H.J. Choi et al. / Journal of Molecular Catalysis B: Enzymatic 70 (2011) 114–118
115
concentration of substrate and the yield of the target compound,
S)-CPPO.
and 2-propanol (95:5, v/v) were used as the mobile phase at
a flow rate of 0.8 mL/min. Relative quantities of 3-CPP and (R)-
and (S)-CPPO were calculated based on the peak area, which was
appropriately calibrated with standards of known concentration.
Enantiomeric excess (e.e.) values were calculated from the alcohol
products.
(
2
. Experimental
2.1. Chemicals
3
-Chloro-1-phenyl-1-propanol (3-CPP), (S)-3-chloro-1-phenyl-
2.4. Selection of suitable ionic liquids and detergents
1
-propanol ((S)-CPPO), NADPH, and NADP+ were purchased
from Sigma–Aldrich Co. (St. Louis, MO, U.S.A.). 1-n-Butyl-
Biotransformation reactions using the E. coli cells harboring
pETR151 plus pACGDH were conducted in ionic liquid/water
systems. The ionic liquids employed in this research included
[Bmim][PF6], [Bmim][NTf2], [Mmim][MeSO4], and [Emim][BF4].
The two-phase system was composed of 800 L of Tris–HCl buffer
(100 mM, pH 7.5) and 200 L of each ionic liquid. To the reaction
system, 30 mM 3-CPP, 1 mM NADPH, 45 mM glucose, 0.5% Tween 40
and permeabilized E. coli cells (corresponding to 3 units reductase)
3
-methylimidazolium
hexafluorophosphate
([Bmim][PF ]),
6
1
-n-butyl-3-methylimidazolium
bis
(trifluoromethylsul-
fonyl) imide ([Bmim][NTf ]), 1-methyl-3-methylimidazolium
2
methylsulfate ([Mmim][MeSO ], 1-ethyl-3-methylimidazolium
4
tetrafluoroborate ([Emim][BF ]) were purchased from C-TRI
4
(
Suwon, Korea). All other chemicals were of analytical grade.
◦
2.2. Reductase and glucose dehydrogenase activity assay
were added, and the reaction mixtures were incubated at 30 C with
shaking at 300 rpm. After 6 h, the quantity of (S)-CPPO generated
was measured via HPLC.
◦
Reductase activity was evaluated at 30 C by measuring the
decrease in absorbance at 340 nm for 10 min with a spectropho-
tometer. The reaction mixture (1 mL) consisted of 1 mM 3-CPP
To select a suitable detergent, the bioconversion reaction was
conducted using various detergents, including Triton X-100, Tween
20, Tween 40, and Tween 80. The two phase system was com-
posed of 800 L of Tris–HCl buffer (100 mM, pH 7.5) and 200 L of
[Bmim][NTf2]. To this reaction system, 50 mM 3-CPP, 1 mM NADPH,
75 mM glucose, 0.5% of various detergents and permeabilized E. coli
cells (3 units reductase) were added, and the reaction mixtures
(
(
100 mM stock in DMSO), 0.2 mM NADPH, 100 mM Tris–HCl buffer
pH 7.5), and 5 L of permeabilized cells. One unit of enzyme was
defined as the quantity of enzyme required to catalyze the oxida-
◦
tion of 1 mol NADPH in 1 min at 30 C.
The oxidation reaction mixture (1 mL) of glucose dehydroge-
+
◦
nase consisted of 10 mM glucose, 0.2 mM NADP , 100 mM Tris–HCl
were incubated at 30 C with shaking at 300 rpm. After 12 h, the
buffer (pH 7.5), and 1 L of permeabilized cells. The reaction rate
quantity of (S)-CPPO produced was measured via HPLC.
was monitored with a spectrophotometer on the basis of the
increase in absorbance at 340 nm for 10 min at 30 C. One unit of
To determine an appropriate concentration of Tween 40, the
bioconversion reaction was conducted with 0–5% of Tween 40. To
the same two-phase system, 100 mM 3-CPP, 1 mM NADPH, 150 mM
glucose, 0–5% of Tween 40 and permeabilized E. coli cells (3 units
reductase) were added and the reaction mixtures were incubated at
◦
enzyme was defined as the quantity required to reduce 1 mol of
NADP+ in 1 min at 30 C.
◦
◦
2.3. Preparation of permeabilized cells and enzymatic coupling
30 C with shaking at 300 rpm. After 24 h, the quantity of (S)-CPPO
reaction
produced was measured by HPLC.
Each sample was centrifuged for 10 min at 12,000 × g and a por-
tion (50 L) of the lower phase was mixed with 150 L of ethyl
acetate, then filtered and dried via evaporating the organic solvent
using Vacuum Centrifugal Evaporator. The sample was analyzed
using an HPLC system as previously described.
Recombinant E. coli BL21 (DE3) cells harboring one plasmid
pETR151, pACGDH, pACR151-GDH) or two plasmids (pETR151
(
◦
plus pACGDH) were cultured at 18 C in 600 mL of LB medium
10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter)
(
containing 100 g/mL of ampicillin (for pETR151) or 170 g/mL
of chloramphenicol (for pACGDH or pACR151-GDH). When the
OD600 nm reached 0.5, isopropyl thio--d-galactoside was added to
a final concentration of 1 mM and cultured for an additional 24 h.
The cultured cells were then harvested via centrifugation (6000 × g,
2.5. (S)-CPPO production by whole cell-[Bmim][NTf2] system
The final bioconversion reaction conditions were as follows:
100 mM 3-CPP, 1 mM NADPH, 150 mM glucose, 4% Tween 40, and
permeabilized E. coli cells containing pETR151 plus pACGDH (30
units reductase activity) were added to the two-phase reaction sys-
tem containing8 mL ofTris–HCl buffer (100 mM, pH7.5) and 2 mLof
1
7
0 min) and resuspended in 12 mL of a 100 mM Tris–HCl buffer (pH
.5). Toluene (0.1%, v/v) was added, and the mixture was shaken
◦
for 10 min at 300 rpm at 30 C. Permeabilized cells were harvested
◦
◦
via centrifugation (6000 × g for 10 min) at 4 C.
[Bmim][NTf2]. The reaction mixture was incubated for 14 h at 30 C
For the coupling reaction using the whole-cell systems, 30 mM
and the pH was monitored and maintained within 7.0–7.5 via the
addition of 1 M NaOH. The quantities of 3-CPP and (S)-CPPO in the
reaction mixture were analyzed by HPLC as previously described.
The partition coefficient of the 3-CPP and (S)-CPPO between
[Bmim][NTf2] and water phases was measured as described in
Supplementary data 1. Based on these results, the concentration of
3-CPP and (S)-CPPO produced during the bioconversion time course
was calculated.
3
-CPP, 1 mM NADPH, 45 mM glucose, and 10 units (calculated on
the basis of reductase activity) of the permeabilized cells har-
boring one plasmid (pETR151 or pACR151-GDH) or two plasmids
(
pERT151 plus pACGDH) were mixed in a total volume of 10 mL of
Tris–HCl buffer (100 mM, pH 7.5), and the mixture was incubated
◦
at 30 C. The pH of the reaction mixture was monitored with a pH
meter and maintained at 7.0–7.5 via the addition of 1 M NaOH.
Four hundred microliter aliquots of the reaction mixture were
sampled, mixed with 1.2 mL of ethyl acetate, and centrifuged for
3. Results and discussion
1
0 min at 12,000 × g. The upper phase (1 mL) was obtained, com-
bined with MgSO , filtered, and then dried via evaporating the
3.1. Enzyme activity assay of E. coli whole cells
4
organic solvent using Vacuum Centrifugal Evaporator (CVE-2000,
EYELA, Tokyo, Japan). The sample was resuspended in ethyl acetate
and analyzed with an HPLC system equipped with a CHIRALPAK IB
column (Daicel Chemical Industries, Ltd., Tokyo, Japan). n-Hexane
In a previous study, the yeast reductase YOL151W was gen-
erated in E. coli BL21 (DE3) cells and the isolated reductase
was employed for the production of (S)-CPPO [20]. In that