
24
24
24
24
24
48
48
25
22
18 (S)
64
63 (R)
88
5
[c]
CF3
60
30
42
24
42
21
45
37
Et[d]
iPr[c]
tBu[c]
tBu[e]
tBu[f]
31(S)
65 (R)
26 (R)
73 (R)
55 (R)
96 (R)
88 (S)
98 (S)
91(S)
93 (S)
66
31
[g]
-
30
127
40
[g]
-
[g]
-
47
1
(a) Conversion was estimated using H NMR spectroscopy by integration of the signal for esters and
alcohols; (b) Conversion (Ecalc) and the enantiomeric ratio (E) were calculated from enantiomeric
excess of substrate ester (ees) and product acid (eep); (c) 50 mg/mL of whole cells expressing esterase
26D; (d) 25 mg/mL of whole cells expressing esterase 26D; (e) 50 mg/mL of whole cells expressing
esterase 26D was added in three portions, 0h, 12 h and 24 h; (f) 150 mg/mL of whole cells expressing
1
esterase 26D; (g) percentage conversion of ester to acid could not be determined by H NMR
spectroscopy analysis.
Having established that 26D could be used for a
transformation which was not readily achieved with
existing biocatalysts, its substrate scope was further
investigated. We had previously examined a panel of
hydrolase enzymes in the kinetic resolution of 2-
phenylalkanols.[23] Excellent enantiopurities were
obtained for a number of alcohol products when
CAL-B was used as the biocatalyst. In contrast,
esterase 26D was a poor biocatalyst for the resolution
of 2-phenylalkanols. A range of ester substrates was
screened, with the substituent on the β-carbon (R1) as
well as the substituent (R2) on the carboxylic acid
moiety of the ester being varied (Table 3). Product ee
was poor to moderate, although in some instances
hydrolysis was seen for substrates (14 and 15) which
were not processed by CAL-B [23] (Table 3, entries 4
and 5), further highlighting the different profile in
terms of the enzyme’s substrate scope.
5
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10.1002/adsc.201801691
Advanced Synthesis & Catalysis
Table 3. Kinetic resolution of 2-phenylalkanols using esterase 26D
% ee
Conversion (%)
R2
E[22]
Entry Compound R1
[b] [22]
1H NMR [a]
Ecalc
Ester
36 (R)
3 (R)
37
Alcohol
18 (S)
19 (S)
40
67
15
48
34
2
2
1
2
3
4
5
Me
Me
Et
Ph
tBu
Ph
tBu
iPr
81
56
55
55
55
11
12
13
14
15
3
40
77
11
Et
iPr
49
19
15
21
29
14
16
10
30
61
91
38
2
5
6
7
8
iPr
iPr
25
16
17
18
Ph
tBu
[c]
25
2
-
Me
Bn
22
1
(a) Conversion was estimated using H NMR spectroscopy by integration of the signal for esters and
alcohols; (b) conversion (Ecalc) and enantiomeric ratio (E) were calculated from enantiomeric excess of
substrate ester (ees) and alcohol product (eep); (c) percentage conversion of ester to alcohol could not be
determined by 1H NMR spectroscopy analysis
We then turned our attention to applying the atypical
substrate acceptance of esterase 26D to
a
pharmaceutical intermediate, since assimilation of a
novel biocatalyst into an industrially relevant
synthetic route would underline its value.
This work was initially extended to cyclic substrates
27-29, as summarised in Table 4. Esterase 26D
hydrolyses acetates of α- and β-tetralols (27 & 28)
and 1-indanol (29), affording high conversion to the
alcohols 31
&
32 but with very poor
enantiodiscrimination.
Following on from this work, the novel acetates 25
and 26 were prepared by acetylation of the
corresponding tetralols 23 and 24, which were
accessed by reduction of the tetralone 22 (Scheme 2).
An intermediate in the synthesis of the Pfizer
antidepressant sertraline, racemic tetralone 22 was
obtained from the condensation of 1-napthol and 1,2-
dichlorobenzene in the presence of a strong Lewis
acid. Enantiopure (4S)-tetralone 22 was also reduced
and acetylated. The tetralin core (α- or β- substituted)
is in itself a privileged moiety in drug discovery,
being present in a number of drugs and bioactive
compounds.[24]
Scheme 2. Synthesis of acetates 25 and 26
When the trans- and cis- acetates 25 and 26 were
exposed to esterase 26D, hydrolysis of the trans-
acetate 25 is more rapid than that of the cis-acetate 26
(Table 4 entry 4 and 5). Excellent enantioselectivity
was observed in the formation of the cis-tetralol 24
(93% ee). As the cis-tetralol 24 product has been used
6
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10.1002/adsc.201801691
Advanced Synthesis & Catalysis
as a synthetic intermediate for sertraline,[25] this
observation is particularly interesting and highlights
the potential of the novel biocatalyst to be utilised for
the resolution of pharmaceutically related
compounds. Absolute stereochemistries of the
biotransformation products were deduced by
comparing the HPLC traces with that of the (4S)-
tetralol and the corresponding acetates.
Table 4. Hydrolysis of acetates 25-29 using esterase 26D.
Conversion
% ee
Time
Entry Substrate
(h)
E[22]
[a] [22]
1H NMR
Ester
30
Alcohol
Ecalc
[b]
1
2
3
4
5
4
1
-
5
10
2
1
27
28
29
25
26
[b]
-
86
50
36
18
32
5
2
22
38
20
4
4
1
24
24
~31[c]
21
55 (1R,4S)
93 (1R,4R)
5
39
(a) Conversion (Ecalc) and the enantiomeric ratio (E) were calculated from enantiomeric excess of
substrate ester (ees) and product alcohol (eep); (b) percentage conversion of ester to alcohol could not be
1
determined by H NMR spectroscopy analysis; (c) full resolution of signals in the HPLC was not
possible
(compound 26) was resolved in excellent
enantiopurity, suggesting the potential of this
biocatalyst to be used in the synthetic route of a
pharmaceutical compound.
Conclusion
In conclusion, the novel esterase 26D acts as a
synthetically valuable biocatalyst displaying a unique
substrate scope, at least within the range of
biocatalysts which we have explored, and to date is
the optimum biocatalyst for the resolution of the
challenging ester substrate 5, highlighting the
importance of metagenomic screening as a valuable
approach for the discovery of novel biocatalysts. The
success in resolving an ester substrate where the
chirality lies in the acid portion (6-10) relative to the
other substrates explored where the chirality lies in
the alcohol (e.g. compounds 11-18 and 25-29) moiety
is unsurprising in an esterase.[26] Furthermore, a
potential intermediate in the synthesis of sertraline
Experimental Section
General
Solvents were distilled prior to use as follows:
dichloromethane was distilled from phosphorus
pentoxide, ethyl acetate was distilled from potassium
carbonate. Hexane was distilled prior to use. Organic
phases were dried over anhydrous magnesium sulfate.
Infrared spectra were recorded neat using a Perkin
Elmer FTIR UATR2 spectrometer.1H (300 MHz) and
13C (75.5 MHz) NMR spectra were recorded on a
7
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10.1002/adsc.201801691
Advanced Synthesis & Catalysis
Bruker Avance 300 MHz NMR spectrometer and all
spectra were recorded at room temperature (̴ 20 °C) in
for 10 min at 4°C to harvest the cells. The cell pellet
was stored at -80°C until ready for expression
analysis.
deuterated chloroform (CDCl3), unless otherwise
stated, using tetramethylsilane (TMS) as an internal
standard and chemical shifts (δH and δC) are reported
in parts per million (ppm) and coupling constants are
expressed in Hertz (Hz). Low resolution mass spectra
were recorded on a Waters Quattro Micro triple
quadrupole spectrometer in electrospray ionisation
(ESI) mode using 50% water/acetonitrile containing
0.1% formic acid as eluent. High resolution mass
spectra (HRMS) were recorded on a Waters LCT
Premier Time of Flight spectrometer in electrospray
ionisation mode (ESI) using 50% water/acetonitrile
containing 0.1% formic acid as eluent. Wet flash
chromatography was performed using Kieselgel
Silica Gel 60, 0.040–0.063 mm (Merck). Thin layer
chromatography (TLC) was carried out on precoated
silica gel plates (Merck 60 PF254). Visualisation was
achieved by UV (254 nm) light detection and KMnO4
staining. Candida Antarctica lipase B (immobilised
on ImmoBead 150) was purchased from Sigma-
Aldrich chemical company. All enzymatic reactions
were performed on a VWR Incubating Mini Shaker
4450. All reagents are analytical grade and purchased
from Sigma-Aldrich, Acros Organics, Fluka or TCI.
Enantiomeric excess values were measured by high
performance liquid chromatography (HPLC) using a
Chiralcel® OB-H column (5 × 250 mm), Chiralcel®
OD-H column (5 × 250 mm), Chiralcel® OJ-H
column (5 × 250 mm) and Chiralpak® IB (5 × 250
mm) purchased from Daicel Chemical Industries,
Japan. Phenomenex Amylose 1 column (5 × 250
mm), Phenomenex Amylose 2 column (5 × 250 mm),
Phenomenex Cellulose 4 column (5 × 250 mm),
purchased from Phenomenex Inc., UK. Mobile phase
and flow rate are included where appropriate, and the
detector wavelength was 209.5 nm. HPLC analysis
was performed on a Waters alliance 2690 separations
module with a PDA detector.
Bioinformatic Dereplication for Novel Activity
The DNA sequences of the positive clone was
bioinformatically aligned using Clustal Omega
[27]
and a consensus sequence was defined using Jalview
Version 2 [28]. A Translated BLAST: blastx search
was conducted against the NCBI Non-redundant
[29]
protein sequence database
.
The cluster
relationship of 26D was inferred using the Neighbor-
Joining method. The distances were computed using
the Poisson correction method and are in the units of
the number of amino acid substitutions per site. The
analysis involved 100 amino acid sequences of the
top BLAST hits by sequence identity. All ambiguous
positions were removed for each sequence pair. This
analysis was conducted in MEGA X.
Lipase-catalysed hydrolysis reactions
In a typical experiment, 50 mg of whole cells
expressing esterase 26D were added to a 15 mL
centrifuge tube (unless otherwise stated) followed by
0.1 M potassium phosphate buffer at pH 7.2 (900
µL/1000 µL). Substrates 1 - 5, 11 - 18 and 25 - 29 (20
mM) were dissolved in 100 µL DMSO (10% v/v) and
then added to the centrifuge tube to make the total
volume 1 mL. The reaction mixture was agitated at
400-450 rpm and incubated at 30 °C for 24 h unless
otherwise stated. Ethyl acetate (3mL) was added to
the centrifuge tube and the reaction mixture was
centrifuged at 5000 rpm for 10 minutes. The layers
were separated, the aqueous layer was washed with
ethyl acetate (2 × 3 mL), the combined organic
extracts were filtered through Celite® and
concentrated under reduced pressure. The entire
reaction sample was analysed by 1H NMR
spectroscopy, reconcentrated and dissolved in a
i
mixture of PrOH /hexane [10:90 (HPLC grade)] and
enantiopurity determined by chiral HPLC analysis.
Compounds 1, 3-5, 7-12, 15, 18, 21, 23-24 and 27-29
were synthesised according to previously reported
methods.[19, 23, 25, 30] Details of novel compounds are
given in the SI.
Expression and purification of lipase/esterase
His-tagged esterase 26D fusion proteins derived from
a tributyrin screen of a pCC1FOS™ (Epicentre)
metagenomic library transformed in EPI300™
Electrocompetent E. coli were prepared for
recombinant protein expression. Single colonies were
Acknowledgements
inoculated
in LB (Luria-Bertani)
medium
supplemented with kanamycin (50 μg/ml),
chloramphenicol (50 μg/ml) and streptomycin (75
μg/ml) and incubated at 37°C with shaking at 180
rpm overnight. Overnight cultures were transferred
(1%) to fresh media with antibiotics and grown to an
OD600nm of 0.8-1.0, at which point the medium was
supplemented with 0.5 mM IPTG (Melford) to induce
recombinant protein expression. After 4 hrs post-
induction, the culture was centrifuged at 12,000 rpm
This publication has emanated from research
conducted with the financial support of the Irish
Research
Council
under
project
number
EPSPG/2016/41, Synthesis and Solid State
Pharmaceutical Centre, funded by Science
Foundation Ireland under grant number 12/RC/2275.
This research was supported in part by grants
awarded to F.O.G. by; Enterprise Ireland (CF-2017-
8
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10.1002/adsc.201801691
Advanced Synthesis & Catalysis
0757-P; IP-2015-0390), the European Commission
(FP7-PEOPLE-2013-ITN, 607786; FP7-KBBE-2012-
6, CP-TP-312184; FP7-KBBE-2012-6, 311975;
OCEAN 2011-2, 287589; EU2020-634486-2015),
Science Foundation Ireland (SSPC-2, 12/RC/2275;
13/TIDA/B2625; 12/TIDA/B2411; 12/TIDA/B2405;
Wiley-VCH, Weinheim, 2006; c) U. T.
Bornscheuer, Encycl. Ind. Biotechnol.
2010, 1-12.
a) D. Romano, F. Bonomi, M. C. de
Mattos, T. de Sousa Fonseca, M. d. C. F.
de Oliveira, F. Molinari, Biotechnol. Adv.
2015, 33, 547-565; b) V. Gotor, V.
Gotor-Fernández, E. Busto, in
Comprehensive Chirality (Eds.: E. M.
Carreira, H. Yamamoto), Elsevier,
Amsterdam, 2012, pp. 101-121; c) C.
Kim, J. Park, M. J. Kim, in
Comprehensive Chirality (Eds.: E. M.
Carreira, H. Yamamoto), Elsevier,
Amsterdam, 2012, pp. 156-180.
a) M. Shang, V. J. Chan, D. W. S. Wong,
H. Liao, MethodsX 2018, 5, 669-675; b)
J. K. Arjun, B. P. Aneesh, T. Kavitha, K.
Harikrishnan, Biotechnol. Lett. 2018, 40,
343-348; c) S. A. Nasseri, L. Betschart,
D. Opaleva, P. Rahfeld, S. G. Withers,
Angew. Chem., Int. Ed. Engl. 2018, 57,
11359-11364.
G. A. Aleku, S. P. France, H. Man, J.
Mangas-Sanchez, S. L. Montgomery, M.
Sharma, F. Leipold, S. Hussain, G.
Grogan, N. J. Turner, Nat. Chem. 2017,
9, 961.
[7]
14/TIDA/2438;
SFI09/RFP/BMT2350),
15/TIDA/2977;
Department of
the
Agriculture and Food (FIRM 11/F009/MabS; FIRM
13/F/516), the Irish Research Council for Science,
Engineering and Technology (GOIPG/2014/647), the
Health Research Board/Irish Thoracic Society
(MRCG-2014-6), the Department of the Marine
(BEAU/BIOD/01), Cystic Fibrosis Foundation, USA
(OG1710). We wish to thank Pat O’Neill, Pfizer for
useful discussions.
[8]
[9]
References
[1]
a) D. P. Gavin, A. R. Maguire, in:
Chirality in Supramolecular Assemblies
(Eds.: F. R. Keene), Wiley-VCH,
Weinheim, 2017, pp 343-406; b) R. E.
Deasy, T. S. Moody, A. R. Maguire,
Tetrahedron: Asymmetry 2013, 24, 1480-
1487.
[2]
[3]
a) R. A. Sheldon, J. M. Woodley, Chem.
Rev. 2018, 118, 801-838; b) M. T. Reetz,
J. Am. Chem. Soc. 2013, 135, 12480-
12496; c) A. S. Bommarius, M. F. Paye,
Chem. Soc. Rev. 2013, 42, 6534-6565.
a) C. C. C. R. d. Carvalho, Microb.
Biotechnol. 2017, 10, 250-263; b) G.
Hughes, J. C. Lewis, Chem. Rev. 2018,
118, 1-3; c) A. S. Bommarius, B. R.
Riebel, in Biocatalysis, WILEY-VCH,
Weinheim, 2004.
[10] N. A. O'Leary, M. W. Wright, J. R.
Brister, S. Ciufo, D. Haddad, R.
McVeigh, B. Rajput, B. Robbertse, B.
Smith-White, D. Ako-Adjei, A.
Astashyn, A. Badretdin, Y. Bao, O.
Blinkova, V. Brover, V. Chetvernin, J.
Choi, E. Cox, O. Ermolaeva, C. M.
Farrell, T. Goldfarb, T. Gupta, D. Haft, E.
Hatcher, W. Hlavina, V. S. Joardar, V. K.
Kodali, W. Li, D. Maglott, P. Masterson,
K. M. McGarvey, M. R. Murphy, K.
O'Neill, S. Pujar, S. H. Rangwala, D.
Rausch, L. D. Riddick, C. Schoch, A.
Shkeda, S. S. Storz, H. Sun, F. Thibaud-
Nissen, I. Tolstoy, R. E. Tully, A. R.
Vatsan, C. Wallin, D. Webb, W. Wu, M.
J. Landrum, A. Kimchi, T. Tatusova, M.
DiCuccio, P. Kitts, T. D. Murphy, K. D.
Pruitt, Nucleic Acids Res. 2016, 44, 733-
745.
[4]
[5]
J. H. Clark, T. J. Farmer, L. Herrero-
Davila, J. Sherwood, Green Chem. 2016,
18, 3914-3934.
a) A. Vimal, A. Kumar, Biotechnol.
Genet. Eng. Rev. 2017, 33, 40-61; b) H.
Demir, C. Tari, J. Sci. Food Agric. 2015,
96, 3575-3582; c) P. Demarche, C.
Junghanns, R. R. Nair, S. N. Agathos,
Biotechnol. Adv. 2012, 30, 933-953.
a) M. Ferrer, R. Bargiela, M. Martínez-
Martínez, J. Mir, R. Koch, O. V.
Golyshina, P. N. Golyshin, Biocatal.
Biotransfor. 2015, 33, 235-249; b) R. J.
Kazlauskas, U. T. Bornscheuer,
[6]
[11] I. A. Castilla, D. F. Woods, F. J. Reen, F.
O'Gara, Mar. Drugs 2018, 16, 21.
[12] a) F. Sarmiento, R. Peralta, J. M. Blamey,
Front. Bioeng. Biotechnol. 2015, 3, 148-
Hydrolases in organic synthesis regio-
and stereoselective biotransformations,
9
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801691
Advanced Synthesis & Catalysis
148; b) A. Trincone, Mar. Drugs 2011, 9,
478-499.
organic synthesis, 2006; b) A. Goswami,
S. G. Van Lanen, Mol. Biosyst. 2015, 11,
338-353.
[13] R. I. Amann, W. Ludwig, K. H. Schleifer,
Microbiol. Rev. 1995, 59, 143-169.
[14] a) W. Gao, K. Wu, L. Chen, H. Fan, Z.
Zhao, B. Gao, H. Wang, D. Wei, Microb.
Cell Fact. 2016, 15, 41; b) W. Gao, H.
Fan, L. Chen, H. Wang, D. Wei,
[19] R. E. Deasy, M. Brossat, T. S. Moody, A.
R. Maguire, Tetrahedron: Asymmetry
2011, 22, 47-61.
[20] a) U. T. Bornscheuer, R. J. Kazlauskas,
Hydrolases in Organic Synthesis: Regio-
and Stereoselective Biotransformations,
2nd Edition, 2006; b) M. Colombo, M.
De Amici, C. De Micheli, D. Pitré, G.
Carrea, S. Riva, Tetrahedron: Asymmetry
1991, 2, 1021-1030; c) J. C. Sih, R. L.
Gu, Tetrahedron: Asymmetry 1995, 6,
357-360.
[21] P. Bisel, L. Al-Momani, M. Müller, Org.
Biomol. Chem. 2008, 6, 2655-2665.
[22] C. S. Chen, Y. Fujimoto, G. Girdaukas,
C. J. Sih, J. Am. Chem. Soc. 1982, 104,
7294-7299.
[23] A. M. Foley, D. P. Gavin, I. Joniec, A. R.
Maguire, Tetrahedron: Asymmetry 2017,
28, 1144-1153.
[24] a) L. Carro, M. Torrado, E. Raviña, C. F.
Masaguer, S. Lage, J. Brea, M. I. Loza,
Eur. J. Med. Chem. 2014, 71, 237-249; b)
K. S. Koblan, S. C. Hopkins, K. Sarma,
F. Jin, R. Goldman, S. H. Kollins, A.
Loebel, Neuropsychopharmacology 2015,
40, 2745-2752.
[25] G. Wang, C. Zheng, G. Zhao,
Tetrahedron: Asymmetry 2006, 17, 2074-
2081.
[26] Nicholas J Turner, L. Humphreys,
Biocatalysis in Organic Synthesis: The
Retrosynthesis Approach, 2018.
[27] F. Sievers, A. Wilm, D. Dineen, T. J.
Gibson, K. Karplus, W. Li, R. Lopez, H.
McWilliam, M. Remmert, J. Söding, J. D.
Thompson, D. G. Higgins, Mol. Syst.
Biol. 2011, 7.
Biotechnol. Lett. 2016, 38, 1165-1171; c)
J. Huang, Y. Y. Huo, R. Ji, S. Kuang, C.
Ji, X. W. Xu, J. Li, Sci. Rep. 2016, 6,
28550; d) T. Buryska, P. Babkova, O.
Vavra, J. Damborsky, Z. Prokop, Appl.
Environ. Microbiol. 2018, 84.
[15] H. A. Bunzel, X. Garrabou, M. Pott, D.
Hilvert, Curr. Opin. Struct. Biol. 2018,
48, 149-156.
[16] a) B. Leis, A. Angelov, M. Mientus, H.
Li, V. T. Pham, B. Lauinger, P. Bongen,
J. Pietruszka, L. G. Goncalves, H. Santos,
W. Liebl, Front. Microbiol. 2015, 6, 275;
b) N. Ziemert, M. Alanjary, T. Weber,
Nat. Prod. Rep. 2016, 33, 988-1005; c) J.
Su, F. Zhang, W. Sun, V. Karuppiah, G.
Zhang, Z. Li, Q. Jiang, World J.
Microbiol. Biotechnol. 2015, 31, 1093-
1102; d) W. Gao, K. Wu, L. Chen, H.
Fan, Z. Zhao, B. Gao, H. Wang, D. Wei,
Microb. Cell Fact. 2016, 15, 41; e) A.
Tchigvintsev, H. Tran, A. Popovic, F.
Kovacic, G. Brown, R. Flick, M.
Hajighasemi, O. Egorova, J. C. Somody,
D. Tchigvintsev, A. Khusnutdinova, T.
N. Chernikova, O. V. Golyshina, M. M.
Yakimov, A. Savchenko, P. N. Golyshin,
K.-E. Jaeger, A. F. Yakunin, Appl.
Microbiol. Biotechnol. 2015, 99, 2165-
2178; f) Z. Fang, J. Li, Q. Wang, W.
Fang, H. Peng, X. Zhang, Y. Xiao, J.
Microbiol. Biotechnol. 2014, 24, 771-
780.
[17] a) F. J. Reen, J. A. Gutierrez-
Barranquero, A. D. Dobson, C. Adams,
F. O'Gara, Mar. Drugs 2015, 13, 2924-
2954; b) J. Rocha-Martin, C. Harrington,
A. D. Dobson, F. O'Gara, Mar. Drugs
2014, 12, 3516-3559; c) A. Navvabi, M.
Razzaghi, P. Fernandes, L. Karami, A.
Homaei, Process. Biochem. 2018, 70, 61-
70.
[28] A. M. Waterhouse, J. B. Procter, D. M.
A. Martin, M. Clamp, G. J. Barton,
Bioinformatics 2009, 25, 1189-1191.
[29] S. F. Altschul, W. Gish, W. Miller, E. W.
Myers, D. J. Lipman, J. Mol. Biol. 1990,
215, 403-410.
[30] a) M. Froimowitz, K.M. Wu, A. Moussa,
R. M. Haidar, J. Jurayj, C. George, E. L.
Gardner, J. Med. Chem. 2000, 43, 4981-
4992; b) M. Lautens, T. Rovis,
[18] a) A. Hidalgo, U. Bornscheuer, Directed
evolution of lipases and esterases for
Tetrahedron 1999, 55, 8967-8976; c) S.
10
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801691
Advanced Synthesis & Catalysis
H. Lee, I. S. Kim, Q. R. Li, G. R. Dong,
M. d. C. F. de Oliveira, T. L. G. d.
Lemos, R. d. A. Marques, M. C. de
Mattos, Appl. Catal., A 2015, 492, 76-82;
g) K. Dong, Y. Li, Z. Wang, K. Ding,
Angew. Chem. Int. Ed. 2013, 52, 14191-
14195.
L. S. Jeong, Y. H. Jung, J. Org. Chem.
2011, 76, 10011-10019; d) H. N. Hoang,
Y. Nagashima, S. Mori, H. Kagechika, T.
Matsuda, Tetrahedron 2017, 73, 2984-
2989; e) R. Liu, Z. H. Lu, X. H. Hu, J. L.
Li, X. J. Yang, Org. Lett. 2015, 17, 1489-
1492; f) T. d. S. Fonseca, M. R. d. Silva,
11
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FULL PAPER
Title: Identification of an Esterase Isolated
Using Metagenomic Technology which Displays
an Unusual Substrate Scope and its
Characterisation
Biocatalyst
as
an
Enantioselective
Adv. Synth. Catal. Year, Volume, Page – Page
Declan P Gavin,a¥ Edel J Murphy,b¥ Aoife M
Foley,a Ignacio Abreu Castilla,c F Jerry Reen,d
David F Woods,c Stuart G Collins,a Fergal
O’Gara,c,e* Anita R Maguiref*
12
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