Rui-Hu et al. / Tetrahedron: Asymmetry 24 (2013) 81–88
87
substrate concentration, and temperature were set at 6.9,
14.4 mmol/L and 33.6 °C, respectively. The maximum predicted
yield was 93.4%.
4.3. General procedure for the light-controlled asymmetric
hydrogenation system
In order to confirm the optimization results, the suggested con-
ditions were performed in triplicate. Under these suggested condi-
tions, the mean value of the (S)-TMSBL yield was 94.5%, which was
in agreement with the predicted value. This optimization strategy
led to an enhancement of the yield from 88.9% to 94.5%. The mod-
els developed were considered to be accurate and reliable for pre-
dicting the production of (S)-TMSBL by photosynthetic bacteria R.
sphaeroides.
An appropriate substrate was added to the above mentioned
asymmetric reduction system and then inoculated into a 200-mL
bubble column photobioreactor containing 100 mL of the 0.1 M
potassium phosphate buffer (KPB, pH 7.0).The reaction mixture
was shaken at 140 rpm at 30 °C under continuous illumination
with
a fluorescent lamp (daylight type, 0–53.6 lmol pho-
tons mꢀ2 sꢀ1). The 5% CO2 gas (v/v, mixed with air) was provided
by a gas cylinder, which was aerated from the photobioreactor bot-
tom at a rate of 0.1 v/v minꢀ1 (volume gas per volume broth per
minute). Finally, the reaction mixture was extracted with ethyl
acetate and the organic phase was dried over anhydrous Na2SO4.
The concentrations of product and substrate were determined,
and the chemical yield and enantiomeric excess (ee) were
evaluated.
3. Conclusion
The light-controlled asymmetric hydrogenation of TMSBO by
photosynthetic bacteria R. sphaeroides is a very promising technol-
ogy for the production of high-quality (S)-TMSBL. The results here-
in clearly indicate that RSM is an effective method for the
optimization of the reaction conditions for maximizing the yield
of (S)-TMSBL. The values of the three main variables, pH (6.9), sub-
strate concentration (14.4 mmol/L), and temperature (33.6 °C),
were found to be optimum for the production of (S)-TMSBL with
high levels of activity and stability.
4.4. Experimental design and data analysis
The experimental design and analysis of results were carried
out using MATLAB 6.5.0 (Mathworks, USA). Response Surface
Methodology (RSM) was used to investigate the effects of substrate
concentration, pH, and temperature on the reaction yield. A 33 fac-
torial design was performed in order to optimize the production of
(S)-TMSBL.
4. Experimental
The following equation describes the regression model utilized
in the factorial planning, including the interaction terms:
4.1. Materials and microorganism
X
X
X
Y ¼ b0 þ
biXi þ
biiX2 þ ꢃ
bijXiXj
i
4-(Trimethylsilyl)-3-butyn-2-one (97% purity), 4-(trimethyl-
silyl)-3-butyn-2-ol (97% purity), and n-decane (>99% purity) were
purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
All other chemicals were from commercial sources and were of re-
agent grade or better. All photosynthetic bacteria were preserved
in our laboratory (Institute of Applied Chemistry, Shanxi
University).
where Y is the predicted response variable {the yield of (S)-TMSBL},
b0 is the intercept coefficient, bi is the coefficient of the linear ef-
fects, bij is the coefficient of interaction and Xi and Xj are the inde-
pendent variables.
4.5. Analysis
4.2. Cell culture and medium
Gas chromatographic analysis was performed using a SHIMA-
DZU model 7900 gas chromatograph (GC) equipped with a GP
CHIRASIL-DEX (25 m ꢄ 0.25 mm; Agilent Technologies Co., Ltd)
and flame ionization detector. The split ratio was 100:1. The injec-
tor and the detector were both kept at 250 °C. The column temper-
ature was held at 71 °C for 1 min, then programed to increase at
1 °C/min to 100 °C, and maintained for 2 min at this temperature.
The carrier gas was nitrogen and its flow rate in the column was
2.5 ml/min. Quantitative data were obtained after integration on
an HP LaserJet 5200L integrator. An internal standard method
was used for the calculations. The retention times for ATMS, n-non-
ane, (R)-1-TMSBL, and (S)-1-TMSBL were 3.374, 5.202, 5.417, and
5.747 min, respectively. JASCO DIP-378 polarimeter was used for
the determination of the enantiomeric excesses (ee). The products
were identified by ICT GC-MS analyses (model 2010, SHIMADZU).
The structures were confirmed by comparison with the mass spec-
troscopic database.
Photosynthetic bacteria R. sphaeroides was cultivated in med-
ium containing (per liter) 0.5 g of KH2PO4, 0.6 g of K2HPO4, 1.0 g
of (NH4)2SO4, 0.2 g of MgSO4ꢂ7H2O, 0.2 g of NaCl, 0.05 g of
CaCl2ꢂ2H2O, 0.1 g of yeast extract, 4 g of malic acid, and 0.2 mL of
trace element solution [2 g of ethylenediaminetetraacetic acid
disodium salt, 2 g of green vitriol, 0.1 g of boric acid, 0.1 g of cobalt
chloride, 0.1 g of zinc chloride, 0.1 g of manganese(II) chloride tet-
rahydrate, 0.02 g of sodium molybdate, 0.02 g of nickelous chlo-
ride, 0.01 g of cupric chloride, and 0.001 g of sodium selenite].
The medium was adjusted to pH 7.2 with 2 M NaOH and auto-
claved for 30 min. A pre-culture was prepared by inoculation of
100 mL of the complex medium with fresh cells from an agar plate
(Swab of inoculation loop). Incubation was performed in a 1000-
mL Erlenmeyer shaking flask. These were grown in the medium
filled with nitrogen under continuous illumination provided by
fluorescence lamps (200 W) at 30 °C. After five days, the cultured
bacteria (OD680 was approximately 1.0, where OD680 is the optical
density at 680 nm, used to indicate the microalgal biomass density
based on turbidimetry) (UltrospecÒ 3300 pro Amersham Biosci-
ences Co., Ltd) were collected by centrifugation (5000 rpm,
20 min), washed twice with phosphate buffer (0.2 mmol/L, pH
7.0) and separated from the aqueous medium by centrifugation
to give a cell wet mass of 2–5 g per 400 mL batch. The ratio ‘cell
wet mass’/‘cell dry mass’ of approximately 4.0 was determined
by lyophilization of the samples of wet cells. The wet cells were
used directly for this reaction.9
The reaction degree and enantioselectivity are indicated by
yield (chemical yield (%)) and ee (%), respectively, defined as
Cp
Co
yield ¼
ꢄ 100
Cs ꢀ CR
Cs þ CR
ee ¼
ꢄ 100
where Co is the initial substrate concentration, Cp is the final prod-
uct concentration, Cs is the final (S)-form product concentration, and
CR is the final (R)-form product concentration.