Cross-linked aggregates of (R)-oxynitrilase: A stable, recyclable biocatalyst for enantioselective hydrocyanation

Article abstract of DOI:10.1021/ol047647z

(Chemical Equation Presented) The (R)-oxynitrilase from almonds was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with 1,2-dimethoxyethane and subsequent cross-linking using glutaraldehyde. The resulting preparation was a highly

Full text of DOI:10.1021/ol047647z

ORGANIC
LETTERS
2005
Vol. 7, No. 2
327-329
Cross-Linked Aggregates of
(R)-Oxynitrilase: A Stable, Recyclable
Biocatalyst for Enantioselective
Hydrocyanation
Luuk M. van Langen,† Rhoderick P. Selassa, Fred van Rantwijk, and
Roger A. Sheldon*
Laboratory of Biocatalysis and Organic Chemistry, Delft UniVersity of Technology,
Julianalaan 136, 2628 BL Delft, The Netherlands
r.a.sheldon@tnw.tudelft.nl
Received November 15, 2004
ABSTRACT
The (R)-oxynitrilase from almonds was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with 1,2-dimethoxyethane
and subsequent cross-linking using glutaraldehyde. The resulting preparation was a highly effective hydrocyanation catalyst under microaqueous
conditions, which suppress the nonenzymatic background reaction. The beneficial effect of these latter conditions on the hydrocyanation of
slow-reacting aldehydes is demonstrated. The oxynitrilase CLEA was recycled 10 times without loss of activity.
The (R)-specific oxynitrilase from almonds (hydroxynitrile
lyase Prunus amygdalus, PaHnL, E.C. 4.1.2.10), which
mediates the hydrocyanation of a wide range of aldehydes
with high (>98%) enantioselectivity,1-3 is an emerging
industrial biocatalyst. It is usually applied in an aqueous-
organic two-phase system, in which the enzyme resides in
the aqueous (working) phase and the reactants and products
reside in the organic (extractive) phase. The resulting low
reactant concentration in the aqueous phase suppresses the
uncatalyzed background reaction that otherwise would erode
the ee (see Figure 1).4 The nonenzymatic reaction is further
suppressed by maintaining the pH in the acidic range.5
Satisfactory results have been achieved in such systems with
a wide range of aldehydes, and a mathematical model that
predicts the final conversion and ee has been drawn up.6,7 It
is obvious that increasing the enzyme loading and reducing
the aqueous phase volume will increase the competitiveness
of the enzymatic reaction and improve the enantiomeric
purity of the product.
Accordingly, enzymatic hydrocyanations have been carried
out in a microaqueous organic medium, in combination with
† Present address: CLEA Technologies, Julianalaan 136, 2628 BL Delft,
The Netherlands.
(1) Brussee, J.; Van der Gen, A. Biocatalysis in the Enantioselective
Formation of Chiral Cyanohydrins, Valuable Building Blocks in Organic
Synthesis. In StereoselectiVe Biocatalysis; Patel, R. N., Ed.; Dekker: New
York, 2000; pp 289-320.
(2) Effenberger, F. Hydroxynitrile Lyases in Stereoselective Synthesis.
In StereoselectiVe Biocatalysis; Patel, R. N., Ed.; Dekker: New York, 2000;
pp 321-342.
(3) Schmidt, M.; Griengl, H. Top. Curr. Chem. 1999, 200, 193-226.
(4) Loos, W. T.; Geluk, H. W.; Ruijken, M. M. A.; Kruse, C. G.; Brussee,
J.; Van der Gen, A. Biocatal. Biotransform. 1995, 12, 255-266.
(5) Niedermeyer, U.; Kula, M. R. Angew. Chem., Int. Ed. Engl. 1990,
29, 386-387.
Figure 1. Competition of enzymatic and uncatalyzed hydro-
cyanation.
10.1021/ol047647z CCC: $30.25
© 2005 American Chemical Society
Published on Web 12/23/2004

an immobilized enzyme, such as defatted almond meal.8-10
We have found that such a microaqueous medium is
particularly advantageous in the hydrocyanation of the
sluggishly reacting 2-chlorobenzaldehyde, but the use of
almond meal imposed a lower limit on the water contents
of 4-8% (v/v).11 Highly concentrated aqueous enzyme
solutions have been used,12 but recycling these is problematic.
The efficient enzymatic hydrocyanation of slow-reacting
aldehydes evidently requires a recyclable, anhydrous enzyme
preparation with a high volumetric activity that allows high
catalyst loadings without introducing extra water into the
system. An immobilization methodology that meets these
requirements is the preparation of cross-linked enzyme
aggregates (CLEAs).13 Further advantages of the latter
methodology are its simplicity, low cost, and fast optimiza-
tion.14 We will show that a CLEA of PaHnL is an efficient
and readily recyclable hydrocyanation catalyst.
We first investigated the precipitation of semipurified (R)-
oxynitrilase from a 50% glycerol solution. The best result,
as regards activity loss, was obtained with 1,2-dimethoxy-
ethane. Upon addition of the latter to a final concentration
of 60% (v/v),13 precipitation was nearly complete (see Figure
2) and approximately 60% of the original activity was
recovered from the precipitate upon redissolution.
upon washing. A dry powder was obtained upon rinsing with
acetonitrile and diethyl ether, followed by vacuum-drying.
The particle size dispersion, as measured by laser diffraction,
was very wide, from 0.5 to 200 µ (average 12 µ, see Figure
3). The activity recovery was disappointingly low when we
Figure 3. Particle size distribution of the (R)-oxynitrilase CLEA.
subjected the CLEA to the standard oxynitrilase activity
assay, the cleavage of mandelonitrile. The recovery amounted
to 5% at room temperature and increased to 9.6% when the
assay was done at 0 °C, indicating that there is an effect of
diffusional limitation in the CLEA particles.15 This issue will
be further discussed later.
The newly prepared CLEA was subjected to an operational
stability test in the hydrocyanation of 2-methylbenzaldehyde
(1a), using a microaqueous 2% diisopropyl ether (DIPE)
medium. The CLEA particles could be easily separated from
the reaction mixture when the reaction was complete. The
biocatalyst was washed with water and reused 10 times
without performance loss (see Figure 4). Rapid deactivation
Figure 2. Precipitation study of (R)-oxynitrilase from almonds with
1,2-dimethoxyethane; activity recovery from the precipitate upon
redissolution (9) and from the supernatant ([) and total activity
(2) vs the precipitant concentration. The starting activity corre-
sponds to 100%.
Subsequent cross-linking of the precipitate with glutar-
aldehyde afforded a CLEA that did not leach any enzyme
Figure 4. Effect of recycling on the performance of the (R)-
oxynitrilase CLEA in the hydrocyanation of 1a.
(6) Willeman, W. K.; Straathof, A. J. J.; Heijnen, J. J. Enzyme Microb.
Technol. 2002, 30, 200-208.
(7) Willeman, W. F.; Gerrits. P. J.; Hanefeld, U.; Brussee, J.; Straathof,
A. J. J.; Van der Gen, A.; Heijnen, J. J. Biotechnol. Bioeng. 2002, 77, 239-
247.
(8) Zandbergen, P.; Van der Linden, J.; Brussee, J.; Van der Gen, A.
Synth. Commun. 1991, 21, 1387-1391.
(9) Kiljunen, E.; Kanerva, L. T. Tetrahedron: Asymmetry 1996, 7, 1105-
1116.
took place when the washing step was omitted, presumably
due to accumulation of 2-methylbenzoic acid, which is a
powerful inhibitor of the oxynitrilase.
The performance of the PaHnL CLEA was assessed in
the hydrocyanation of three aldehydes that are known to react
(10) Lin, G.; Han, S.; Li, Z. Tetrahedron: Asymmetry 1998, 9, 1835-
1838.
(11) Van Langen, L. M.; Van Rantwijk, F.; Sheldon, R. A. Org. Proc.
Res. DeV. 2003, 7, 828-831.
(12) Po¨chlauer, P.; Wirth, I.; Mayrhofer, H.; Neuhofer, R. PCT Int. Appl.
WO 01/44487A1, 2001; Chem. Abstr. 2001, 135, 45292u.
(13) Cao, L.; Van Rantwijk, F.; Sheldon, R. A. Org. Lett. 2000, 2, 1361-
1364.
(14) Schoevaart, R.; Wolbers, M. W.; Golubovic, M.; Ottens, M.;
Kieboom, A. P. G.; Van Rantwijk, F.; Van der Wielen, L. A. M.; Sheldon
R. A. Biotechnol. Bioeng. 2004, 87, 754-762.
(15) Janssen, M. H. A.; Van Langen, L. M.; Pereira, S. R. M.; Van
Rantwijk, F.; Sheldon, R. A. Biotechnol. Bioeng. 2002, 78, 425-432.
328
Org. Lett., Vol. 7, No. 2, 2005

quite sluggishly. 2-Chlorobenzaldehyde (1b) was selected,
as it is a chiral intermediate in the industrial synthesis of
the antithrombotic agent clopidogrel. 2-Nitrobenzaldehyde
(1c) is an interesting reactant due to the electronegativity
and steric demands of the nitro substituent. We investigated
the hydrocyanation of trans-cinnamaldehyde (1d) because
the adduct (2d) may be a useful intermediate in the synthesis
of ACE inhibitors.16 Three reaction systems were em-
ployed: the CLEA in microaqueous (2%, v/v) DIPE and the
CLEA, as well as free enzyme in a biphasic DIPE-H2O
(1:1) medium (see Table 1).
performed in microaqueous medium, but the increase in the
product ee shows that the nonenzymatic reaction is almost
completely suppressed under these conditions. 2b with 95%
ee was obtained from the reaction in microaqueous medium
at 0 °C, which compares well with the 91% ee that we
achieved in the presence of 8% water11 and is one of the
highest ever measured.17
Similar trends were observed in the hydrocyanation of 1c,
but the product ee was considerably lower in all reaction
systems and could not be improved beyond 65%. It would
seem that this mediocre enantioselectivity is inherent to the
enzyme and is not caused by a background reaction. The
hydrocyanation of 1d, besides being slow, also suffered from
an unfavorable equilibrium,18 which was remedied by
increasing the excess of HCN to 6-fold. The product ee was
high and little affected by the reaction temperature.
A comparison of the reactions with the CLEA and the
free enzyme shows that the latter is 1.5-2 times more active;
hence, at least 60% of the active sites in the CLEA are
catalytically competent. The question arises why the activity
recovery, as determined by photometric assay, is only 5%.
We ascribe this latter result to the low (0.2 mM) reactant
concentration in the assay, which provides only a small
driving force for diffusion and makes the latter rate-limiting.
The increase in apparent activity recovery at 0 °C also
indicates that diffusion limitation is involved, as the reaction
rate tends to be more temperature-dependent than the
diffusion rate.15
Table 1. Hydrocyanation of Aldehydes 1b-d in the Presence
of (R)-Oxynitrilasea
bio-
temp
time conversion ee
reactant catalyst (°C)
mediumb
(min)
(%)
(%)
1b
1b
1b
1b
1b
1b
1c
1c
1c
1c
1d
1d
1d
1d
CLEA
CLEA
CLEA
CLEA
free
25
0
25
0
25
0
25
0
0
0
25
0
25
25
microaqeous
microaqeous 120
2-phase
2-phase
2-phase
2-phase
microaqeous
microaqeous 210
2-phase
2-phase
microaqeous 120
microaqeous 240
2-phase
2-phase
60
99
98
99
99
99
99
99
99
99
100
90
96
80
86
92
95
56
76
52
77
62
65
38
40
99
99
84
92
120
90
60
60
60
free
CLEA
CLEA
CLEA
free
CLEA
CLEA
CLEA
free
120
130
In conclusion, we have shown that the PaHnL CLEA is a
stable and recyclable biocatalyst. The application of the
CLEA in microaqueous medium clearly is a superior
biocatalyst for the enantioselective hydrocyanation of slow-
reacting aldehydes.
180
150
a Reaction conditions: 0.2 M aldehyde and 0.6 M HCN (1.2 M with
1d) in DIPE (1 mL), 90 U of free enzyme, or 12 mg of CLEA.
b Microaqueous: 20 µL of 20 mM citrate buffer pH 5.5 added to the above
solution. 2-Phase: 1 mL of citrate buffer.
Acknowledgment. This work was financially supported
by Calaire Chimie, Tessenderlo Group (Calais, France). The
authors thank Dr. J. F. Cavalier and Mr. A. Lavieville
(Calaire Chimie) and Dr. M. Belmans and Dr. L. Kemps
(Tessenderlo Chemie) for valuable discussions and assistance.
The hydrocyanation of 1b took place with full conversion
in our reaction systems. A comparison of the activities of
the free enzyme and the CLEA in 50% aqueous DIPE
showed that the CLEA was somewhat less active than the
free enzyme (see later). The biocatalyst formulation had little
effect on the enantiomeric purity of the product, which was
only a modest 56 and 77% at 25 and 0 °C, respectively.
Hence, there is a considerable contribution by the uncatalyzed
background reaction in 50% aqueous DIPE. A further
reduction in rate was observed when the reaction was
Supporting Information Available: Detailed descrip-
tions of experimental procedures. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL047647Z
(17) Glieder, A. Lecture presented at the 6th International Symposium
on Catalysis Applied to Fine Chemicals, April 6-10, Delft, The Netherlands.
(18) Warmerdam, E. G. J. C.; Van den Nieuwendijk, A. M. C. H.; Kruse,
C. G.; Brussee, J.; Van der Gen, A. Recl. TraV. Chim. Pays-Bas 1996, 115,
20-24.
(16) Herold, P.; Indolese, A. F.; Studer, M.; Jalett, H. P.; Siegrist, U.;
Blaser, H. U. Tetrahedron 2000, 56, 6497-6499.
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