Enzymatic Production of l-Menthol by a High Substrate Concentration Tolerable Esterase
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Spots were visualized by treatment with 5% (w/v) vanillin/
sulphuric acid solution and heating of the dried plates. The
1H NMR spectra were recorded in CDCl3 on a Bruker
Avance 500 spectrometer (at 500 MHz, Bruker, Karlsruhe,
Germany). Mass spectra were recorded on an Agilent 5975
MSD mass spectrograph (electron impact, 70 eV, Agilent,
USA). Optical rotations were determined on a Jasco P-1030
polarimeter.
thesis of compound 5 was performed as described by
Omata.[9]
dl-Menthyl chloroacetate (1):[20] This compound was ob-
1
tained as a colourless liquid; yield: 8.64 g (75%); H NMR
(CDCl3, 500 MHz): d=0.77 (d, 3H, J=7.0 Hz), 0.83–0.92
(m, 7H), 0.99–1.08 (m, 2H), 1.40–1.54 (m, 2H), 1.67–1.71
(m, 2H), 1.85–1.89 (m, 1H), 2.00–2.03 (m, 1H), 4.04 (dd,
2H, J=17.2 Hz, 14.6 Hz), 4.77 (dt, 1H, J=10.9 Hz, 4.4 Hz).
dl-Menthyl acetate (2):[21] This compound was obtained as
a yellow liquid; yield: 9.02 g (91%); 1H NMR (CDCl3,
500 MHz): d=0.76 (d, 3H, J=7.0 Hz), 0.82–1.10 (m, 9H),
1.32–1.39 (m, 1H), 1.43–1.53 (m, 1H), 1.64–1.70 (m, 2H),
1.83–1.90 (m, 1H), 1.96–2.03 (m, 4H), 4.68 (dt, 1H, J=
10.9 Hz, 4.4 Hz); MS (EI): m/z=198 (M+), 183, 165, 138,
123, 109, 95, 81, 67, 55, 43.
Screening Procedure of Microbial Strains
Different soil samples were collected firstly from the nature,
and then enrichment cultured using dl-menthyl acetate as
the sole carbon source. Those strains capable of hydrolyzing
dl-menthyl acetate were rapidly screened through TLC anal-
ysis. Those with obvious spots of the menthol were marked
for further confirmation by GC. The best strains were fur-
ther compared and used in the subsequent study.
dl-Menthyl butyrate (3):[22] This compound was obtained
1
as a colourless liquid; yield: 2.77 g (61%); H NMR (CDCl3,
500 MHz): d=0.76 (d, 3H, J=7.0 Hz), 0.82–1.10 (m, 12H),
1.34–1.39 (m, 1H), 1.43–1.53 (m, 1H), 1.62–1.70 (m, 4H),
1.83–1.90 (m, 1H), 1.96–2.01 (m, 1H), 2.26 (t, 2H, J=
7.5 Hz), 4.68 (dt, 1H, J=10.9 Hz, 4.4 Hz); MS (EI): m/z=
226 (M+), 155, 138, 123, 109, 95, 81, 71, 55, 43.
Enzyme Preparation
Cultivation of Bacillus subtilis ECU0554: B. subtilis
ECU0554 was grown aerobically in a simply optimized
medium with the following composition (per liter): glycerol,
30.0 g; peptone, 5.0 g; yeast extract, 5.0 g; NaCl, 1.0 g;
KH2PO4, 0.5 g; K2HPO4, 0.5 g; MgSO4, 0.2 g; pH 7.0. The
microorganisms were cultivated in 500-mL flasks with
100 mL medium at 308C for 18 h with shaking at 180 rpm.
After cultivation, the microbial cells were harvested by cen-
trifuge under 48C at 10,000ꢂg for 10 min and washed twice
with physiological saline and stored at 48C for further use.
Crude enzyme preparation: The harvested cells of B. sub-
tilis ECU0554 were resuspended in sodium phosphate buffer
(pH 7.0, 50 mM) and disrupted 3 times by a high-pressure
homogenizer (AH110B, ATS Engineering Inc.). Cell debris
was removed by centrifugation (12,000ꢂg, 48C, 20 min) and
the resultant supernatant was used as a crude extract. Am-
monium sulphate was added slowly to the crude extract with
stirring at 08C. The protein that precipitated between 60%
and 90% saturation of ammonium sulphate was collected by
centrifugation (12,000ꢂg, 48C, 20 min) and dialyzed against
10 mM sodium phosphate buffer (pH 7.0). Then the dialy-
sate was dried with a freeze dry system, and the resultant
crude esterase was stored at 48C.
dl-Menthyl benzoate (4):[23] This compound was obtained
1
as a colourless solid; yield: 10.81 g (83%); H NMR (CDCl3,
500 MHz): d=0.80 (d, 3H, J=7.0 Hz), 0.88–0.97 (m, 7H),
1.07–1.18 (m, 2H), 1.52–1.58 (m, 2H), 1.70–1.75 (m, 2H),
1.94–1.99 (m, 1H), 2.11–2.15 (m, 1H), 4.68 (dt, 1H, J=
10.9 Hz, 4.4 Hz), 7.42–7.45 (m, 2H), 7.53–7.56 (m, 1H),
8.04–8.06 (m, 2H); MS (EI): m/z=260 (M+), 162, 147, 138,
123, 105, 95, 81, 67, 55, 41, 29.
dl-Menthyl succinate (5):[24] This compound was obtained
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as a colourless solid; yield: 5.25 g (82%); H NMR (CDCl3,
500 MHz): d=0.74 (d, 3H, J=7.0 Hz), 0.82–1.08 (m, 9H),
1.35–1.39 (m, 1H), 1.43–1.52 (m, 1H), 1.64–1.70 (m, 2H),
1.83–1.89 (m, 1H), 1.96–2.00 (m, 1H), 2.58–2.62 (m, 2H),
2.67–2.72 (m, 2H), 4.71 (dt, 1H, J=10.9 Hz, 4.4 Hz).
Synthesis of (RS)-1-Phenylethyl Acetate (6) and
(RS)-3-Chloro-1-phenyl-1-propyl Acetate (7)
The (RS)-1-phenylethanol (6.11 g, 0.05 mol) or (RS)-3-
chloro-1-phenyl-1-propanol (8.53 g, 0.05 mol), sodium ace-
tate (0.1 equiv.) and acetic anhydride (1.5 equiv.) were
added into chloroform (40 mL), and the mixture was re-
fluxed. The reactions were performed until (RS)-1-phenyl-
General Procedure for Chemical Synthesis of
Racemic Menthyl Esters
AHCTUNGTREGeNNUN thanol or (RS)-3-chloro-1-phenyl-1-propanol was completely
consumed (as monitored by TLC). The product was extract-
ed into ethyl acetate (50 mLꢂ3), then washed with an aque-
ous solution of saturated sodium bicarbonate (100 mLꢂ3)
and dried over anhydrous sodium sulphate. The organic sol-
vent was evaporated under reduced pressure and the prod-
uct was dried under vacuum.
For synthesis of compounds 1, 2 and 4, chloroacetyl chloride
(1.1 equiv.), acetyl chloride (1.1 equiv.) and benzoyl chloride
(1.2 equiv.) were added dropwise to a mixture of dl-menthol
(7.81 g, 0.05 mol) and pyridine (0.2 equiv.) in dry chloroform
(40 mL) on an ice bath, and then the solution was stirred at
room temperature. For synthesis of compound 3, the dl-
menthol (3.12 g, 0.02 mol) and n-butyric anhydride (1.2
equiv.) were added into dry toluene (20 mL), and the mix-
ture was refluxed. The reactions were performed until men-
thol was completely consumed (as monitored by TLC). The
product was extracted into ethyl acetate (100 mLꢂ3), then
washed with an aqueous solution of saturated sodium bicar-
bonate (100 mLꢂ3) and dried over anhydrous sodium sul-
phate. The organic solvent was evaporated under reduced
pressure and the product was dried under vacuum. The syn-
(RS)-1-Phenylethyl acetate (6):[25] This compound was ob-
1
tained as a colourless liquid; yield: 7.34 g (89%); H NMR
(CDCl3, 500 MHz): d=1.53 (d, 3H, J=6.6 Hz), 2.07 (s, 3H),
5.88 (q, 1H, J=6.6 Hz), 7.24–7.40 (m, 5H).
(RS)-3-Chloro-1-phenyl-1-propyl acetate (7):[26] This
compound was obtained as a colourless liquid; yield: 9.50 g
1
(89%). H NMR (CDCl3, 500 MHz): d=2.07 (s, 3H), 2.15–
2.21 (m, 1H), 2.35–2.42 (m, 1H), 3.40–3.45 (m, 1H), 3.52–
3.57 (m, 1H), 5.92 (dd, 1H, J=8.1 Hz, 5.7 Hz), 7.27–7.36
(m, 5H).
Adv. Synth. Catal. 2009, 351, 405 – 414
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
411