K. Faber et al.
FULL PAPER
Biocatalytic Procedures
cells of Lactobacillus paracasei DSM 20207, which was obtained
from 0.33 L of culture (about 2.5 g wet cell paste) was suspended
in 6 mL of BIS-TRIS buffer (50 m, 10–2 MgCl2, pH 6). The
cells were broken by treatment with energy for 20 min (1 s pulse
followed by 2 s rest period for cooling) with an amplitude of 30%
(corresponding to 60 W). During cell disruption the suspension was
externally cooled with ice. Afterwards the crude cell lysate was cen-
trifuged at +4 °C and 18000 rpm (38.000g) for 30 min for the re-
moval of cell debris.
Bacterial Strains: All strains were obtained from the Deutsche
Sammlung für Mikroorganismen und Zellkulturen (Braunschweig,
Medium for Active Strains: Lactobacillus paracasei DSM 20008,
DSM 20207, DSM 2649, Lactobacillus sakei DSM 20017, Lacto-
bacillus halotolerans DSM 20190, Lactobacillus delbrueckii DSM
20074, Lactobacillus confusus DSM 20196 and Lactobacillus oris
DSM 4864 were grown on medium #11 as suggested by DSMZ
were sterilized in five separate groups: group I: Pepticase (10 gL–1,
Sigma), bacteriological peptone (10 gL–1, Oxoid), yeast extract
(5 gL–1, Oxoid); group II: Glucose (20 gL–1, Fluka); group III:
Tween 80 (polyoxyethylene-sorbitan-monooleate, 1 gL–1, Aldrich);
group IV: K2HPO4 (2 gL–1, Merck); group V: sodium acetate tri-
hydrate (8.3 gL–1, Fluka), ammonium citrate (2 gL–1, Fluka),
MgSO4·7H2O (0.20 gL–1, Fluka), MnSO4 (0.05 gL–1, Fluka).
Assay Procedure for Racemization Activity: Cell-free extract of
Lactobacillus paracasei DSM 20207 (400 µL) was placed into an
Eppendorff vial (1 mL) followed by addition of (S)-3-phenyllactic
acid (1; 3 mg), which was dissolved in BIS-TRIS buffer (50 m,
10–2 MgCl2, pH 6, 50 µL) and the pH was adjusted to 6. A de-
fined amount of stock solution of the cofactor or inhibitor, respec-
tively, dissolved in BIS-TRIS buffer (50 m, 10–2 MgCl2, pH 6)
was added, to reach a final concentration of 2 and 5 m. For
reasons of comparison, parallel test reactions were performed un-
der standard conditions in the absence of additive. The mixtures
were shaken at 42 °C and 130 rpm on a rotary shaker for a defined
time. For work-up, the samples were acidified with 3 HCl
(1 drop), the products were extracted with diethyl ether, and, after
centrifugation, the organic phase was dried with anhydrous sodium
sulfate. The organic phase was evaporated under reduced pressure
and the residue was dissolved in the HPLC eluent (without tri-
fluoroacetic acid). The determination of racemization activity was
carried out by HPLC analysis. For details see the Supporting Infor-
mation.
Strain Maintenance: Lactobacilli were maintained on agar plates
using the above described medium with the addition of agar. The
following components of the medium were sterilised in five separate
groups: group I: Pepticase (10 gL–1, Sigma), bacteriological pep-
tone (10 gL–1, Oxoid), yeast extract (5 gL–1, Oxoid); group II: glu-
cose (20 gL–1, Fluka); group III: Tween 80 (polyoxyethylene-sorbi-
tan-monooleate, 1 gL–1, Aldrich); group IV: K2HPO4 (2 gL–1,
Merck), agar (18 gL–1, Oxoid); group V: NaOAc·3H2O (8.3 gL–1,
Fluka), ammonium citrate (2 gL–1, Fluka), MgSO4·7H2O
(0.20 gL–1, Fluka), MnSO4 (0.05 gL–1, Fluka). The pH was ad-
justed to 6.2–6.5. Agar plates (40–45 plates out of 1 L medium)
were prepared using the warm sterilised medium. The plates were
kept in an incubator for 48 h at 30 °C (Lactobacillus paracasei
DSM 20008, DSM 20207, DSM 2649, Lactobacillus sakei DSM
20017, Lactobacillus halotolerans DSM 20190, Lactobacillus con-
fusus DSM 20196) and at 37 °C (Lactobacillus delbrueckii DSM
20074, Lactobacillus oris DSM 4864); long-term storage was at
+4 °C.
Acknowledgments
This study was performed in cooperation with BASF AG (Ludwig-
shafen) within the Research Centre Applied Biocatalysis. Financial
support by the FFG, the City of Graz and the Province of Styria
is gratefully acknowledged. H. Zollner (Graz) is cordially thanked
for his helpful advice with regard to enzyme inhibition.
Growth of Microorganisms: Strains were grown in flask cultures
without shaking at 30 °C (Lactobacillus paracasei DSM 20008,
DSM 20207, DSM 2649, Lactobacillus sakei DSM 20017, Lacto-
bacillus halotolerans DSM 20190, Lactobacillus confusus DSM
20196) and at 37 °C (Lactobacillus delbrueckii DSM 20074, Lacto-
bacillus oris DSM 4864). After transfer from agar plates, the micro-
organisms were grown for 3 d. Then, the cells were harvested by
centrifugation (18000g), washed twice with BIS-TRIS buffer
(50 m, 10–2 MgCl2, pH 6), lyophilized, and stored at +4 °C. Ap-
proximately 1.5–2 g of lyophilized cells was obtained from 1 L of
medium.
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General Procedure for the Biocatalytic Racemization: Lyophilized
cells (50 mg) were rehydrated in aqueous BIS-TRIS buffer (50 m,
10–2 MgCl2, 0.5 mL, pH 6) for 1 h at 42 °C with shaking at
150 rpm. Substrates 1–13 (5 mg) were added, followed by shaking
of the reaction mixture at 150 rpm and 42 °C for 24 h. Then, the
reaction mixture was acidified with 2 HCl (1 drop) and the cells
were removed by centrifugation. The supernatant was extracted
with ethyl acetate and the organic phase was dried with anhydrous
sodium sulfate. The determination of racemization was carried out
by analysis of the enantiomeric excess by HPLC or GC on a chiral
stationary phase. For HPLC determination, the organic phase was
evaporated under reduced pressure and the residue was dissolved
in HPLC eluent (without trifluoroacetic acid and formic acid). For
details see the Supporting Information.
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Cell Disruption: Cell disruption was carried out using a digital ul-
trasonifier (Branson, 250 W). For cell breakage a portion of wet
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