Kinetics of acetophenone reduction to (R)-1-phenylethanol
3
ꢁ
1
ꢁ1
Cell cultivation
mmol l to 17 mmol l . Reaction progress curves
were measured at similar conditions but the biocata-
Pichia capsulata cells were cultivated in a 4-l bioreac-
tor (Biostat-B, Sartorius, Goettingen, Germany).
Volumetric ratio of inoculum and the growth
medium was 1:9. The inoculum was prepared in a
ꢁ1
lyst and glucose concentrations were 12 g l
1
and
mmol l , respectively. The initial acetophenone
ꢁ
7
concentration was in the range of 0.50–9.3 mmol l
ꢁ
1
.
The reaction medium was sampled at appropriate
time intervals and analyzed for glucose, acetophe-
none, and (R)-/(S)-1-phenylethanol content using
HPLC and GC analysis, respectively.
48-h cultivation on the YPD medium in shaken flasks.
Details of its preparation are described elsewhere
(Homola et al. 2015). The bioreactor cultivation was
ꢀ
performed at 24 C, pH 5.8, and the initial glucose
ꢁ1
concentrations of 10 g l using the YPD medium.
Stirrer frequency and aeration intensity were used as
manipulated variables to control the dissolved oxygen
concentration at 1–2% of equilibrium value. The
produced cells were harvested by centrifugation,
washed thrice, and centrifuged again. Dry mass of
the wet cells was determined using an automated dry-
residue meter MLS 50-3 HA 160 (Kern, Balingen,
Germany). A sample (ꢃ20 ml) was dried at the
Analytical methods
The substrate and products of the biocatalytic
reaction were determined using gas chromatography.
Acetophenone, (R)-1-phenylethanol, and (S)-1-phe-
nylethanol were extracted from 750 ml of a reaction
medium with the same volume of ethyl acetate
containing 2-phenylethanol as the internal standard.
The 2-ml extraction tubes were stirred for 10 min
using a vortex mixer and the resulting liquid phases
ꢀ
temperature of 105 C. The process was programmed
to stop when the mass of the sample changed by
50.001 g in four consecutive minutes. Each experi-
ment was triplicated. The differences between indi-
vidual measurements were typically 52%. Specific
alcohol dehydrogenase activity of the whole-cell
biocatalyst [(R)-1-phenylethanol produced per dry
were separated by centrifugation for 10 min at
ꢀ
18,000 rpm and 5 C. The upper phase was dried
over anhydrous sodium sulfate, filtered through a
PTFE filter (0.45 mm) and analyzed using an Agilent
7
890A GC System (Palo Alto, CA) equipped with a
ꢀ
ꢁ
1
flame ionization detector set to 250 C. The analytes
were separated on a chiral Cyclodex-B column
mass] was 1.50 U g and the enantiomeric excess of
the R-form was 94% (Homola et al. 2015).
(
30 m ꢄ 0.320 mm, 0.25 mm film thickness, J&W
Scientific, Santa Clara, CA). After 0.3 min at
Activity determination
ꢀ
1
1
10 C, the column temperature was raised to
ꢁ1
ꢀ
ꢀ
50 C at 10 C min . Retention times of acetophe-
The acetophenone reduction reaction was performed
in 50 ml Beckman tubes so that the amount of dry
biomass in a 100-mM phosphate buffer with pH 6
none, (S)-1-phenylethanol, and (R)-1-phenylethanol
were 3.07, 3.67, and 3.74 min, respectively.
Contents of (S)-1-phenylethanol, (R)-1-phenyletha-
nol, and acetophenone were calculated on the basis
of the internal standard. To determine the glucose
concentration, the reaction medium was first rapidly
ꢁ
1
was 70 g l and the initial concentrations of acet-
ꢁ1
ophenone and glucose were 21.4 mmol l
and
17 mmol l , respectively. Tubes with the reaction
medium were stirred with a magnetic stirrer
ꢁ
1
1
cooled and centrifuged for 10 min at 18,000 rpm and
ꢀ
(
300 rpm) and temperature was maintained at
ꢀ
5
C. The separated liquid phase was properly
2
5 C using a water bath. Concentrations of acet-
diluted and filtered through a cellulose-acetate filter
(0.2 mm). The sample was then analyzed using a
HPLC Agilent 1100 Series equipped with a refractive
index detector. The column used was a Polymer IEX
H form 8 mm (250 mm ꢄ 8 mm, Watrex, Prague,
Czech Republic) and the volumetric flow rate of the
ophenone and phenylethanol were determined using
gas chromatography. Whole-cell activity was deter-
mined from the initial rate of the 1-phenylethanol
production and the enantiomeric excess was calcu-
lated on the basis of the amount of (R)-1-phenyl-
ethanol produced. Specific activity of the whole-cell
biocatalyst was defined per 1 g of dry biomass. The
mean error of the activity determined from the rate of
mobile phase, 9 mM H
SO
solution in distilled
2
4
ꢁ1
water, was 0.8 ml min . The column and detector
ꢀ
ꢀ
(
R)-1-phenylethanol formation was 1.5%.
temperatures were 22 C and 35 C, respectively.
The retention times were 5.98 min for glucose and
1
4.40 min for ethanol.
Kinetic experiments
Initial reaction rates were measured in the same way
as described earlier for the activity determination
except for the pH being set to 5 and the initial
acetophenone concentration varying from ꢃ1.5
Kinetic parameter estimation
Reaction kinetics of (R)-1-phenylethanol formation
was described using a model consisting of one