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nary ease of addition and removal, the “teabag” approach is
a very user friendly mode to screen optimal reaction condi-
tions, process parameters, and/or catalyst combinations.
Whether the SpinChem reactor or the stirred-tank reactor is
recommended for a scaled-up application of “teabag”-encapsu-
lated catalyst depends on the equipment available. Although
the SpinChem reactor is the most elegant way to mix with the
least mechanical force, it is limited by availability and maxi-
mum catalyst loading. As an alternative, our model stirred-tank
reactor of a round-bottomed flask and a stirrer bar is easy to
set up and ubiquitously available.
Preparation of alginate immobilizates
Alginate was weighed into a small beaker and deionized water
À1
(
10 mL) was added to achieve a concentration of 20–30 gL . The
heated mixture (45–558C) was stirred for 30 min. Frozen cell pellet
(10 g) was resuspended in deionized water (10 mL) under gentle
stirring (258C). The alginate solution was allowed to cool to 30–
3
2
58C and mixed with the cell suspension. For bead formation,
0 mL of the mixture was dropped into stirred CaCl (25–100 mm)
2
solution using a dispenser pipette. The beads were allowed to fur-
ther harden in the CaCl solution overnight at 48C (protocol was
2
[24]
modified from the method published by Hartmeier). Before use,
the beads were taken from the solution and dried for several sec-
onds between two paper towels.
To prove “teabag” encapsulation as a new alternative for
multi-step syntheses using whole-cell catalysts, the SpinChem
reactor was chosen for a one-pot two-step reaction on a prepa-
rative scale. The reaction was performed under microaqueous
“Teabag” preparation
À1
conditions and afforded a concentration of 32.9 gL (1R,2R)-1-
“Teabags” were prepared from PVDF western blotting membranes
with a cut-off of 0.2 mm (Bio-Rad Laboratories GmbH, Munich, Ger-
many). The ring structure consisted of four adjacent compart-
ments, each filled with a quarter of the total catalyst loading, cov-
ering the inner wall of the SpinChem chamber. Precise manufactur-
ing details for this ring can be found in Supporting Information SI-
phenylpropane-1,2-diol at a very good stereoselectivity (98.6%
target isomer content) and with a reasonable space–time yield
À1 À1
(
8.2 gL d ). This demonstrates that, despite the presence of
a diffusion limitation by the applied “teabag” membrane,
thresholds of industrial feasibility can still be exceeded.
1
2
. For 5 mL scale transformations, membrane material of 1.4
.7 cm was cut, folded, and sealed to give a small bag to be filled
In summary, an alternative way to implement whole-cell cat-
alysis for use in the emerging SpinChem technology was pre-
sented and its applicability was confirmed by the production
of a model fine chemical on a gram scale. Additionally, the
combination of “teabag” encapsulation and whole-cell catalysis
was also proven to be scalable to classical reactor concepts.
Therefore, this technique has become even more appealing to
users with and without a profound experience in biocatalysis
and the handling of biological systems. It combines the ad-
vantage of (i) facile and flexible catalyst combination screening,
with 51.5 mg lyophilized whole-cell catalyst.
Benzoin condensation on 5 mL scale in an overhead-shaken
vial
Benzaldehyde was weighed into a graduated flask that was filled
with MTBE to give a 200 mm benzaldehyde solution. For “teabag”-
catalyzed reactions, the solution was 202 mm to compensate for
dilution by the addition of buffer as described below. For the use
of alginate immobilizates in free suspension, alginate beads
(
ii) investigation of ideal reaction parameters and operation
À1
(
250 mg, which correspond to 10.3 mgmL cell dry weight within
modes, (iii) simple catalyst recovery, exchange, and recycling,
and (iv) applicability to various scales and reactor types.
the reaction; see Supporting Information SI-7) were weighed into
mL glass vials. Benzaldehyde/MTBE solution (5 mL, 200 mm) was
added to start the reaction. If “teabags” were used (filled with
1.5 mg catalyst), benzaldehyde/MTBE solution (4.95 mL, 202 mm)
was added into 8 mL glass vials and mixed with TEA buffer
51.4 mL, 1m, pH 10.0). If freely suspended cells were used, lyophi-
8
5
Experimental Section
(
lized cells (51.5 mg) were put into the 8 mL glass vial and reaction
solution (5 mL) composed of benzaldehyde, MTBE, and buffer was
added to start the reaction as described for the use of catalytic
“teabags”. All reactions were incubated with overhead shaking
(30 rpm, 308C).
Chemicals
All chemicals were purchased in high chemical grade. Aldehydes
and benzoin were received from Fluka (Buchs, Switzerland), methyl
tert-butyl ether (MTBE) from Carl Roth (Karlsruhe, Germany), benzyl
alcohol from Sigma Aldrich (Munich, Germany), and alginic acid
sodium salt from AppliChem (Darmstadt, Germany). (R)-HPP and
[6,24]
PPD were synthesized in-house as described elsewhere.
Benzoin condensation on 140 mL scale by using the Spin-
Chem reactor
For alginate-bead-catalyzed reactions, each compartment of the
SpinChem (type S4530) was filled with beads (1.75 g in each com-
partment, in total corresponding to a dry catalyst load of
Preparation of lyophilized cells
À1
E. coli SG13009 cells that contained overexpressed BAL were
10.3 mgmL reaction volume). Substrate stock solution (benzalde-
lyophilized from a frozen cell pellet that was produced by fermen-
hyde in MTBE; 140 mL, 200 mm) was put into the prewarmed Spin-
Chem reactor (type R100), and the reaction was incubated while
stirring (500 rpm, 308C; IKA stirrer RW20.n). During the investiga-
tion of the impact of the stirring speed on the reaction rate, the
beads were recycled twice between the benzoin condensation re-
actions. To avoid product carryover, the immobilizate was washed
(35 min) in water-saturated MTBE after each reaction, and excess
MTBE was removed by spinning the chamber above the liquid for
[38,39]
tation as described elsewhere.
E. coli BL21 (DE3) cells that con-
tained RasADH were cultivated for 48 h in a shake flask at 208C in
an autoinduction medium according to a recipe published else-
[40]
where. DNA and amino acid sequences of both enzymes are
listed in Supporting Information SI-6. Cell pellets were lyophilized
for at least two days, and the lyophilized cells were stored as crude
powder at À208C.
ChemCatChem 2016, 8, 607 – 614
612
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