Use of ionic liquids ti increase the yield and enzyme stability in the β-galactosidase catalysed synthesis of N-acetyllactosamine

Article abstract of DOI:10.1021/op0255231

The use of ionic liquids as alternative solvents for enzyme catalysis was investigated. β-Galactosidase from Bacillus circulans catalyses the synthesis of N-acetyllactosamine starting from lactose and N-acetylglucosamine in a transglycoslyation reaction. The addition of 25% v/v of 1,3-di-methyl-imidazol-methyl sulfate [MMIM] [MeSO₄] as a water-miscible ionic liquid suppresses the secondary hydrolysis of the formed product, resulting in doubling the yield to almost 60%. The enzyme can be reused several times after ultrafiltration of the reaction mixture without loss of activity. Results of different amounts of ionic liquids in the reaction medium on the thermostability of the galactosidase as well as on oxidoreductases are presented as well.

Full text of DOI:10.1021/op0255231

Organic Process Research & Development 2002, 6, 553−557
Use of Ionic Liquids to Increase the Yield and Enzyme Stability in the
â-Galactosidase Catalysed Synthesis of N-Acetyllactosamine
†
‡
,†
Nicole Kaftzik, Peter Wasserscheid, and Udo Kragl*
Rostock UniVersity, Deptartment of Chemistry, 18051 Rostock, Germany, and Institute of Technical and Macromolecular
Chemistry, RWTH Aachen, 52066 Aachen, Germany
Abstract:
solvents for biocatalytic reactions as well so that some
problems such as substrate solubility, yield, enzyme stability
or selectivity may be solved.
The use of ionic liquids as alternative solvents for enzyme
catalysis was investigated. â-Galactosidase from Bacillus cir-
culans catalyses the synthesis of N-acetyllactosamine starting
from lactose and N-acetylglucosamine in a transglycoslyation
reaction. The addition of 25% v/v of 1,3-di-methyl-imidazol-
Generally, there are three modes to operate ionic liquids
in a biocatalytic process: (i) use as a pure solvent, (ii) use
as a cosolvent in aqueous systems, or (iii) as a cosolvent in
a biphasic system. After trials first using ethylammonium
4
methyl sulfate [MMIM] [MeSO ] as a water-miscible ionic
8
liquid suppresses the secondary hydrolysis of the formed
product, resulting in doubling the yield to almost 60%. The
enzyme can be reused several times after ultrafiltration of the
reaction mixture without loss of activity. Results of different
amounts of ionic liquids in the reaction medium on the
thermostability of the galactosidase as well as on oxidoreduc-
tases are presented as well.
nitrate in mixtures with water more than 15 years ago, in
the last two years a number of papers were published on
first results of the use of pure ionic liquids as reaction
medium for enzymatic reactions. In these examples ionic
liquids such as [BMIM] [PF
6 4
] or [BMIM] [BF ] have been
used to replace organic solvents in protease- and lipase-
9
-15
catalysed reactions.
A biphasic system containing an ionic
liquid for in situ product extraction for a whole-cell process16
and also the use of simple ionic liquids for protein renatur-
ation have been described as well.
1
7
Introduction
During the past decade ionic liquids have gained increas-
ing attention for performing all types of reaction with
sometimes remarkable results.1 Ionic liquids are low-melting
Results and Discussion
Enzyme Activity in the Presence of Ionic Liquids. The
,2
1
8
influence of different ionic liquids on the activity of
hydrolases (â-galactosidase from Bacillus circulans, lipases
from different origins) and oxidoreductases (formate dehy-
drogenase (FDH) from Candida boidinii, alcohol dehydro-
genase (YADH) from yeast, carbonylreductase (CPCR) from
Candida parapsilosis) was investigated. The results of the
lipase-catalysed resolution of phenylethanol in pure ionic
liquids with improved enantioselectivity compared to methyl-
tert-butyl ether (MTBE) as reference solvent have been
(<100 °C) salts which represent a new class of nonmolecular,
ionic solvents. They have many properties which make them
of fundamental interest to all chemists, since both the ther-
modynamics and kinetics of reactions carried out in ionic
liquids are different to those in conventional molecular sol-
vents. Therefore, there are many good reasons to study ionic
liquids as alternative solvents in enzyme catalysis as well.
3
Besides the engineering and environmental advantage of
their nonvolatile nature, the investigation of new biphasic
reactions is of special interest. In transition metal-catalyzed
reactions, for example, it has been found in many cases that
ionic liquids can act as a superior solvent in comparison to
water and common organic solvents, especially when ionic
complexes are used as catalysts.4 In these cases, significant
enhancement of catalyst activity and stability has been
possible. Catalysts for enantioselective reactions have been
1
9
published elsewhere. For the other enzymes the activity in
dependence on the content of ionic liquid in the aqueous
buffer solution were studied using established photometric
enzyme assays. The results obtained for the FDH assay and
the â-galactosidase assay are summarized in Table 1.
-6
(8) Magnuson, D. K.; Bodley, J. W.; Evans, D. F. J. Solution Chem. 1984, 13,
583.
(9) Erbeldinger, M.; Mesiano, A. J.; Russel, A. J. Biotechnol. Prog. 2000, 16,
1129.
7
recycled using ionic liquids as well. Due to their special
(10) Lau, R. M.; van Rantwijk, F.; Seddon, K. R.; Sheldon, R. A. Org. Lett.
properties and possible advantages ionic liquids may be ideal
2000, 2, 4189.
(11) Kim, K.-W.; Song, B.; Choi, M.-Y.; Kim, M.-J. Org. Lett. 2001, 3, 1507.
*
Author to whom correspondence may be sent. Fax: +49 381/498 6452.
(12) Laszlo, J. A.; Compton, D. L. Biotechnol. Bioeng. 2001, 75, 181.
(13) Lozano, P.; de Diego, T.; Guegan, J.-P.; Vaultier, M.; Iborra, J. L.
Biotechnol. Bioeng. 2001, 75, 563.
(14) Itoh, T.; Akasaki, E.; Kudo, K.; Shirakami, S. Chem. Lett. 2001, 262.
(15) Park, S. Kazlauskas, R. J. J. Org. Chem. 2001, 66, 8395.
(16) Cull, S. G.; Holbrey, J. D.; Vargas-Mora, V.; Seddon, K. R.; Lye, G. J.
Biotechnol. Bioeng. 2000, 69, 227.
(17) Summers, C. A.; Flowers, R. A., II. Protein Sci. 2000, 9, 2001.
(18) Ionic liquids are commercially available, e.g. from Solvent Innovation
GmbH Cologne (www.solvent-innovation.com).
E-mail: udo.kragl@chemie.uni-rostock.de.
†
Rostock University.
Institute of Technical and Macromolecular Chemistry.
‡
(
(
(
(
(
1) Wasserscheid, P.; Keim, W. Angew. Chem., Int. Ed. Engl. 2000, 39, 2226.
2) Welton, T. Chem. ReV. 1999, 99, 2071.
3) Seddon, K. R. J. Chem. Technol. Biotechnol. 1997, 68, 351.
4) Wasserscheid, P.; Eichmann, M. Catal. Today 2001, 66, 309.
5) Wasserscheid, P.; Waffenschmidt, H.; Machnitzki, P.; Kottsieper, K. W.;
Stelzer, O. Chem. Commun. 2001, 451.
(
(
6) B o¨ hm, V. P. W.; Hermann, W. A. Chem. Eur. J. 2000, 6, 1017.
7) Song, C. E.; Roh, E. J. Chem. Commun. 2000, 837.
(19) Sch o¨ fer, S.; Kaftzik, N.; Wasserscheid, P.; Kragl, U. Chem. Commun. 2001,
425.
1
0.1021/op0255231 CCC: $22.00 © 2002 American Chemical Society
Vol. 6, No. 4, 2002 / Organic Process Research & Development
•
553
Published on Web 06/27/2002

Table 1. Enzyme activity in the presence of ionic liquid, %
of residual activity in comparison to the activity in pure
buffer solution
Scheme 1. Enzyme-catalyzed synthesis of
N-acetyllactosamine 3; the enzyme E is â-galactosidase (A)
transgalactosylation, (B) hydrolysis of 1 (side reaction), (C)
hydrolysis of the product 3 (secondary hydrolysis)
FDH
50
â-galactosidase
Ionic liquid [% v/v]
25
75
25
50
75
[
[
[
[
[
[
[
[
MMIM] [MeSO
4
]
65
-
73
-
55
-
-
-
3
98
-
-
-
-
-
-
-
74
-
14
-
-
-
-
-
-
10
-
-
-
-
6
-
-
-
Et
Et
PrNH
3
NH] [MeSO
4
]
3
NMe] [MeSO
] [NO
BMIM] [BF
EMIM] [PhCO
BMIM] [F
BMIM] [OctSO
4
]
82
-
-
3
3
]
-
4
]
-
31
-
2
]
-
3
CSO
3
]
38
-
-
4
]
-
35
The best results were obtained for FDH and â-galactosi-
dase in mixtures of the ionic liquid 1,3-dimethyl-imidazol-
methyl sulfate [MMIM][MeSO ] with buffer. The YADH-
catalysed oxidation of ethanol was only possible in the
presence of 25% v/v of [BMIM][F CSO ] with a residual
4
3
3
Scheme 1 shows the reaction scheme of the transglyco-
sylation described here. Starting with lactose 1 transgalac-
tosylation results in the transfer of the galactose moiety of 1
to N-acetylglucosamine 2 to afford N-acetyllactosamine 3
activity of less than 5%. However, it must be pointed out
that possible impurities in the ionic liquids which are difficult
to analyse and to remove may be the reason for the loss of
enzyme activity. For the CPCR-catalysed reduction aceto-
phenone was used as substrate. The low solubility of aceto-
phenone in aqueous media can be increased 2- to 3-fold in
the presence of 25% (v/v) [MMIM] [MeSO4]. However, thus
far no activity could be found for CPCR in the presence of
the ionic liquids investigated. This is surprising because for
this enzyme a good tolerance against organic solvents has
(A). The galactosyl donor 1 is hydrolysed to glucose 4 and
galactose 5 in a side reaction (B). The desired product 3 is
also a substrate for the â-galactosidase. When the reaction
is performed in water, the yield is limited to 30% due to the
secondary hydrolysis of 3 in a subsequent reaction by the
enzyme (C). However, it is difficult to separate the product
from the starting disaccharide and also to determine the
optimum reaction time to terminate the reaction to obtain
high yield of the desired product. As we have previously
reported this can be advantageously realised in a continuously
operated stirred tank reactor with retention of the enzyme
2
0,21
been reported.
For FDH which can be used for cofactor
regeneration in the CPCR-catalysed reaction even higher
contents of ionic liquids were possible without reduction of
the enzyme’s activity.
â-Galactosidase-Catalyzed Synthesis of N-Acetyllac-
tosamine. In the following we focused our work on the
behaviour of â-galactosidase in the presence of ionic liquids.
The use of enzymes for the synthesis of di- and oligosac-
34
by an ultrafiltration membrane.
There have been studies to increase the yield of N-
acetyllactosamine by addition of organic cosolvents such as
2
-ethoxy ethyl ether and trimethyl phosphate, but starting
2
2-24
charides has been established more than a decade ago.
The commercially available â-galactosidase from B. circu-
with lactose as a cheap galactose donor with 2-ethoxy ethyl
ether only a yield higher than 20% could be achieved. Higher
yields of 30-40% are only possible with more expensive
2
5
lans shows a remarkable â-1,4 selectivity and oligosac-
charide synthesis results in high regioisomeric purity of the
synthesized product.2 Oligosaccharides such as N-acetyl-
lactosamine (LacNAc) have attracted much interest in
immunological and pharmacological research because of their
35,36
donors such as p-nitrophenylgalactoside as substrates.
6,27
Another approach is the addition of high salt concentra-
tions. Kren and co-workers reported the increase of the yield
from 2 to 15% by addition of salt concentrations of up to 1
significance as fundamental structures of glycoproteins and
37
mol/L.
glycolipids.2
8-30
D-Galacto-oligosaccharides have been syn-
Influence of Ionic Liquids on Yield and Selectivity of
Disaccharide Synthesis. In the following the results of the
synthesis of N-acetyllactosamine in the presence of ionic
liquids are presented. To compare the results with those
published earlier, 25 °C was chosen as reaction temperature.
Accordingly, the concentrations were in the same order of
thesized mainly by utilizing the transglycosylation activity
24,27,31-33
of â-galactosidase of various origins.
(20) Zelinski, T.; Kula, M. R. Biocatal. Biotrans. 1996, 15, 57.
(21) Zelinski, A.; Liese, A.; Wandrey, C.; Kula, M. R. Tetrahedron: Asymmetry
1
999, 10, 1681.
(
(
(
22) ] Ajisaka, K.; Fujimoto, H.; Nishida, H. Carbohydr. Res. 1988, 180, 35.
23) Nilsson, K. G. I. Carbohydr. Res. 1987, 167, 95.
24) Wong, C. H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry;
Pergamon Press: Oxford, 1994.
(31) Nilsson, K. G. I. Trends Biotechnol. 1988, 6, 256.
(32) van Rantwijk, F.; Woudenberg-van Oosterom, M.; Sheldon, R. A. J. Mol.
Catal. B: Enzym. 1999, 6, 511.
(33) Sauerbrei, B.; Thiem, J. Tetrahedron Lett. 1992, 33, 201.
(34) Hermann, G.; Kragl, U.; Wandrey, C. Angew. Chem., Int. Ed. Engl. 1993,
32, 1342.
(
25) â-Galactosidase from B. circulans is commercially available from Daiwa
Kasei, Osaka, Japan.
(26) Sakai, K.; Katsumi, R.; Ohi, H.; Usui, T.; Ishido, Y. J. Carbohydr. Chem.
1
992, 11, 553.
(
(
(
(
27) Crout, D. H. G.; Vic, G. Curr. Opin. Chem. Biol. 1998, 2, 98.
28) Lasky, L. A. Science 1992, 258, 964.
29) Kobata, A. Eur. J. Biochem. 1992, 209, 483.
30) Feizi, T. Nature 1985, 314, 53.
(35) Yoon, J. H.; Rhee, J. S. Carbohydr. Res. 2000, 327, 377.
(36) Farkas, E.; Thiem, J. Eur. J. Org. Chem. 1999, 3073, 3.
(37) Rajnochova, E.; Dvorakova, J.; Hunkova, Z.; Kren, V. Biotechnol. Lett.
1997, 19, 869.
554
•
Vol. 6, No. 4, 2002 / Organic Process Research & Development

Figure 1. Concentration as function of time for the synthesis
of N-acetyllactosamine 3 in a batch reactor without (open
symbols) and with ionic liquid (filled symbols); starting con-
centrations: (a) 62 mmol/L lactose 1, 600 mmol/L N-acetyl-
Figure 3. Water activity (a
w
) as function of water content [%
v/v)] in different water-ionic liquid mixtures at 25 °C; (A)
MMIM] [MeSO ], (B) [BMIM] [OctSO ].
(
[
4
4
glucosamine 2, 2 mg/mL â-galactosidase, 195 mmol/L K
2
HPO
, pH 7.3, 23 °C; (b) 62 mmol/L lactose 1,
00 mmol/L N-acetylglucosamine 2, 2 mg/mL â-galactosidase,
95 mmol/L K HPO , 65 mmol/L KH PO , 25% (v/v) [MMIM]
], pH 7.3, 23 °C. The amount of 3 (b) and 1 ([)
4
,
transgalactosidation product. The final product/educt ratio
of disaccharide of 2:1 obtained is another advantage facilitat-
ing product isolation.
6
6
1
2 4
5 mmol/L KH PO
2
4
2
4
[
MeSO
4
To present a real alternative to synthesis in water or
organic solvents, it is important to demonstrate that ionic
liquids can be recycled after the reaction and product
separation is possible. If the ionic liquid used in this
experiment is not miscible with organic solvents, the product
isolation is possible via simple extraction. However, in this
case that cannot be realized, because of the low solubility
of the saccharides in organic solvents. Product isolation is
possible by using chromatography methods as published
produced/hydrolyzed as a function of time were examined, and
samples were analyzed by HPLC.
3
8
earlier. The product isolated within this method still
contains small amounts of the ionic liquid, but modern
nanofiltration membranes allow the separation of the non-
3
9,40
charged product from the ionic liquid.
Because of the
high cost of enzymes it is also important to separate the
enzyme so that it can be reused several times. In addition to
4
1
Figure 2. Improved selectivity in ionic liquids for the enzy-
matic synthesis of N-acetyllactosamine (LacNAc) due to sup-
pression of the secondary hydrolysis. Conditions: 62 mmol/L
the immobilisation of enzymes, ultrafiltration is a good
method to separate a soluble enzyme from the reaction
34,42
mixture.
The â-galactosidase is stable under the condi-
Lac, 600 mmol/L GlcNAc, 25% (v/v) [MMIM][MeSO
4
], 2 g/L
tions employed, allowing its repeated use after filtration with
an ultrafiltration membrane. In this way, enzyme stability
can be investigated in the presence of ionic liquid under
reaction conditions. After three times of repeated use in the
4
â-galactosidase, 25 °C, pH 7.3. Selectivity is the ratio of LacNAc
3
formed to lactose 1 consumed. The product (conversion ×
selectivity) is the yield of the reaction.
magnitude. Table 1 shows a decrease of â-galactosidase
activity with increasing ionic liquid content. Therefore, its
amount was limited to 25% (v/v). Under these conditions
the maximum activity was obtained in the presence of 25%
presence of [MMIM] [MeSO ], no reduction of activity was
4
3
observed.
The somewhat lower reaction rate of the hydrolysis of
both lactose and N-acetyllactosamine could be correlated with
(
v/v) [MMIM][MeSO
symbols) is compared to that with the addition of ionic liquid
solid symbols). In buffer the concentration of N-acetyllac-
4
]. In Figure 1 a reaction in buffer (open
a reduction of water activity (a ) caused by the ionic liquid.
w
In the presence of 25% (v/v) [MMIM] [MeSO ] for a a
4
w
(
value of 0.83 was obtained. As shown in Figure 3, the water
tosamine goes through a maximum which corresponds to a
yield of 30% caused by the secondary hydrolysis. In the
activity in the presence of 25% (v/v) [MMIM] [MeSO ] is
lower than in the presence of 25% (v/v) [BMIM] [OctSO ].
4
4
44
4
presence of [MMIM][MeSO ] the secondary hydrolysis is
suppressed, resulting in final product concentrations of 36
mmol/L corresponding to a yield of 58%. As can be seen
from Figure 1 the reaction velocity of product formation is
not influenced by the presence of the ionic liquid. In Figure
(38) Range, G.; Kr a¨ hmer, R.; Welzel, P. Tetrahedron 1997, 53, 1695.
(39) Kr o¨ kel, J.; Kragl, U. Manuscript in preparation.
(
40) Dudziak, G.; Fey, S.; Hasbach, L.; Kragl, U. J. Carbohydr. Chem. 1999,
18, 41-49.
(
41) Hernaiz, M. J.; Crout, D. H. G. J. Mol. Cat B: Enzym. 2000, 10, 403.
42) Kragl, U.; G o¨ dde, A.; Wandrey, C.; Kinzy, W.; Cappon, J. J.; Lugtenburg,
J. Tetrahedron: Asymmetry 1993, 4, 1193.
(
2
the selectivity is shown as a function of conversion. The
enzyme shows a better selectivity in ionic liquid over a long
time. There is no need to determine the optimum reaction
time to terminate the reaction to obtain high yields of the
(43) Kragl, U. Kaftzik, N.; Sch o¨ fer, S. H.; Eckstein, M.; Wasserscheid, P.;
Hilgers, C. Chimica Oggi (Chem. Today) 2001, 7/8, 22-24.
44) B o¨ smann, A.; Datsevich, L.; Jess, A.; Lauter, H.; Schmitz, C.; Wasserscheid,
(
P. Chem. Commun. 2001, 2494.
Vol. 6, No. 4, 2002 / Organic Process Research & Development
•
555

Figure 5. Time courses of â-galactosidase inactivation at 50
C in: (A) 100% (v/v) H O, (B) 100% (v/v) [MMIM] [MeSO ],
C) 100% (v/v) [BMIM] [MeSO ], (D) 100% (v/v) [MNEt
MeSO ], (E) 100% (v/v) saturated NaCl solution. The enzy-
matic stability was determined by measuring the residual
hydrolytic activity with o-nitrophenylgalactoside in water as
described in the Experimental Section. The initial activity was
measured at 23 °C and was set to 100% for water as standard.
Figure 4. Conversion as function of time for the synthesis of
N-acetyllactosamine in the presence of different ionic liquids
resulting in different water activities; starting concentrations:
°
(
[
2
4
4
3
]
4
6
2 mmol/L lactose, 600 mmol/L N-acetylglucosamine, 2 mg/
mL â-galactosidase, 195 mmol/L K HPO , 65 mmol/L KH PO
pH 7.3; 23 °C. (A) 20% (v/v) [MMIM] [MeSO ]. (B) 20% (v/v)
BMIM] [OctSO ].
2
4
2
4
,
4
[
4
This might be due to the stronger interaction between the
methyl sulfate ions and the water molecules. The lower water
activity may be the reason for suppression of the secondary
hydrolysis as shown in Figure 4. However, the effects ob-
served may be also ascribed to interactions of the components
of the ionic liquid with charged groups in or near the active
site of the enzyme. Studies about the reasons for the
improved yield revealed that simple salts such as sodium
chloride can be used in a similar way. A saturated solution
aqueous medium at 80 °C, whereas in 100% (v/v) [MMIM]-
[MeSO ] the enzyme retained 71% of its initial hydrolytic
4
activity after 12 h of incubation. Unfortunately, these
conditions do not offer further advantages for the synthesis
of N-acetyllactosamine besides an increased enzyme stability.
At higher ionic liquid content the enzyme activity strongly
decreases. This might be due to mass-transport limitations
due to the higher viscosity of the reaction medium, even at
higher temperatures.
w
of sodium chloride has an a value of 0.75 and a 25% (w/
w) solution of 0.88, but the use of sodium chloride is limited
by its solubility in the reaction media, while [MMIM]
Conclusions and Outlook
[
4
MeSO ] is totally miscible with water without limitations.
These results clearly demonstrate that ionic liquids can
be used advantageously in enzymatic transglycosylation
reactions. Not only can they replace organic solvents in these
reactions, but they can also offer the advantage of altering
the reactions conditions in an advantageous way. From initial
investigation it becomes obvious that ionic liquids offer a
useful method to control the water activity in water-rich
environments in a different manner from simple salts. It
should be noted that the ionic liquids are probably not fully
dissociated when placed in an aqueous environment. Thus,
the conductivity of these solutions is quite low. Ion pairs
and larger aggregates may also be the reason for the
improved solubility of hydrophobic compounds in mixtures
of water and ionic liquids. The influence of water activity
in nonmiscible ionic liquids has been demonstrated re-
Another important advantage of the ionic liquid as cosolvent
is the increase in the solubility of hydrophobic components.
Solvent Effect on Enzyme Stability. It has been shown
that the stability of enzymes is markedly increased under
low water conditions,45 making it possible to perform
biotransformations at temperatures higher than those used
in conventional aqueous solutions. The most common cause
for the inactivation of enzymes at elevated temperatures is
the loss of the native, catalytically active conformation.
Recently the work of Turner et al.46 demonstrated that the
temperature at which a protein undergoes thermal denatur-
ation (T
d
) is strongly dependent on the amount of water
47
associated with the protein. It was found that the thermal
w
denaturation could be correlated with the water activity (a )
of the sample.4
8-50
With lowering the water activity of the
5
1,52
cently.
d
medium T increases. After 60 h of incubation in different
ionic liquids (100% (v/v)), water, and saturated sodium
chloride solution at 50 °C, the â-galactosidase remained
active only in [MMIM][MeSO
On the other hand it is very likely that there are
interactions between the compounds of an ionic liquid and
charged groups of the enzyme as well. The interaction might
be the reason for changes in the enzyme’s selectivity and
stability. Here the effects observed resemble to the of the
ectoines, a class of zwitterionic compounds found to protect
4
] and water (Figure 5). A
rapid enzyme inactivation is observed in a predominantly
(45) Adlercreutz, P.; Mattiasson, B. Biocatalysis 1987, 1, 99.
(46) Turner, N. A.; Duchateau, D. B.; Vulfson, E. N. Biotechnol. Lett. 1995,
53
and stabilise proteins.
1
7, 371.
47) Volkin, D. B.; Staubli, A.; Lange, R.; Klibanov, A. M. Biotechnol. Bioeng.
991, 37, 843.
(
1
(51) Eckstein, M.; Wasserscheid, P.; Kragl, U. Biotechnol. Lett. 2002, 24, 763.
(52) Eckstein, M.; Sesing, M.; Kragl, U.; Adlercreutz, P. Biotechnol. Lett. 2002,
24, 867.
(
(
(
48) Halling, P. J. Enzymol. Microb. Technol. 1984, 6, 513.
49) Halling, P. J. Trends Biotechnol. 1989, 7, 50.
50) Kuhl, P.; Halling, P. J. Biochim. Biophys. Acta 1991, 1078, 326.
(53) Lippert, K.; Galinski, E. A. Appl. Microbiol. Biotech. 1992, 37, 61.
556
•
Vol. 6, No. 4, 2002 / Organic Process Research & Development

Experimental Section
Synthesis of N-Acetyllactosamine in a Batch Reactor.
All reactions were carried out in 1.5-mL flasks at room
temperature. Enzyme solution (0.2 mL, 10 mg/mL in buffer)
was added to a mixture containing 0.05 mL of buffer
Materials. â-Galactosidase was purchased from Daiwa
Kasei, Osaka, Japan. All other chemicals were from Fluka,
Buchs, Switzerland. The ionic liquids were kindly donated
by the group of Dr. Wasserscheid, RWTH Aachen. CPCR
and FDH was a gift from Professor Kula, Heinrich-Heine-
University, D u¨ sseldorf.
2 4 2 4
(65 mmol/L KH PO , 195 mmol/L K HPO , pH 7.3),
N-acetylglucosamine solution (0.5 mL, GlcNAc 1.2 mol/L
in buffer), and lactose solution (0.25 mL, 250 mmol/L in
buffer). For the reactions in the presence of ionic liquid 0.2
mL of enzyme solution (10 mg/mL in buffer) was added to
Enzyme Assay Formate Dehydrogenase. The reduction
of â-nicotinamide adenine dinucleotide (NAD ) with simul-
+
taneous oxidation of formate was used. Aqueous sodium
a mixture containing 0.05 mL of buffer (65 mmol/L KH
2
-
formate solution (0.1 mL, 2.4 mol/L) and enzyme solution
PO , 195 mmol/L K HPO , pH 7.3), 0.5 mL of N-acetyl-
4
2
4
(
0.1 mL, FDH 0.7 mg/mL, 8.4 U, containing 6 mmol/L
glucosamine solution (GlcNAc 1.2 mol/L in 2:1 buffer/ionic
liquid), and 0.25 mL of lactose solution (250 mmol/L in 2:1
buffer/ionic liquid). The reactions were terminated by thermal
denaturation of the enzyme (10 min at 100 °C). The samples
were filtered (Minisart RC 4 from Sartorius) to remove
proteins and analysed by HPLC using a Aminex HPX-87H
column from BioRad, Munich, Germany, 0.006 mol/L
sulfuric acid as eluent with a flow of 0.8 mL/min at 65 °C;
detection was done using an UV detector at 208 nm and a
RI detector.
NAD) were added to 1 mL of buffer (50 mM triethanolamine
hydrochloride, 1 mmol/L dithiothreitole, HCl, pH 7). The
increase of NADH was measured at 25 °C using a spectro-
photometer at 340 nm. For the reactions in the presence of
different % v/v of ionic liquid the amount of buffer was
reduced and replaced by the ionic liquid.
Enzyme Assay â-Galactosidase. Hydrolysis of o-nitro-
phenylgalactoside to o-nitrophenol und galactose was used.
Aqueous o-nitrophenylgalactoside solution (0.1 mL, 30
mmol/L) and aqueous enzyme solution (0.1 mL of â-galac-
tosidase 10 mg/mL) were added to 1 mL of buffer (65
Recovery and Repeated Use of â-Galactosidase by
Ultrafiltration. Six milliliters of enzyme solution (10
mg/mL in buffer) was added to a substrate solution contain-
2 4 2 4
mmol/L KH PO , 195 mmol/L K HPO , pH 7.3). The
increase of o-nitrophenol was measured at 25 °C using a
spectrophotometer at 405 nm. For the reactions in the
presence of different % v/v of ionic liquid the amount of
buffer was reduced and replaced by the ionic liquid.
Enzyme Assay Yeast Alcohol Dehydrogenase. The
oxidation of ethanol with simultaneous reduction of â-nico-
ing 1.5 mL of buffer (65 mmol/L KH
2 4
PO , 195 mmol/L
K
2
HPO , pH 7.3), 15 mL of N-acetylglucosamine solution
4
(GlcNAc 1.2 mol/L in 2:1 buffer/ionic liquid) and 7.5 mL
of lactose solution (250 mmol/L in 2:1 buffer/ionic liquid)
and mixed in an ultrafiltration cell (Schleicher and Schuell,
V
max: 80 mL, Pmax: 6 bar, equipped with a membrane YM
+
tinamide adenine dinucleotide (NAD ) was used. Aqueous
3, cutoff 3000 g/mol) for 10 min. The ultrafiltration cell was
kept at 23 °C. By pressurizing the cell with argon the solution
containing product and nonreacted substrates is separated
from the enzyme. The retentate is concentrated to 6 mL.
Twenty-four milliliters of fresh substrate solution is added
to the remaining solution in the filtration cell and treated as
the first batch. The filtrate is diluted with water and analyzed
by HPLC. Part of the solution obtained was purified as
+
ethanol solution (0.1 mL, 7.2 mol/L), NAD solution (0.1
mL, 1.2 mmol/L), and enzyme solution (0.01 mL, YADH
30 U/mL) were added to 0.99 mL of buffer (65 mmol/L KH
2
-
PO , 195 mmol/L K HPO , pH 7.3). The increase of NADH
4
2
4
was measured at 25 °C using a spectrophotometer at 340
nm. For the reactions in the presence of different % v/v of
ionic liquid the amount of buffer was reduced and replaced
by the ionic liquid.
38,54
desribed in the literature.
The resulting LacNAc (20 mg)
Enzyme Assay CPCR. Reduction of acetophenone under
oxidation of â-nicotinamide adenine dinucleotide (NADH)
was used. â-Nicotinamide adenine dinucleotide solution (0.1
mL, NADH 2 mmol/L in buffer) and enzyme solution (0.02
mL, CPCR 2-3 U/mL in glycerin) were added to 0.88 mL
of acetophenone solution (10 mmol/L acetophenone, 100
mmol/L TEA/NaOH, pH 7.0). The decrease of NADH was
measured at 37 °C using a spectrophotometer at 340 nm.
For the reactions in the presence of different % v/v of ionic
liquid the amount of buffer was reduced and replaced by
the ionic liquid.
was identical to a commercial sample. Impurities of ionic
liquid still present did not interfere with the analytical
methods.
Acknowledgment
Generous donations of CPCR and FDH by the Group of
Professor M.-R. Kula, Institute of Enzymetechnology, Hein-
rich-Heine-University D u¨ sseldorf, Germany are gratefully
acknowledged. Part of the work was financially supported
by the “Fonds der Chemischen Industrie”.
Measurement of Water Activity. The water activity (a
in different water/ionic liquid mixtures was measured with
an a measuring instrument from Novasina (Novasina AW
SPRINT, Axair Ltd. Switzerland).
w
)
Received for review February 18, 2002.
OP0255231
w
(54) Range, G. PhD Dissertation, Uni Leipzig, 1996.
Vol. 6, No. 4, 2002 / Organic Process Research & Development
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