Efficient biocatalysis with immobilized enzymes or encapsulated whole cell microorganism by using the SpinChem reactor system

Article abstract of DOI:10.1002/cctc.201300599

The chambers of Trivial Pursuit: Locking immobilized enzymes or encapsulated whole cells into a compartment that is connected to the stirrer enables efficient reactions and facilitates the reuse of biocatalysts by using this SpinChem system. R-ATA=(R)-amine transaminase. Copyright

Full text of DOI:10.1002/cctc.201300599

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DOI: 10.1002/cctc.201300599
Efficient Biocatalysis with Immobilized Enzymes or
Encapsulated Whole Cell Microorganism by Using the
SpinChem Reactor System
Hendrik Mallin,[a] Jan Muschiol,[a] Emil Bystrçm,*[b] and Uwe T. Bornscheuer*[a]
Nowadays, biocatalysis is an es-
tablished method for the enzy-
matic synthesis of chiral building
blocks for organic compounds
and
pharmaceuticals,
com-
pounds for the flavor and fra-
grance industry, the production
of bulk chemicals, and the modi-
fication of lipids for the food
industry.[1]
Biocatalysis
has
become
highly competitive with classical
(asymmetric) chemical routes
that use transition-metal cata-
lysts, especially in combination
with new methods for enzyme
discovery and protein engineer-
ing,[2] as recently shown for the
synthesis of the drug Sitaglip-
tin.[3] The cost-effective applica-
tion of enzymes, in particular for
the synthesis of cheap products,
requires immobilization of the
Figure 1. Top: Schematic representation of the three reactor setups that were investigated. Bottom: Photograph
of the SpinChem device (reflux cooler and oxygen supply only for BVMO reaction).
biocatalyst (or the encapsulation of whole cells) to enhance
their long-term stability[4,5] and facilitate their reuse. At the
same time, immobilization of the biocatalyst should enable the
use of established reactor setups, such as fixed-bed reactors
(FBRs), instead of simple stirred-tank reactors (STRs, Fig-
ure 1).[1b,6] FBRs are used, for instance, for the large-scale pro-
duction of chiral amines[7] or emollient esters for the cosmetic
sector[8] by using lipase catalysts. However, several disadvan-
tages are encountered with FBRs, which depend on, for exam-
ple, the length, diameter, and particle size in the reactor, the
flow rate, the pressure drop within the column, and reactant
and pH gradients, as well as inactivation profiles after extend-
ed use. In contrast, the more operationally simple STR encoun-
ters mechanical challenges for the carrier, which results in
abrasion of the biocatalyst material and severe damage of en-
capsulated whole cells beside the fact that the recycling of the
immobilized biocatalyst is rather laborious.
Herein, we have investigated the use of an alternative setup
for the application of immobilized enzymes and encapsulated
whole cells. This SpinChem reactor (SCR; SpinChem is a regis-
tered trademark by Nordic ChemQuest AB, Umeꢀ, Sweden) en-
ables the simultaneous stirring and efficient percolation of
a liquid through packed particle beds, which is implemented
by a hollow stirring device that allows the solid reaction cham-
ber to be located inside the stirring element itself. The SCR can
be seen as an evolution of the standard basket reactor.[9,10] The
basket reactor, first published by Carberry in 1964, is a setup in
which four baskets rotate inside a well for gas/solid reactions.
This concept was later developed as the “annular spinning
basket reactor” by Mahoney et al. in 1978. However, in the
SpinChem reactor, the solid phase (such as an immobilized
enzyme) is present in the stirring element itself in up to four
separate compartments, which provides greater mixing and
flexibility compared to the basket reactors.
[a] H. Mallin,+ J. Muschiol,+ Prof. Dr. U. T. Bornscheuer
Institute of Biochemistry
Dept. of Biotechnology & Enzyme Catalysis
Greifswald University
Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany)
Fax: (+49)3834-86-794367
E-mail: uwe.bornscheuer@uni-greifswald.de
[b] Dr. E. Bystrçm
Nordic Chemquest AB
Box 7958, S-907 19 Umeꢀ (Sweden)
E-mail: emil.bystrom@nordicchemquest.com
[+] These authors contributed equally to this work.
Supporting information for this article, including all of the experimental
details, is available on the WWW under http://dx.doi.org/10.1002/
cctc.201300599.
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In this way, a variety of heterogeneous operations (catalysis,
solid-phase reactions, scavenging, etc.) can be performed in an
efficient and convenient fashion, because the material is con-
tained in the overhead stirring device and is not subject to me-
chanical wear or filtration problems. By rotating the SCR, the
liquid inside is “thrown out” through a centrifugal effect and
the new liquid will be drawn into the SCR from both the
bottom and the top (Figure 1). The main advantages of the
SpinChem system are easier downstream processing and
simple recycling of the biocatalyst, because the compartment
that contains the immobilized enzyme can be easily separated
from the bulk reaction solution.
Because Baeyer–Villiger monooxygenases work best at lower
substrate concentrations, only 1.96 gLÀ1 (0.02m) cyclohexa-
none was used for the CHMO-catalyzed reaction. To ensure an
optimal mass transfer, we first determined the optimal stirring
speed for the SCR, which was found to be 500 rpm for all
three reactions that were studied (the range 100–1000 rpm
was investigated; see the Supporting Information, Table S6).
In the transaminase-catalyzed kinetic resolution (Scheme 1,
Figure 2, and Table 1), the SCR and the STR gave the same con-
To verify the properties of the SCR compared to established
reactor systems for biocatalysis, we have investigated 1) the ki-
netic resolution of (R,S)-1-phenylethylamine by using an immo-
bilized (R)-transaminase from Gibberella zeae (GibZea)[11] and
2) the kinetic resolution of (R,S)-1-phenylethanol by using an
immobilized Candida antarctica lipase B (CAL-B,[12] Novozyme
435, N435) in n-hexane (Scheme 1).
Figure 2. Kinetic resolution of (R,S)-1-phenylethylamine to afford (S)-1-phe-
nylethylamine by using the immobilized GibZea (R)-transaminase.
versions after 6 h, whereas the FBR gave a 1.2-fold-lower con-
version. For the lipase-catalyzed kinetic resolution (Scheme 1,
Figure 3, and Table 1), almost-identical conversions (close to
50%) were determined after only 4 h, even at a substrate con-
centration of 1m. The production of e-caprolactone catalyzed
Table 1. Conversions that were achieved with three different reactor sys-
tems.
Scheme 1. Biocatalytic reactions that were studied by using the different re-
actor systems. R-ATA=(R)-amine transaminase, CHMO=cyclohexanone
monooxygenase.
Enzyme
Conversion [%]
STR
SCR
FBR
transaminase[a]
lipase[b]
37Æ8.0
45Æ1.0
36Æ6.1
37Æ11
46Æ1.0
35Æ6.0
30 Æ4.3
n.d.
4Æ0.2
Furthermore, 3) calcium-alginate-encapsulated Escherichia
coli whole cells that harbor the cyclohexanone monooxyge-
nase (CHMO) from Acinetobacter calcoaceticus NCIMB 9871[13]
were used for the production of e-caprolactone from cyclohex-
anone (Scheme 1). Stability has been a particularly challenging
issue for O2-consuming enzymes, which still has to be ad-
dressed. Furthermore, in FBRs, the O2 supply is a difficult issue
and, thus, an alternative reactor system is sought.
CHMO[c]
[a] After 6 h; [b] after 4 h; [c] after 24 h; n.d. =not determined.
by the CHMO also showed the same conversions (35%) after
24 h in both the SCR and the STR (Scheme 1, Figure 4, and
Table 1). In contrast, for the FBR, a significant nine-fold-lower
conversion was obtained. This dramatic slowdown could be
explained by the decreased oxygen supply in the column.
Thus, SCR and STR enable similar conversions for CHMO-,
lipase-, and transaminase-catalyzed reactions, thus indicating
that, for these reactors, mass transfer is not a limiting issue.
Next, reuse and downstream processing were studied in the
SCR and the STR under identical conditions. In the case of the
SCR, this study was simply performed by taking the stirrer out
of the reactor and washing it three times in small beakers
The reactions in the SCR and the STR were performed with
a volume of 0.5 L in a New Brunswick BioFlo 110 Fermentor/
Bioreactor (total volume: 0.9 L). In the FBR reactions a reservoir
with a volume of 0.5 L was used. In all three setups, we used
identical amounts of enzyme (based on units of activity; for
details, see the Supporting Information). For the lipase and
transaminase reactions, we operated at high substrate concen-
trations of 122.17 gLÀ1 (1m) and 16.12 gLÀ1 (0.133m), respec-
tively.
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Figure 3. Transesterification of (R,S)-1-phenylethanol into the corresponding
(R)-acetate by using immobilized lipase CAL-B in the SCR and the STR.
Figure 5. Recycling study with immobilized GibZea (R)-transaminase (2 h per
batch) in the SCR and the STR; 100% relative activity refers to 19% conver-
sion of (R)-1-phenylethylamine in the first batch.
Figure 4. Formation of e-caprolactone by using alginate-encapsulated rest-
ing E. coli cells that harbored a CHMO.
under stirring for 30 s. Then, the biocatalysts that were present
in the SpinChem compartment were ready to use in the next
cycle. In the STR, the reaction solution was first filtered, fol-
lowed by washing the immobilized catalyst before the next
cycle was started. These recycling studies revealed that, for the
transaminase reaction, the SpinChem system was superior:
93% relative activity was recovered in the SCR compared to
only 62% in the STR (Figure 5).
Figure 6. Recycling study with immobilized lipase CAL-B (2 h per batch) in
the SCR and the STR; 100% relative activity refers to 33% (SCR) and 39%
conversions (STR) of (R,S)-1-phenylethanol in the first batch.
between the fourth and fifth cycles, the encapsulated cells
were stored overnight at 48C (Figure 7). The cells in the SCR
showed no significant loss of activity, whereas, in contrast, only
half of the relative activity was determined for the cells in the
STR. In addition, for successful recycling of the alginate cap-
sules, the presence of 10 mm CaCl2 in the reaction and wash-
ing solution was necessary. Without this additive, the beads
completely dissolved after the second cycle, owing to removal
of the Ca2+ ions from the calcium-alginate complex (data not
shown).
This result suggested that the immobilized transaminase
was better protected from mechanical forces in the SpinChem
device. In the lipase-catalyzed kinetic resolution, the SCR was
slightly superior until the fourth cycles; after the sixth cycle,
both systems gave similar conversions (Figure 6).
Notably, CAL-B is a highly robust lipase and, hence, losses in
activity are difficult to observe because the immobilized bio-
catalyst is typically stable for several months under process
conditions. Furthermore, this initial decrease in activity could
be caused by accumulation of the reaction compounds on the
carrier or in the enzyme environment. In the oxidation reac-
tions that were catalyzed by CHMO whole cells, the SCR was
clearly superior and showed 41% residual activity after six
cycles (versus 14% relative activity in the STR). Furthermore,
In conclusion, we have developed an alternative reactor con-
cept for the use of immobilized enzymes or whole cells in bio-
catalysis. The SCR was shown to be equivalent to—or even su-
perior to—conventional setups. In particular, recycling experi-
ments in consecutive batch reactions are facilitated and the ac-
tivity loss was reduced in the SCR. The SCR does not require
special laboratory equipment because, in essence, only the stir-
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Acknowledgements
We thank the “Bundesministerium fꢁr Bildung und Forschung” for
financial support within the “Biokatalyse 2021” cluster (FKZ:
0315175A) and the “Deutsche Forschungsgemeinschaft” (Bo1862/
6-1) for financial support. We are also grateful to the mechanical
workshop at Greifswald University for their support in construct-
ing the reactor setups.
Please note: Minor changes have been made to this manuscript since its
publication in ChemCatChem. The Editor.
Keywords: biocatalysis · enzymes · immobilization · kinetic
resolution · microreactors
Received: July 23, 2013
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Zelinski, Angew. Chem. 2004, 116, 806–843; Angew. Chem. Int. Ed. 2004,
Published online on October 11, 2013
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