Immobilization of benzaldehyde lyase and its application as a heterogeneous catalyst in the continuous synthesis of a chiral 2-hydroxy ketone

Article abstract of DOI:10.1016/j.tetasy.2004.07.039

Hexahistidine-tagged benzaldehyde lyase from E. coli SG13009/BAL _HIS was immobilized by means of metal ion affinity binding to a nickel(II)-nitrilotriacetic acid derivatized carrier and applied as a heterogeneous biocatalyst in the synthesis of (R)-2-hydroxy-1-phenyl-propanone. The applicability of the immobilization by metal ion affinity binding was proven in repetitive batch reactions and in a continuously operated plug flow reactor.

Full text of DOI:10.1016/j.tetasy.2004.07.039

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2955–2958
Immobilization of benzaldehyde lyase and its application as
a heterogeneous catalyst in the continuous synthesis of a chiral
2-hydroxy ketone
Nils Kurlemann and Andreas Liese*
Institute of Biochemistry, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, 48149 Mu¨nster, Germany
Received 12 May 2004; accepted 1 July 2004
Available online 11 September 2004
Abstract—Hexahistidine-tagged benzaldehyde lyase from E. coli SG13009/BALHIS was immobilized by means of metal ion affinity
binding to a nickel(II)–nitrilotriacetic acid derivatized carrier and applied as a heterogeneous biocatalyst in the synthesis of (R)-2-
hydroxy-1-phenyl-propanone. The applicability of the immobilization by metal ion affinity binding was proven in repetitive batch
reactions and in a continuously operated plug flow reactor.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
O
O
O
BAL-Ni-NTA
H
+
+ ThDP/Mg2+
buffer/DMSO
Enantiopure 2-hydroxy ketones are important building
blocks in the synthesis of biologically active compounds.
Both (R)-2-hydroxy-l-phenyl-propanone 4 [(R)-HPP,
Scheme 1] and its derivatives are applied as pharmaceu-
tical intermediates.1,2 Chemical routes towards hydroxy
ketones include the stereoselective oxidation of enol
H
OH
2
1
3
O
BAL-Ni-NTA
+ ThDP/Mg2+
buffer/DMSO
3
ethers with OsO4 and the regio- and enantioselective
OH
hydrogenation of 1,2-diketones.4 The 2-hydroxy ketones
are also synthesized by other biocatalytic methods, such
as the oxidation of 1,2-diols into 2-hydroxy ketones by
glycerol dehydrogenase5 and the microbial reduction
of 1,2-diketones.6 An efficient approach is the stereo-
selective condensation of aldehydes catalyzed by the
thiamine diphosphate (ThDP)-dependent enzymes benz-
aldehyde lyase and benzoyl formate decarboxylase
yielding enantio-complementary 2-hydroxy ketones.7,8
Benzaldehyde lyase (BAL, EC 4.3.2.38) from Pseudo-
monas fluorescens Biovar 1 is able to catalyze the enan-
tioselective C–C-bond formation between benzaldehyde
1 and acetaldehyde 2, leading to (R)-2-hydroxy-l-phe-
nyl-propanone 4 with an ee greater than 99%.9,10 Benz-
oin 3 is formed as an intermediate in low concentrations.
4
Scheme 1. Enantioselective synthesis of (R)-2-hydroxy-1-phenyl-prop-
anone with histidine-tagged benzaldeyde lyase (BAL–Ni–NTA) immo-
bilized on Ni–NTA–sepharose as a heterogeneous catalyst.
uous operation modes, the catalyst must be retained in-
side the reactor. This can be achieved by immobilizing
soluble catalysts, that is, enzymes on a solid phase or
by membrane retention.11,12 In general, the immobiliza-
tion of enzymes possesses many advantages such as sta-
bilization, reusability of the enzymes and simplified
isolation of the product.13 One approach to controlled
immobilization is to attach a tag––a peptide sequence
with an affinity to a material––to the N- or C-terminus
of a protein and take advantage of the specific interac-
tion of the tag with a surface bound group.14 Ideally
the catalyst is reversibly bound to the solid phase. This
enables the removal of the exhausted catalyst from the
carrier without having to dismantle the reaction vessel,
that is, a chromatography column. The solid carrier is
To improve its cost effectiveness a catalyst must be
recovered after a discontinuous reaction. During contin-
*
Corresponding author. Tel.: +49 0251 83 33042; fax: +49 0251 83
32122; e-mail: aliese@uni-muenster.de
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.07.039

2956
N. Kurlemann, A. Liese / Tetrahedron: Asymmetry 15 (2004) 2955–2958
kept inside the reactor while replacing the catalyst,
which considerably reduces set-up times. This should
be advantageous in fields requiring high-priced compo-
nents such as in the case of micro structured reactor sys-
tems: a reversible linking of enzymes can be achieved by
site. The immobilization of the enzyme on the Ni–
NTA was almost quantitative. In the supernatant, a
residual fraction of 1% protein was detected.
For continuous operation of a reactor containing an
immobilized catalyst, the binding must be strong enough
to avoid wash out of the catalyst. To ensure this, no
other compound with heteroatoms in the reaction mix-
ture should bind to the Ni–NTA carrier. A binding of
neither HPP nor benzaldehyde to the Ni–NTA resin
could be detected. On incubation with 5mM thiamine
diphosphate (a 10-fold excess compared to the reaction
conditions), a protein concentration of 2% was observed
in the supernatant. This points to an unspecific binding
of ThDP to Ni–NTA resulting in a desorption of a small
amount of enzyme.
complex formation between metal ions, that is, Ni2+
,
Cu2+ or Ti2+ and the functional groups of certain amino
acids like histidine and arginine.15 The hexahistidine-tag
(His6-tag) interacts with the vacant coordination posi-
tions of nickel or copper chelates. Two neighbouring
histidine amino acids attach strongly and reversibly to
nickel(II)-ions,16 which are bound to nitrilotriacetic acid
(NTA)-derivatized agarose resin.
The His6-tag was designed for protein purification
through chromatography using Ni–NTA agarose beads
as a packed bed. The tag is widely used in this field but
also offers the possibility of employing the carrier bound
enzymes as immobilized catalysts. In the field of carbo-
hydrate chemistry, a few applications involving multiple
enzymes immobilized on Ni–NTA agarose beads acting
as heterogeneous biocatalysts have been reported.17,18
To compare the ligase activities of immobilized BAL
and homogeneously dissolved BAL, the biotransforma-
tion of acetaldehyde and benzaldehyde to (R)-HPP was
performed with dissolved BAL and Ni–NTA bound
BAL in batch reactors (Fig. 1).
Benzaldehyde lyase with a hexahistidine tag was bound
to Ni–NTA–Superflow (highly cross-linked agarose,
Qiagen, Germany). The synthesis of (R)-2-hydroxy-l-
phenyl-propanone 4 starting from benzaldehyde 1 and
acetaldehyde 2 was carried out with immobilized benzal-
dehyde lyase in repetitive batch reactions and in a con-
tinuously operated plug flow reactor. To the best of
our knowledge, this is the first example of an Ni–NTA
immobilized enzyme being applied in the enantioselec-
tive C–C bond formation.
100
80
batch with dissolved BAL
60
batch with BAL-Ni-NTA (1)
repetitive batch with
BAL-Ni-NTA (2)
40
20
0
0
1
2
3
4
2. Results and discussion
t / h
After purification,19 the recombinant His6-tagged en-
zyme benzaldehyde lyase was immobilized on Ni–
NTA–Superflow20 (Scheme 2).
Figure 1. Repetitive batch synthesis of (R)-2-hydroxy-l-phenyl-prop-
anone with immobilized benzaldehyde lyase (BAL–Ni–NTA) in KPG-
stirred tank reactors (10mg BAL [(1) and (2): 10mg BAL on 1mL
Ni–NTA–Superflow], 50mM triethanolamine buffer, pH= 8, 0.5mM
ThDP, 0.5mM MgSO4, 30vol% DMSO, 10mM benzaldehyde, 60mM
acetaldehyde).
HO
(HIS)a
O
-O
Ni2+
O
N
O-
N-
N-
O
NH
The levels of conversion reached in the first batch reac-
tion involving immobilized BAL (1) are comparable to
those of the experiment applying dissolved enzyme. This
demonstrates that the immobilization process does not
alter the enzyme activity noticeably. The decrease in
conversion observed during the second batch reaction
with immobilized BAL (2) is probably due to material
losses, which occurred during filtration and recovery
of the catalyst rather than to any enzyme deactivation.
H
N
O
N
O
-O
HO
N
HN
n
(HIS)b
O
BAL
a = 0-4, b = 4-a
Scheme 2. Structure of the chelate complex formed by Ni–NTA and
two histidine molecules from hexahistidine-tagged benzaldehyde lyase.
As a model for the plug flow reactor with heterogene-
ously immobilized biocatalyst, a packed bed of 7mL
in a flash chromatography column was established with
BAL–Ni–NTA agarose and perfused with substrate-
containing solution. The experiment was continuously
operated for 95h (Fig. 2). The given residence time s is
the quotient of the reactor volume and the flow rate.
Both the purification and immobilization processes are
based on the strong affinity of the imidazole group of
histidine molecules to nickel(II)–nitrilotriacetic acid.
The formation of the chelate complex is reversible.
The histidine linker can be replaced by pure imidazole,
which possesses a stronger affinity to the Ni2+ binding

N. Kurlemann, A. Liese / Tetrahedron: Asymmetry 15 (2004) 2955–2958
2957
continuous experiment involving the same reaction con-
ditions, the residual protein in the packed bed reactor
was quantified after 185 residence times. The BAL was
eluted from the column with 250mM imidazol solution
(add 250mM imidazol to 50mM potassium phosphate
buffer, pH= 7, 0.1mM ThDP, 2mM MgSO 4). The pro-
tein concentration corresponded to 78% of the total
(R)-2-hydroxy-
1-phenyl-propanone
benzaldehyde
benzoin
15
10
5
0.7 h
τ
7 h 1.4 h
0.35 1.4 h 0.14 h
0.07 h
amount of enzyme suggesting
a retention factor
Rf >99.8% per residence time. The progressive decrease
in enzyme activity is probably both due to thermal deac-
tivation of the BAL and a slow process of washing out.
0
0
50
100
150
3. Conclusion
number of residence times Θ = t / τ
Figure 2. Continuous synthesis of (R)-2-hydroxy-l-phenyl-propanone
in a plug flow reactor, the residence time is indicated in the graphic
(50mg BAL on 5mL Ni–NTA–Superflow, 50mM triethanolamine
buffer, pH= 8, 0.5mM ThDP, 0.5mM MgSO 4, 30vol% DMSO,
13mM benzaldehyde, 60mM acetaldehyde).
The recombinant hexahistidine-tagged enzyme benzal-
dehyde lyase was attached to Ni–NTA–Superflow by
metal ion affinity binding, which proved to be a facile
method for enzyme immobilization and thereby genera-
tion of a heterogeneous biocatalyst. The immobilized
enzyme was applied in repetitive batch reactions to cat-
alyze an enantioselective C–C-bond formation by syn-
Different residence times were applied by changing the
flow rate, yielding different levels of space–time yield
(sty) and conversion (Fig. 3).
thesizing
(R)-2-hydroxy-l-phenyl-propanone
from
benzaldehyde and acetaldehyde. The immobilized benz-
aldehyde lyase was employed as a heterogeneous biocat-
alyst in a packed bed in a continuously operated plug
flow reactor for over 140h and yielded (R)-2-hydroxy-
120
100
l-phenyl-propanone in up to 102gLÀ1 dÀ1
.
space-time yield
conversion
80
Acknowledgements
60
40
20
0
We thank Dipl.-Chem. S. Lutz from the Research Cen-
¨
ter Julich for generously providing the E. coli SG13009/
BALHis cells.
0.0
0.5
1.0
1.5
7.0
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was carried out in a round-bottomed flask by suspending
1mL (5mL) Ni–NTA–Superflow (highly cross-linked
agarose, Qiagen, Hilden, Germany) in 3mL (15mL)
triethanolamine (TEA) buffer (50mM, pH= 8) containing
ThDP (0.5mM), MgSO4 (0.5mM) and 30vol% DMSO.
After the addition of 10mg (50mg) lyophilized BAL, the
suspension was stirred for 1h with a KPG stirrer (a
magnetic stirrer would disrupt the resin). After filtration,
the immobilized BAL was washed several times with
buffer solution and directly added to the batch or
plug flow reactor. The determination of selectivity and
conversion rates were performed via HPLC monitoring
using a 25 · 0.4cm RP-8 column (Merck, Darmstadt,
Germany): 40% acetonitrile in aq 0.2% TEA-buffer,
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20. The purification of benzaldehyde lyase was carried out
as described previously.19 The immobilization of BAL
s(benzoin) = 11.2min.