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NADPH). The cell suspension was sonicated (total pulse time:
5 min; pulse on: 1 s; pulse off: 4 s; amplitude 30%; cooling with
ice), and the resulting mixture was centrifuged to remove cell
debris (14000 rpm for 20 min at 48C). The crude extract solution
was heat-treated at 708C for 20 min to inactivate non-thermosta-
ble proteins, then precipitated proteins were removed by another
centrifugation step (14000 rpm for 20 min at 48C) and lyophilized.
WIC cells were employed. Experiments in micro-aqueous (mAq)
conditions showed the high tolerance of the catalyst for organ-
ic solvents, whereby reaction rates depend on the water con-
centration. Owing to the low enantioselectivity of the catalyst,
it is suitable as an enzymatic racemization catalyst as shown
for the racemization of (S)-1b and (S)-1g. To improve the
speed of racemization, addition of the ketone (e.g., 0.1 equiva-
lent) of the alcohol to be racemized turned out to be advanta-
geous. Utilization of a polyvinylidene difluoride (PVDF) mem-
brane in a TeaBag construction for compartmentalization of
the two reactions of a bis-enzymatic DKR was favorable. Al-
though for efficient enzymatic racemization a biocatalyst with
even higher activity is still required, a proof of concept
showed the successful combination of enzymatic racemization
in aqueous phase combined with acylation in cyclohexane
containing the lipase in a TeaBag.
General procedure used for reactions in buffer with lyophi-
lized cells of E. coli/TeSADH WIC
Reactions were conducted in 2 mL glass vials. To lyophilized cells
of E. coli/TeSADH WIC and NADP+ were dissolved in the Tris-HCl
buffer (50 mm, pH 8.0, 10 mm ZnCl2, 0.1 mm dithiothreitol), followed
by addition of substrates and co-substrates (for oxidation reaction
only). Reactions were shaken at 508C on a thermoshaker and
stopped by extraction with MTBE (0.5 mL). The organic layer was
separated by centrifugation (1 min, 14000 rpm), dried over anhy-
drous Na2SO4, and analyzed by GC-MS and GC on a chiral station-
ary phase. Compounds 1b–h, 1j, 1k, 1m, 1o, and 1s were derivat-
ized prior to chiral GC chromatography with acetyl chloride and
pyridine. All reactions were made in triplicate (standard deviation
was in general <2%).
Experimental Section
General information
All reagents were purchased from commercial sources (Sigma–Al-
drich, Roth, Alfa Aesar, Acros Organics, Lancaster, Oxoid) and used
without further purification. The gene coding TeSADH W110A I86A
C295A (TeSADH WIC) optimized for expression in E. coli was pur-
chased from Invitrogen (GeneArt) and cloned into a pET21a vector
and used for transformation in E. coli BL21(DE3) cells. Reactions
were analyzed by gas chromatography by using an Agilent 7890A
equipped with a chiral coating Chirasil-Dex CB or Restek Rt-BDEXse
column. GC-MS analysis was performed by using an Agilent 7890A
with 5975C detector equipped with an Agilent HP-5 column. NMR
spectra were recorded with a Bruker AVANCE III 300 MHz spectrom-
eter. All of the reaction products were known compounds and
were identified by comparison of MS spectra with those in the
NIST database. DKR reference products were obtained by Fisher
esterification. TeaBags were prepared in accordance with an al-
ready described procedure.[40] For more detailed experimental in-
formation see the Supporting Information.
General procedure used for mAq reactions with lyophilized
cells of E. coli/TeSADH WIC
Reactions were conducted in 2 mL glass vials. Lyophilized cells of
E. coli/TeSADH WIC were rehydrated in Tris-HCl buffer (50 mm,
pH 8.0, 10 mm ZnCl2, 0.1 mm dithiothreitol, NADP+) for 10 min at
room temperature and 300 rpm on a thermoshaker. Then, the or-
ganic solvent was added, followed by addition of substrates and
co-substrates (for oxidation reaction only). Reactions were shaken
at 508C on a thermoshaker. The reactions were cooled, the organic
phase was separated and the remaining cell pellet was washed
with MTBE (0.5 mL), followed by centrifugation (1 min, 14000 rpm).
Combined organic phases were dried over anhydrous Na2SO4 and
analyzed by GC-MS and GC on a chiral stationary phase. Com-
pounds 1b and 1g were derivatized prior to chiral GC chromatog-
raphy with acetyl chloride and pyridine. All reactions were made in
triplicate (standard deviation was in general <2%).
General procedure for the preparation of lyophilized cells of
E. coli/TeSADH WIC
TeSADH W110A I86A C295A was expressed in BL21(DE3) E. coli
cells. Cultures were grown in lysogeny broth (LB) medium (300 mL,
5 g NaCl, 5 g yeast extract, 10 g tryptone) containing ampicillin
disodium salt (100 mgLÀ1) in a 1 L baffled Erlenmeyer flask at 378C
and 120 rpm. When the culture reached an OD600 of 0.6–0.8, ex-
pression was induced with isopropyl b-d-1-thiogalactopyranoside
(IPTG, 0.1 mm) and the cultures were shaken for 14 h at 308C. The
cells were harvested by centrifugation (5000 rpm for 10 min),
washed with buffer (50 mm Tris-HCl pH 8) and suspended in
15 mL of buffer (50 mm Tris-HCl pH 8, 10 mm ZnCl2, 0.1 mm dithio-
threitol, 0.4 mm NADP+, 0.3 mm NADPH), followed by overnight
lyophilization.
General procedure used for DKR reactions with CFE of
TeSADH WIC and Novozym 435
Reactions were conducted in 2 mL or 10 mL glass vials with given
concentrations of CFE of TeSADH WIC or E. coli/TeSADH WIC and
Novozym 435. Novozym 435 was transferred to a PVDF TeaBag and
sealed. Lyophilized cells or CFE were rehydrated in Tris-HCl buffer
(50 mm, pH 8.0, 10 mm ZnCl2, 0.1 mm dithiothreitol, 1 mm NADP+)
for 10 min at 228C. Resulting mixtures were preheated to the des-
ignated temperature and reactions were started by addition of
substrates and acyl donors. Then, a TeaBag with Novozym 435 was
attached in the vials in such a way so as to be soaked by the or-
ganic phase, and also to prevent any contact with the buffer phase
during the reaction. Reactions were performed at 508C on a rotary
thermostated shaker (6.4 mL, 120 rpm). Reactions were stopped by
separating the organic layer and extraction of the water phase
with MTBE (2ꢁ reaction volume). Combined organic fractions were
dried over anhydrous Na2SO4 and analyzed by GC-MS and GC on a
chiral stationary phase.
General procedure for the preparation of lyophilized CFE of
TeSADH WIC
E. coli/TeSADH WIC cultures were grown as described above and
after centrifugation and washing were suspended in 30 mL of
buffer (50 mm Tris-HCl pH 8, 10 mm ZnCl2, 0.4 mm NADP+, 0.3 mm
ChemCatChem 2018, 10, 1 – 7
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