G.-S. Nguyen et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 88–94
enzymes, each displaying moderate enantioselectivity, were iden-
tified from that study; a thermostable esterase A from Pyrubaculus
calidifontis and the metagenome-derived esterase Est8. Interest-
ingly, both belong to the family of hormone-sensitive-lipase-like
enzymes and share a high sequence similarity.
In this paper, we report the further investigation of hydrolytic
enzymes for the hydrolysis of TAs, using two complementary
approaches. First, the genome sequence of the actinomycete N.
farcinica IFM10152 [16], was screened for the presence of esterase
genes encoding enzymes containing the GGG(A)X motif, which is
known to confer activity of these enzymes towards TAEs [16]. The
cloning, expression and characterisation of activity and enantios-
electivity, with particular focus on chiral tertiary alcohols, for two
of these esterases (EstA1 and EstA2), is described herein.
Second, we used the new ␣/-Hydrolase Fold Enzyme Family
3DM Database (ABHDB), which is a structure-based classification
of 12,431 available sequences of ␣/-hydrolase fold enzymes, to
search for enzymes with high similarity to esterase A from P. calid-
therefore show potential for improved enantioselectivity in the
enzymes [17] and was recently applied as a tool for the predic-
tion of key residues for the engineering of the enantioselectivity
of an esterase [18] and for the guidance in library generation
for protein engineering [19,20]. These complementary approaches
have proved to be successful in unearthing new esterase activi-
ties towards sterically demanding TA esters of relevance to both
flavour/fragrance and pharmaceutical chemistry.
amide and subsequent arylation (818 mg, 62%) [21]. The spectro-
scopic data matched literature data. Gossonorol (2g) was prepared
2-one as described [4]. The spectroscopic data matched literature
data [4]. Preparation of 2-acetoxy-2-methyl-1-phenylbutane-
1-one (1f) and gossonoryl acetate (1g) was made according to
Bäckvall et al. [22].
2.2.1. Acetoxy-2-methyl-1-phenylbutane-1-one (1f)
(650 mg, 82% yield). 1H NMR: ı = 0.99 (t, 3H, J = 7.5 Hz, CH3), 1.69
(s, 3H, CH3), 1.98 (s, 3H, CH3), 2.00–2.3 (m, 2H, CH2), 7.4 (m, 3H,
H–Ar), 8.03 (d, 2H, H–Ar). 13C NMR: ı = 7.6, 21.2, 21.3, 30.6, 87.0,
128.29, 128.33, 132.33, 134.9, 179.0, 199.1. MS (EI): 177 (M+-43),
159, 145, 132, 115, 105, 73, 43.
2.2.2. Gossonoryl acetate (1g)
1H NMR:ı = 1.51(s, 3H, C(CH3)2x1), 1.64 (s, 3H, C(CH3)2x1), 1.82
(s, 3H, CCH3), 1.85–2.02 (4H, m, CH2CH2), 2.05 (s, 3H, COCH3), 2.3 (s,
3H, Ar–CH3), 5.04 (t, 1H, CCH), 7.2 (m, 5H, H–Ar). 13C NMR: ı = 17.49,
20.95, 22.23, 22.51, 24.91, 25.61, 42.22, 83.86, 123.7 124.47, 128.83
131.75 137.27, 141.96, 169.67. MS (EI): 201, 91, 77, 69, 43, 41.
2.3. Cloning of GGG(A)X motif esterases from N. farcinica
From the genomic DNA of N. farcinica, the GGG(A)X motif
esterases EstA1, EstA2, and EstA3 (accession numbers: Q5YP18,
Q5YQP8, Q5YPM0 respectively) were amplified using the following
oligonucleotide primers with ligation independent cloning (LIC)
specific sites EstA1: 5ꢀ-CCAGGGACCAGCAATGGACAACGTGGTCG-
AAGCGCCCTCG-3ꢀ and 5ꢀ-GAGGAGAAGGCGTTATGCACGGCAAGCT-
GTCGAGGGGACT-3ꢀ; EstA2: 5ꢀ-CCAGGGACCAGCAATGACCATCCG-
ATACGACACCACCGTC-3ꢀ and 5ꢀ-GAGGAGAAGGCGTTAGCTGGTCC-
GCCAGCCGAAGTCGACT-3ꢀ; EstA3: 5ꢀ-CCAGGGACCAGCAATGGT-
2. Experimental
2.1. General
All chemicals were purchased from Fluka (Buchs, Switzerland),
Sigma (Steinheim, Germany) and Merck (Darmstadt, Germany),
unless stated otherwise. NMR spectroscopy experiments were per-
formed on an ARX300 (300.13 MHz for 1H and 75.5 MHz for 13C,
Bruker, Karlsruhe, Germany), using the ı scale (ppm) for chemical
shifts; 13C-spectra were edited using DEPT techniques. J values are
given in Hz. Mass spectra were recorded on a QP2010 GC–MS device
(electron impact, 70 eV, Shimadzu, Japan). The 3DM database [17] is
aBlue singles and BL21 (DE3) cells were obtained from Novagen.
PCR primers designed towards the Nocardia farcinica esterase were
ordered from MWG-Eurofins (Germany). Synthetic genes were
obtained from GenScript USA Inc. (New Jersey, USA) and trans-
formed into competent E. coli BL21 cells. KOD hot start DNA
polymerase was obtained from Novagen. The pET-YSBLIC3C vector
was obtained from the Structural Biology Laboratory (University
of York). Restriction enzymes were purchased from New England
Bio Labs. PCR clean-up and mini-prep kits were purchased from
Qiagen, and a gel extraction kit from Sigma Aldrich. Isopropyl -
d-thiogalactopyranoside (IPTG) was purchased from Melford Labs.
The homology models of EstA4, EstA5, EstA6 were created using
hit was discovered based on the template of the protein with the
PDB code c2c7bA.
GGCAACGATCGACATCACGACC-3ꢀ
and
5ꢀ-GAGGAGAAGGCGT-
TAGCAGTCCCACGGCTGGGACTGGACT-3ꢀ in a standard reaction
mixture (0.4 M of each primer, 0.2 mM dNTPs, 1 unit of KOD hot
start DNA polymerase, 1 mM MgSO4, 10× buffer, 50 ng template
DNA, 10% (v/v) DMSO, and water to 50 L) using a tempera-
ture cycling program of: 4 min at 94 ◦C, followed by 35 cycles
of: 1 min at 94 ◦C, 1 min at 55 ◦C, 1.5 min at 72 ◦C, with a final
extension step for 3 min at 72 ◦C. The band from agarose gel
a spectrophotometer. The purified PCR product was cloned into
the cleavable N-terminally his-tagged pET-YSBLIC3C vector using
a ligation independent cloning protocol as described previously
[23,24]. Transformation of the plasmid into chemocompetent E. coli
NovaBlue cells, was carried out by incubating on ice for 5 min,
followed by a heat-shock at 42 ◦C for 30 s, and then incubation on
ice for a further 5 min before the addition of 0.5 mL of LB medium
and incubation at 37 ◦C for 1 h. Transformed cells were plated onto
LB agar plates containing kanamycin (100 g mL−1) and incubated
at 37 ◦C overnight. Several colonies were picked and the plasmids
isolated. Sanger sequencing (Technology Facility, Department of
Biology, University of York) of cloned inserts was used to verify
that genes contained no mutations.
2.4. Expression and purification of recombinant esterases
2.2. Synthesis of tertiary alcohol esters
500 mL cultures of E. coli BL21 (DE3) cells containing the trans-
formed plasmid were grown in LB media containing appropriate
antibiotics (EstA1: kanamycin 100 g mL−1, EstA4 and EstA5 ampi-
cillin 100 g mL−1 and chloramphenicol 50 g mL−1) at 37 ◦C until
an OD600 nm 0.5, before inducing with IPTG (0.1 mM) and growing
(R,S)-2-(Pyridin-2-yl)but-3-yn-2-yl acetate (1d), (R,S)-2-
(pyridin-4-yl)but-3-yn-2-yl acetate (1b) and (R,S)-2-(tert-
butylcarbamoyl)-1,1,1-trifluorobut-2-yl
prepared as described [13,15].
acetate
2-Hydroxy-2-methyl-1-
(1e)
were