Immobilized whole cells as effective catalysts for chiral alcohol production

Article abstract of DOI:10.1071/CH09112

Recombinant Escherichia coli overexpressing the gene LbADH, which encodes for an alcohol dehydrogenase from Lactobacillus brevis, was successfully transformed and cultured. The cells are able to catalyze the reduction of pro-chiral ketones, e.g. ethyl acetoacetate into R-()ethyl hydroxybutyrate (EHB) with high conversion and enantiomeric excess 99%. Immobilizing the whole cells in alginate beads leads to a catalyst with improved stability and ease of handling while maintaining the high activity of the free cells. The whole-cell catalyst was tested in a stirred batch reactor (CSTR) and in a continuously operated packed-bed reactor. An Mg²⁺ concentration of 2 mM was crucial for maintaining the activity of the biocatalyst. After a partial optimization of the process conditions, a productivity of 1.4 g_EHB g_wcw^-1 h^-1 could be maintained in a continuous flow reactor over a prolonged period of time. CSIRO 2009.

Full text of DOI:10.1071/CH09112

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2009, 62, 1034–1039
www.publish.csiro.au/journals/ajc
Immobilized Whole Cells as Effective Catalysts
for Chiral Alcohol Production
A
A,B
Jeck Fei Ng and Stephan Jaenicke
ADepartment of Chemistry, National University of Singapore, 3 Science Drive 3,
Singapore 117543, Republic of Singapore.
B
Corresponding author. Email: chmsj@nus.edu.sg
Recombinant Escherichia coli overexpressing the gene LbADH, which encodes for an alcohol dehydrogenase from Lacto-
bacillus brevis, was successfully transformed and cultured. The cells are able to catalyze the reduction of pro-chiral
ketones, e.g. ethyl acetoacetate into R-(−)ethyl hydroxybutyrate (EHB) with high conversion and enantiomeric excess
>99%. Immobilizing the whole cells in alginate beads leads to a catalyst with improved stability and ease of handling
while maintaining the high activity of the free cells. The whole-cell catalyst was tested in a stirred batch reactor (CSTR)
and in a continuously operated packed-bed reactor. An Mg2 concentration of 2 mM was crucial for maintaining the
+
−
1
−1
h could
wcw
activity of the biocatalyst. After a partial optimization of the process conditions, a productivity of 1.4 gEHB g
be maintained in a continuous flow reactor over a prolonged period of time.
Manuscript received: 24 February 2009.
Manuscript accepted: 18 June 2009.
Introduction
S)-alcohols in a process characterized by Prelog’s rule.[8] How-
(
Most biologically active compounds including many pharma-
ceuticals have chiral molecular structures with one or more
stereogenic centres. Because of safety, therapeutic, and regu-
latory concerns, it is necessary to evaluate and test every drug in
ever, enzymes isolated from the Lactobacillus strains L. kefir
or L. brevis catalyze the formation of (R)-alcohols from a wide
range of substrates in good yields and high enantiomeric excess
(ee) (94–99%).[
9,10]
[1]
their enantiomerically pure forms. The ready access to chiral
synthons is therefore very important. Optically active secondary
alcohols are widely used by the pharmaceutical industry for
the introduction of chirality.[ Routes to single enantiomers
of small molecules can be classified into four groups: chiral
pool synthesis, resolution, biological, and chemical asymmetric
Free enzymes have disadvantages that make them less suit-
able for technical applications, notably insufficient long-term
stability, and limited substrate acceptance.[ When isolated
enzymes are used for oxidation or reduction reactions, the neces-
sity to provide the required coenzymes constitutes a major
drawback. The cost for stoichiometric amounts of (reduced)
nicotinamide adenine dinucleotide (NADH) and nicotinamide
adenine dinucleotide phosphate (NADPH) is prohibitive, but
even when the cofactors are regenerated, the required processes
are complex, proving a significant economic obstacle against
using ADH for commercial syntheses.[
11]
2]
[
3]
techniques. Asymmetric reduction of the corresponding pro-
chiral ketones proved to be one of the most effective and promis-
ing routes to chiral alcohols.[ The environmentally friendly
biocatalytic approach using oxidoreductases is attractive com-
pared with chemical methods involving transition metal catalysts
and organic solvents. The biocatalyst, used either as the isolated
enzyme or as the whole cell, catalyzes the reduction of pro-chiral
4]
12]
Cofactor regeneration can be achieved in different ways, such
as coupled enzyme,[
13,14]
coupled substrate,
[15]
as well as by
[5]
[16]
ketones with remarkable chemo-, regio-, and stereoselectivity.
electro-enzymatic, photochemical, and chemical approaches.
Whole cell fermentation offers an alternative to the use of iso-
lated enzymes. The whole cell biocatalyst acts as a mini-reactor
with all the necessary cofactors and sequences of enzymes avail-
able within one cell. The same enzyme, ADH, catalyzes the
reduction of the substrate and the oxidation of the cosubstrate
isopropanol to regenerate the NAD(P)H as shown in Fig. 1.[
In addition, isopropanol acts as the cosolvent and increases the
solubility of the organic substrate in the aqueous phase in which
the biocatalytic reaction is carried out.
Functional group protection/deprotection strategies are usually
not necessary in reactions catalyzed by enzymes, in contrast
to chemical catalysts. Moreover, biocatalytic reductions can be
carried out at ambient temperature and atmospheric pressure.[
Thus, biotransformations can simplify manufacturing processes,
making them economically more attractive and at the same time
more environmentally acceptable.[7]
6]
17]
Alcohol dehydrogenases (ADHs) catalyze the reversible
reduction of functional groups such as ketones and ketoesters to
chiral alcohols, and provide access to either the (R) or (S)-form,
depending on the enzyme used.The most commonly usedADHs
are those available from yeast, horse liver, and Thermoanaero-
bium brockii. All these enzymes reduce the ketone by trans-
ferring the pro-R hydrogen to the re-face of substrate forming
Furthermore, the enzymes are more stable within the cell
where they are in their natural environment.[ The wild forms
of microorganisms frequently contain several enzymes that can
act on the same substrate, usually with different stereoselectiv-
ity. This compromises the ee. In a study by Ribeiro et al. on
18]
©
CSIRO 2009
10.1071/CH09112
0004-9425/09/091034

RESEARCH FRONT
Immobilized Whole Cells as Effective Catalysts
1035
Cell membrane
100
O
HO H
ADH
ADH
8
0
R1
R2
R1
R2
ϩ
NAD(P)H
NAD(P)
Acetone
Isopropanol
co-substrate)
60
(
4
2
0
0
0
Fig. 1. Whole-cell reduction: substrate and cosubstrate enter the cell where
the enzymatic reduction takes place under regeneration of the cofactor
NAD(P)H.
0 mg mLϪ1
0 mg mLϪ1
60 mg mLϪ1
80 mg mLϪ1
100 mg mLϪ1
2
4
the reduction of ethylacetoacetate by different wild type micro-
organisms, the highest ee was 81% with a Hansenula sp.[19]
This situation improved dramatically with the development of
‘
designer bugs’ in which only those enzymes specific for the
[20]
desired biotransformation are overexpressed.
0
40
80
120
160
200
Continuously operated processes are favoured on an indus-
trial scale because they offer higher volumetric productivity and
avoid the setup times associated with batch reactions. However,
less than a quarter of all biotechnological processes currently
conducted on an industrial scale are operated in a continu-
Time [min]
Fig. 2. Effect of cell loading on the bioreduction of ethyl acetoacetate.
[
21]
ous mode.
Schroer et al. demonstrated the first continuously
operated biotransformation process catalyzed by recombinant
Escherichia coli using a membrane reactor.[ By applying the
coupled substrate approach, these authors successfully produced
the reaction rate. However, 3% and 4% sodium alginate solu-
tions are more viscous, making the extrusion of the solution into
the hardening bath more difficult. 2% Sodium alginate solution
was preferred over 1% for further studies because of the higher
mechanical strength which increases the potential for reusability.
17]
(
R)-methyl-3-hydroxybutyrate at a maximum space time yield
−1
−1
of 700 g L per day.They used 50 gwcw L biomass in a 0.05 L
reactor and retained the cells by an ultrafiltration membrane with
a molecular weight cutoff of 10 kDa. However, problems such as
membrane failure are common for membrane reactors, and the
higher cost of the ultrafiltration membrane adds to the cost of
production. Immobilization of the biocatalyst in a porous carrier
allows for an effective continuous production of chiral alcohols
at a much lower biomass concentration. Immobilization of the
cells on a support is promising for cell reuse, because it elim-
inates the costly processes of cell recovery by centrifugation
or cross-flow filtration, and moreover, offers stability advan-
Cell Concentration
The bioreduction of EAA was investigated as a function of the
cell loading within the 2% sodium alginate beads. The result
is shown in Fig. 2. A higher concentration of cells resulted in
a lower conversion of EAA. Under these conditions, the cells
agglomerate, and this reduces the surface area available for the
bioreduction. Only cells near the surface take part in the reaction,
whereas cells deeper inside the beads cannot be reached by the
substrate. Smaller pores might also lead to additional diffusion
limitations.
tages over free cells.[22,23]
The principal methods for whole
cell immobilization are adsorption, covalent binding, encapsu-
lation, entrapment, crosslinking, or combinations thereof.[
24–26]
The successful immobilization of a variety of cells in different
Influence of the Cations for the Hardening Bath
matrices has been reported.[
27–29]
The present study describes
Itisknownthattheaffinityofalginatesforalkalimetalsincreases
the optimization of the immobilization of recombinant E. coli
in calcium alginate beads and the reaction conditions for the
chiral reduction of various β-ketoesters to the corresponding
alcohol, in particular the reduction of ethylacetoacetate (EAA)
to (R)-ethyl-3-hydroxybutyrate (EHB).
in the order Mg2 << Ca < Sr < Ba
. Calcium and
2+ [30]
+
2+
2+
barium ions were chosen as the divalent ions in the hardening
bath to study the effect on the mechanical strength of the beads.
The result is shown in Fig. 3. Both the calcium alginate and
the barium alginate immobilized biocatalyst gave similar results
◦
when tested over 15 cycles at 37 C. Calcium alginate dissolves
Results and Discussion
in sodium citrate solution, with the citrate acting as a chelating
Concentration of Sodium Alginate
2+
agent for Ca ions. However, barium alginate is tough enough
Different concentrations (1%, 2%, 3%, and 4%) of sodium
alginate were used to prepare the immobilized biocatalyst. The
conversion of EAA to EHB was independent of the concentra-
tion of sodium alginate. With all of the immobilized biocatalysts,
to remain in bead shape after stirring in citrate buffer for 24 h.
However, the increased robustness of the barium alginate beads
did not lead to a better performance during the recycling, and
in both cases, the biocatalysts lost ∼25% of their initial activ-
ity after 15 cycles of re-use. It might be that cell leaching only
occurred within the beads’ outer surfaces, or the decrease of
activity is simply due to the deactivation of the biocatalyst.
∼98% conversion of 100 mM EAA could be achieved within
h reaction time. Because EAA is a rather small molecule, dif-
fusion through the alginate network does not noticeably affect
3

RESEARCH FRONT
1
036
J. F. Ng and S. Jaenicke
100
100
8
6
4
2
0
0
0
0
0
8
6
4
2
0
0
0
0
0
Free cell
Calcium alginate
Barium alginate
Calcium alginate
0
2
4
6
8
10
12
14
16
0
2
4
6
8
10
12
14
16
Cycle
Cycle
Fig. 5. Process stability for free and immobilized cells during the contin-
uous reduction of ethyl acetoacetate in a batch-wise reaction.
Fig. 3. Effect of divalent ions used in the hardening bath.
00
1
Table 1. Experimental details for the continuous bioreduction of ethyl
acetoacetate (EAA)
8
6
4
2
0
0
0
0
0
Tubular reactor
Reactor volume
Flow rate
Residence time (τ)
EAA:IPA
16 mm ID × 480 mm
0.096 L
−
1
0.032 L h
3 h
1:2
◦
Temperature
Product monitoring
37 C
pH 6
pH 7
Optical rotation (Perkin–Elmer 341) and GC
Agilent with capillary column HP-5)
(
0
1
2
3
4
5
6
Cycle
recycling experiment, and it was found that it had decreased sig-
nificantly, from 0.10 g to 0.018 g. In contrast, ∼78% of the cells
still remained entrapped in the calcium alginate after 14 cycles.
Additionally, the immobilized biocatalyst showed a higher activ-
Fig. 4. Effect of pH during the immobilization of the cells in calcium
alginate.
−1
ity as expressed in the productivity of 24.7 mmolEHB g cell
−
1
Effect of pH in the Preparation of Calcium Alginate
Immobilized Cells
compared with only 10.6 mmolEHB g cell for the free cells.
The best performance was obtained in solution buffered at near
neutral pH. The optimum pH was further fine tuned in the prepa-
ration of calcium alginate immobilized cells.Acetate buffer pH 6
andTRIS-HCl buffer pH 7 were used as the buffers in dissolving
sodium alginate and preparing the cell slurry. The bioreduction
was carried out in buffered solutions with the respective pH at
Continuous Reduction of EAA in a Plug-Flow Reactor
The bioreduction with calcium alginate immobilized recombi-
nant E. coli overexpressing LbADH was carried out under the
operational condition shown in Table 1.
To establish the optimum concentration of EAA for highest
productivity, EAA concentrations from 0.3 M to 1 M were fed
◦
3
7 C. Fig. 4 shows the results. Initially, cells immobilized at pH
−1
into the reactor. As shown in Table 2, 0.7 M of EAA (91 g L
)
6
and pH 7 gave similar performance; however, the activity of
is the optimum concentration at 3 h retention time in the reac-
tor. Higher concentrations may lead to substrate inactivation of
the enzyme, or to physiological changes in the cell wall due to
the high content of organic solvents in the medium. The maxi-
cells immobilized at pH 7 decreased more rapidly than that of
cells immobilized at pH 6 and was lower by ∼20% for the 6th
cycle of re-use. Slightly acidic conditions were thus identified
as optimal for the bioreduction with whole E. coli cells.
−1
−1
mal productivity was 1.4 gEHB g
h
at a conversion of 81%.
wcw
However, the activity of the catalyst started to decrease grad-
ually after 18 h. Further optimization of the reaction medium
Reusability
was carried out. When 2 mM Mg2 was added to the substrate
solution, the reactor could be operated for 30 h before eventu-
ally the activity of the catalyst gradually decreased as shown
+
The stability and the reusability of the biocatalyst were stud-
ied and the results are shown in Fig. 5. The conversion of EAA
to EHB decreased drastically after the free cells had been re-
cycled six times, and less than 5% of the initial activity remained
after the 14th run. However, the cells immobilized in calcium
alginate demonstrated excellent stability over many cycles, and
the conversion of the EAA was more than 70%, even after 15
cycles. Part of the drop in activity of the free cells may be
caused by inefficient recovery of the biocatalyst by centrifuga-
tion after the reaction.The cell mass was checked at the end of the
in Fig. 6. Mg2 is present in the active site of LbADH, and it is
+
2+
known that the binding of Mg causes structural changes which
activate the enzyme.[ The effect is element specific, and no
31]
2+
positive effect on the activity was observed when Ca instead of
2+
Mg was co-fed with the substrate solution. The performance
of the continuous process under optimized condition is shown
in Table 3.

RESEARCH FRONT
Immobilized Whole Cells as Effective Catalysts
1037
Table 2. Optimization of the substrate concentration
Conclusions
Whole recombinant E. coli cells overexpressing LbADH were
successfully immobilized using alginate as immobilization
matrix. Cells immobilized by the optimized protocol show a
better stability than the free cells, so that the activity of the bio-
catalyst is maintained over more cycles. The recombinant E. coli
shows excellent activity for the reduction of several ketoesters
to the corresponding chiral alcohols with high conversion and
ee. Immobilization does not affect the activity of the cells for
the catalytic reduction. The continuous bioreduction of EAA
in a plug flow reactor using the immobilized cells is a simple
and economic approach, and we demonstrated a productivity of
Wet cell EAA concentration Flow rate Maximum
Productivity
−
1
−1
−1
−1
h ]
wcw
mass [g]
[mol L
]
[L h
]
conversion [gEHB g
1
1
1
1
.67
.67
.67
.98
0.3
0.5
0.7
1.0
0.034
0.032
0.032
0.026
98%
97%
81%
70%
0.8
1.2
1.4
1.2
1
1
.4
.2
1
−
1
−1
h with low biomass concentration.
wcw
1.4 gEHB g
Experimental
Transformation of E. coli with Plasmids Bearing
the Gene Encoding for LbADH
0
0
0
0
.8
.6
.4
.2
0
Plasmid pBtac-Lbadh (X-zyme, Düsseldorf, Germany) bearing
the adh gene from Lactobacillus brevis as well as an ampicillin
resistance gene was employed as expression vector for the E.
coli BL21 Star (DE3) (Invitrogen, Carlsberg, USA) host cells.
Competent E. coli cells were transformed by adding 5 µL of the
plasmid solution into an Eppendorf tube containing the E. coli in
0
10
20
30
40
50
◦
a buffer.The cells were incubated for 20 min at 4 C to induce the
Time [h]
◦
uptake of plasmids. The cells were then heated in a 42 C water
bath for 90 s to inactivate the nucleases, and then cooled to 4 C
◦
Fig. 6. Stability of the continuous bioreduction of ethyl acetoacetate. (ꢀ),
2
+
2+
again for 5 min before 400 µL of Hanahan’s SOC medium was
2
mM Mg ; (ꢁ), 2 mM Ca ; (◦), without divalent ions. EHB, R-(−)ethyl
added.[ The cells were then incubated at 37 C for 1 h with
32]
◦
hydroxybutyrate.
light shaking to allow the cells to repair the cell membranes.
2
5 µL of cell suspension was streaked on a Luria-Bertani (LB)-
Table 3. Optimized reaction conditions for continuous production of
−1
agar plate containing 100 mg L of ampicillin and the plates
were incubated overnight at 37 C. The ampicillin present in the
R-(−)ethyl hydroxybutyrate
◦
LB agar selects for cells that were successfully transformed.
A colony of transformed E. coli was selected randomly from a
LB agar plate and used to inoculate 100 mL LB medium con-
Ethyl acetoacetate concentration
Conversion
Biomass concentration
Space time yield
Productivity
Enantiomeric excess
0.7 M
0.81
17 gwcw L
25 g L
1.4 gEHB g
>99%
−
1
−1
taining 100 mg L ampicillin in a 250 mL Erlenmeyer flask.
−
1
−1
1
h
◦
This preculture was incubated overnight at 30 C with stirring at
− −1
h
wcw
150 rpm. Aliquots of this preculture were used for subsequent
cell cultivation.
Cell Cultivation
0
.5 mL of the preculture was further cultured in 500 mL ‘terrific
Bioreduction of Other Ketoesters and Acetophenone
−
1
◦
broth’medium containing 200 mg L of ampicillin at 37 C and
00 rpm. Antifoam (Anti-foam 204, Sigma) in ppm level was
Recombinant E. coli overexpressing an ADH of L. brevis is able
to convert several ketoesters to the corresponding chiral alco-
hols with good ee (Table 4). However, the branched molecule,
methyl 4-methyl-3-oxopentanoate, was not accepted as substrate
3
added to reduce foaming in the aerated fermentation. Culturing
was continued until the OD660 reached to ∼0.6–0.8 which took
∼
6 h. At this point, isopropylthiogalactoside (IPTG) was added
(
entry 5). This is most likely caused by the more bulky residue
to a final concentration of 0.2 mM to induce gene expression.
Culturing was continued for a total of 24 h. The cells were har-
attached to the carbonyl group of this ketoester. The activity of
the cells for the reduction of simple ketones was also investi-
gated. Acetophenone is reduced at a much lower rate compared
withtheotherketoesters.ThisconfirmstheobservationbyWeck-
becker et al., who reported that the activity of LbADH towards
EAA was 4.36 relative to that of acetophenone which was taken
◦
vested by centrifugation (2860g, 20 min) and were stored at 4 C
as a 50 g L cell suspension in 50 mM potassium phosphate
buffer pH 6.0.
−
1
[
11]
Immobilization of Recombinant E. coli in Calcium
Alginate Gel
as 1. In all cases, the recombinant cells produce alcohols with
the same absolute configuration at the optically active centre.
This is generally the (R)-form, except for the case of methyl 4-
chloro-3-oxobutanoate where the product is (S) as a consequence
of the Cahn-Ingold-Prelog nomenclature rules. The immobi-
lized biocatalyst performed generally as well as the free cells
in terms of conversion and stereoselectivity, and no problems
due to diffusion limitations within the beads were observed.
2.0 g alginic acid sodium salt from brown algae (Fluka) was
heated in 80 mL of 50 mM acetate buffer pH 6 to give a clear
solution. The alginate solution was cooled to room temperature
with stirring. 2.0 g of wet cells was washed once with 50 mM
acetate buffer pH6 and resuspended in 20 mL of the same buffer
and added to the alginate solution to give a final concentration

RESEARCH FRONT
1
038
J. F. Ng and S. Jaenicke
Table 4. Different substrates for reduction by recombinant Escherichia coli cells
Substrate Conversion % immobilized free cells Enantiomeric excess %
Entry
1
O
O
97.5
98.3
93.7
97.3
98.4
94.1
>99 (R)
>99 (R)
>99 (R)
>99 (S)
O
Methyl 3-oxobutanoate
O
O
2
3
4
O
Ethyl 3-oxobutanoate
O
O
O
Methyl 3-oxopentanoate
O
O
Cl
Ethyl 4-chloro-3-oxobutanoate
100
100
O
O
O
5
6
O
–
–
–
Methyl 4-methyl-3-oxopentanoate
O
54.9
54.9
>99 (R)
1
-Phenylethanone
acetophenone)
(
−
2
% (w/v) sodium alginate solution containing 20 mg mL 1 of
cells. The homogeneous mixture was extruded drop-wise into
00 mL of 0.1 M calcium chloride solution via a BD spinal
syringe needle 20GA 3.5 IN by using a syringe pump at a flow
cloudy cell suspension. The optical density of the suspension
was measured at 660 nm. The cell density corresponding to the
OD was determined from a calibration curve.
2
−1
rate of 2.0 mL min . The calcium chloride hardening bath was
stirred at 100 rpm during the addition. After finishing the addi-
tion, the formed beads with immobilized cells were kept stirring
for another hour to ensure complete hardening. After that, the
beads were filtered off and washed with 100 mL of 0.1 M cal-
cium chloride solution. They were then resuspended in 100 mL
Batch Bioreduction of EAA
For bioreductions with free cells, 0.1 g of cell pellet was weighed
in a conical flask and suspended in 25 mL of 50 mM potassium
phosphate buffer pH 6.0. For immobilized cells, the beads were
washed once with 50 mM acetate buffer pH 6. Beads containing
0.1 gofcells(basedoncellloading)wereweighedandsuspended
◦
of the same solution and kept at 4 C overnight for complete cur-
ing. This procedure yields uniform calcium alginate beads with
in 25 mL of the same buffer. For both cases, ethyl acetoacetate
and isopropanol were added to the buffer to a final concentration
a diameter of ∼3 mm.
of 100 mM and 200 mM, respectively. The flask was shaken at
◦
1
20 rpm at 37 C for 3 h. The cells were separated from the reac-
Effect of Different pH (Preparation of Immobilized Cell)
tion mixture by filtration (immobilized cells) or centrifugation
(free cells). The remaining reaction mixture was extracted thrice
with an equal volume of diethyl ether. The solvent was evapo-
rated and the ee of the product was determined by GC using a
Supelco BetaDex 325 chiral column.
Two different buffers were used in dissolving the sodium alginate
and suspending the cells, namely a 50 mM acetate buffer for pH
and a 50 mMTris buffer for pH 7.The procedure was otherwise
the same as for the preparation of calcium alginate immobilized
cells described above.
6
Acknowledgement
Determination of Cell Loading
This work was funded by NUS research grant R-143–000–374–112. We
are grateful to Professor Christian Wandrey and his research group (Jülich,
Germany) for the gift of the plasmid, and for initiating us to the know-how
of recombinant technology. The help of Miss Ida Wang, exchange student
from the University of British Columbia, Vancouver, Canada, in the early
part of this project is gratefully acknowledged.
The calcium alginate beads were dissolved as described by Xue
[
33]
et al.
A known amount of immobilized E. coli (10 beads)
was weighed and added to a 0.06 M trisodium citrate solution
pH 7.8–8.2 in a 5 mL volumetric flask. The beads were stirred
at 200 rpm until they were completely dissolved and yielded a

RESEARCH FRONT
Immobilized Whole Cells as Effective Catalysts
1039
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