Substrate sorption into the polymer matrix of Novozym 435 and its effect on the enantiomeric ratio determination

Article abstract of DOI:10.1016/S0957-4166(03)00577-9

In the enantioselective esterification of 4-methyloctanoic acid with ethanol by immobilised Candida antarctica lipase B (Novozym 435), the enantiomeric excesses determined during the course of the reaction deviated strongly from the theoretical values, le

Full text of DOI:10.1016/S0957-4166(03)00577-9

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 2699–2704
Substrate sorption into the polymer matrix of Novozym 435®
and its effect on the enantiomeric ratio determination
Nicole W. J. T. Heinsman,a,b,† Carin G. P. H. Schroe¨n,b Albert van der Padt,b,‡
Maurice C. R. Franssen,a,* Remko M. Boomb and Klaas van‘t Rietc
aLaboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands
bDepartment of Food Technology and Nutritional Sciences, Food and Bioprocess Engineering Group, Wageningen University,
Bomenweg 2, 6703 HD Wageningen, The Netherlands
cTNO Strategy and Planning, P.O. Box 6070, 2600 JA Delft, The Netherlands
Received 10 June 2003; accepted 16 July 2003
Abstract—In the enantioselective esterification of 4-methyloctanoic acid with ethanol by immobilised Candida antarctica lipase B
(Novozym 435®), the enantiomeric excesses determined during the course of the reaction deviated strongly from the theoretical
values, leading to unacceptably large confidence intervals for the enantiomeric ratio (E value). This observation was in contrast
to our previous findings for transesterification and hydrolysis reactions with this enzyme. Herein, the three reactions are
compared; the anomalous results in the esterification reaction appear to be caused by adsorption of 4-methyloctanoic acid inside
the enzyme beads. We found that on average 1.19 g of 4-methyloctanoic acid was incorporated per g of Novozym 435®. If the
concentration of this substrate was adjusted accordingly in the calculations, the resulting E values showed acceptable confidence
intervals. In previous research on transesterification reactions in excess apolar solvent (comparable affinity for the beads), sorption
does not play an important role because only small amounts of substrate were lost. For hydrolysis reactions, sorption takes place
but the acid is released from the beads upon titration and no effect on the E value is found. However, for esterification reactions,
sorption should not be neglected since there is no driving force to release the acid from the beads.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
enantiomers. Several methods exist for the determina-
tion of E.11–16
Nowadays, lipases are well-accepted catalysts in
organic chemistry, both in aqueous and non-aqueous
media. Working with these enzymes in organic solvents
has several potential advantages, e.g. the possibility to
shift the thermodynamic equilibrium to favour esterifi-
cation over hydrolysis.1–5 In this way, the kinetic reso-
lution of acids or alcohols can be performed by
lipase-mediated esterification in organic solvents.6–10
Recently, we have studied the kinetic resolution of
some branched chain fatty acids and their esters.17–19
Novozym 435® (immobilised Candida antarctica lipase
B, CALB) showed the best results (E=23) in the trans-
esterification reaction of 4-methyloctanoic acid methyl
ester to its butyl ester.17 Also this enzyme was success-
fully applied in hydrolysis reactions.19 The same
enzyme is now used for the esterification of 4-methyloc-
tanoic acid with ethanol in a solventless system (see
Scheme 1).19 The effect of substrate sorption is investi-
gated in detail and the results are compared to those
obtained for hydrolysis and transesterification.
To evaluate a kinetic resolution in terms of enantiose-
lectivity, the enantiomeric ratio (E) has been defined.
This parameter corresponds to the ratio of the specific-
ity coefficients, Vmax/KM, of the individual substrate
2. Results and discussion
* Corresponding author. Tel.: +31-317-482976; fax: +31-317-484914;
e-mail: maurice.franssen@wur.nl
2.1. Determination of the enantiomeric ratio in the ester-
ification of 4-methyloctanoic acid
† Current address: Quest International Nederland BV, PO Box 2,
1400 CA Bussum, The Netherlands.
‡ Current address: Friesland Coberco Dairy Foods, PO Box 87, 7400
AB Deventer, The Netherlands.
Esterification of 4-methyloctanoic acid was performed
using a range of concentrations varying from 2:1 up to
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0957-4166(03)00577-9

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N. W. J. T. Heinsman et al. / Tetrahedron: Asymmetry 14 (2003) 2699–2704
Scheme 1.
Since in our case, swelling may explain the poor fits
obtained, an attempt was made to measure the swelling
quantitatively. Different techniques were used to mea-
sure the volume increase that results from the diffusion
of ester or acid into the macroporous beads of the
polymer on which C. antarctica lipase B is immobilised.
1:20 mol fatty acid to mol ethanol (see Table 2 for the
exact range). Novozym 435® was able to catalyse this
reaction efficiently in the absence of solvent and in the
presence of a large amount of polar substrate (ethanol,
log P=−0.24).19 Except for the 2:1 ratio, all the reac-
tions reached approximately 80% conversion of 4-
methyloctanoic acid after 100 h. The enantiomeric
excess of the substrate was determined from the GC-
data and plotted versus the measured conversion.
In order to monitor changes in the structure of the
enzyme beads or increase in volume, Novozym 435®
was equilibrated with pure 4-methyloctanoic acid or its
ethyl ester. The hydrophobic material was studied
under a microscope. Because of sorption in the pores,
the hydrophobic beads changed from opaque into a
larger translucent structure (Fig. 2). At the same time,
the mean diameter increased. A reliable mean value of
the diameter was obtained by taking the average of
approximately 300 beads of both enzyme beads equili-
brated with 4-methyloctanoic acid or its ethyl ester, and
‘dry’ Novozym 435® beads. In Figure 3, the distribution
of the diameter was plotted for the three different cases.
For the wetted enzyme beads, the distribution shifted
towards the right, which implies an increase in diame-
ter. The Sauter mean diameter (d32) of the beads wetted
in 4-methyloctanoic acid and in 4-methyloctanoic acid
ethyl ester does not differ significantly since the confi-
dence intervals overlap (Table 1). Assuming spherical
The enantiomeric ratio, the equilibrium constant and
their confidence intervals were calculated with a com-
puter program13 that uses the Chen equation for com-
petitive Michaelis Menten kinetics for equilibrium
reactions.12 Figure 1 shows the calculated E values and
their confidence intervals for all fatty acid to ethanol
ratios studied. In most of the cases, there was poor
agreement between the Chen-model and the experimen-
tal data, resulting in large confidence intervals for the
estimated E values. For the ratio 1:8, a confidence
interval is shown from 0 to 140 because in this case the
Chen equation could not be used to fit the data. The
poor fits could not be attributed to enzyme inactiva-
tion, since the reuse of CALB showed the same enzyme
activity (results not shown).
It may be that a part of the substrate is not readily
available for the reaction, for instance because of
swelling of the enzyme support in the presence of
organic solvent. If this is the case, this leads to an
overestimation of the conversion. The liquid inside the
beads can act as a ‘dead’ volume if diffusion is slow
compared to the reaction time. However, an analysis of
the characteristic times for the processes that take place
revealed that slow diffusion cannot explain the effects
on the E-value in Figure 1 (results not shown). This
means that there must be another reason for our
anomalous results. Physical adsorption of the substrate
to the polymer beads would be a possibility. We con-
sulted the manufacturer of the enzyme carrier (Bayer
AG) and they were kind enough to confirm our suspi-
cion that sorption of the substrate to the carrier can
take place (Rudolf Wagner, personal communication).
This is also true for other hydrophobic molecules. In
these cases, part of the liquid is not available for
reaction. Therefore, we decided to study the swelling
behaviour in more detail and quantify these effects.
Figure 1. Esterification of 4-methyloctanoic acid with etha-
nol, catalysed by Novozym 435® at different ratios varying
from 2:1 up to 1:20 (mol fatty acid: mol ethanol). The
estimated E values and their confidence intervals were calcu-
lated using the equation of Chen et al.11,12 in the program
SIMFIT by Jongejan.13
2.2. Swelling of enzyme beads
Swelling of polymer materials in the presence of sol-
vents is a common and widely known phenomenon.20–25

N. W. J. T. Heinsman et al. / Tetrahedron: Asymmetry 14 (2003) 2699–2704
2701
Figure 2. (a) ‘dry’ Novozym 435®, (b) swollen Novozym 435® equilibrated in 4-methyloctanoic acid.
To determine the time needed for absorption/swelling,
the swelling of five different beads was taped on video.
For each bead, the relative volume is plotted against
time in Figure 4. Within 2 min, the volume reached its
maximum.
beads the volume of both the swollen and ‘dry’ beads
could also be calculated. From these calculations, it can
be concluded that the volume increased with 67 17 nL.
Assuming this increase is caused by sorption of acid
and no contraction takes place, the volume increase
(175–250%) is caused by 7.2 2.6 mmol of fatty acid per
gram of enzyme beads.
Since our esterification reactions took place on a time
scale of more than 100 h, it can be concluded that at
the initial stage of the esterification reaction the
enzymes were saturated with 4-methyloctanoic acid.
Although Figure 4 shows a wide range of maximal
relative volumes, the absolute volume increase, DV=
72 4.6 nL, equalled the value calculated from Table 2.
The large confidence interval is a consequence of the
fact that only five beads were investigated.
Table 1. Sauter mean diameter (d32) and volume of wetted
and ‘dry’ Novozym 435®
d32 (mm)
Mean volume (nL)
Dry beads
5.07×102 0.21×102
58.5 4.0
Beads wetted in acid 6.50×102 0.28×102 125.3 12.4
Beads wetted in ester 6.15×102 0.16×102 111.4 5.9
Figure 3. Frequency distribution of the calculated diameters from ‘dry’ CALB and CALB wetted either in 4-methyloctanoic acid
or in 4-methyloctanoic acid ethyl ester.

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Figure 4. Swelling of Novozym 435® due to the sorption of
4-methyloctanoic acid. The swelling was recorded for five
single beads (ꢀ, ꢁ, ꢂ, ", 2).
Figure 5. Esterification of 4-methyloctanoic acid with etha-
nol, catalysed by Novozym 435® at different ratios varying
from 2:1 up to 1:20 (mol fatty acid:mol ethanol). The esti-
mated E values and their confidence intervals were calculated
using the equation of Chen et al.11,12 in the program SIMFIT
by Jongejan.13 Note the difference in scale in Figure 1 and
Figure 5.
Table 2. Ratios of substrates used in the experiments
Ratio A:Ea
(mol:mol)
4-MOA
(mmol)
EtOH
(mmol)
(g)
(g)
role. Apparently, the affinity of the carrier for the
substrate is larger than for the product.
2:1
1:1
1:2
1:4
1.743
1.580
1.263
0.927
0.607
0.517
0.377
0.295
11.01
9.98
7.98
5.86
3.84
3.27
2.38
1.86
0.265
0.475
0.749
1.086
1.430
1.508
1.689
1.701
5.75
10.31
16.26
23.57
31.04
32.73
36.66
36.92
Figure 5 shows that with decreasing 4-methyloctanoic
acid concentrations, the enantiomeric ratio increases up
to an optimum of E=57 at 0.11 mol/mol. When the
4-methyloctanoic acid concentration is further
decreased (ratios: 1:10, 1:15 and 1:20 (mol:mol)) the
enantiomeric ratio drops. These results suggest that the
conformational stability of the C. antarctica lipase is
dependent on the protein-solvent interaction and the
enzyme structure.26–29 It may be that at relatively high
4-methyloctanoic acid concentrations the enzyme is in a
more hydrophobic environment leading to a more rigid
enzyme, thus enhancing the enantioselectivity. The rea-
son why the enantioselectivity decreases again if the
4-methyloctanoic acid concentration is lowered further
is not known. Berglund et al.30 observed a drop in E
value upon decreasing the concentration of 2-methylde-
canoic acid in the Candida rugosa lipase-catalysed ester-
ification of 1-heptanol, which was caused by the fact
that heptanol influences VSmax and VmRax in different
ways. So, in our system, two effects (conformational
versus kinetic) may be operating simultaneously, which
may be the reason for the observed optimum in E
value.
1:8
1:10
1:15
1:20
a A=4-methyloctanoic acid, E=ethanol.
2.3. Correction of the enantiomeric ratio
As described previously, during esterification of 4-
methyloctanoic acid, a part of the substrate diffuses
into the enzyme beads, causing swelling of these parti-
cles. The reaction under study is an equilibrium reac-
tion, therefore, the driving force for the release of this
absorbed substrate from the bead is considerably
smaller (if even present) than would be the case for
reactions that go to completion. This part of the sub-
strate is assumed not to take part in the reaction.
Therefore, the amount of substrate sorbed by Novozym
435® beads must be subtracted from the initial sub-
strate concentration so that the ‘true’ conversion can be
calculated. The average value of 7.2 mmol per g lipase
was used to correct the conversion for sorption and to
estimate the actual E values (Fig. 5). The data could
now be described adequately by the Chen equation.
Figure 5 shows the E value as a function of the
4-methyloctanoic acid concentration with relatively
small confidence intervals. This means that the sub-
strate sorption is playing a crucial role here; product
sorption, although possible, does not play a significant
2.4. Comparison with transesterification and hydrolysis
Previously, we presented an enantiomeric ratio of 23 in
the transesterification of 4-methyloctanoic acid methyl
ester to its butyl ester,17 also catalysed by Novozym
435c. In this reaction, the mixture for transesterification
consisted of 500 mM n-butanol and 100 mM 4-methyl-
octanoic acid methyl ester in an excess of octane (1:10).

N. W. J. T. Heinsman et al. / Tetrahedron: Asymmetry 14 (2003) 2699–2704
2703
A comparison of the estimated E for the plot of the
4.2. Enzyme
measured ees versus c and the measured ees versus eep
showed an E value of 23 for both plots. Based on these
findings, it is expected that substrate sorption did not
play an important role in this case. Both octane and
4-methyloctanoic acid are expected to sorb equally well
(personal communication, Bayer AG). Also because the
octane is in large excess, the loss of substrate is so small
that it apparently does not influence the results.
Candida antarctica lipase B (Novozym 435®) is a product
of NOVO Nordisk A/S Denmark. The support material
consisted of a divinylbenzene-crosslinked, hydrophobic
macroporous polymer based on methyl and butyl
methacrylic esters. The lipase is adsorbed onto the
surface of the support material by hydrophobic interac-
tion.
In hydrolysis reactions, the fatty acid production is
commonly measured using in-line titration. Although
4-methyloctanoic acid ethyl ester sorbs into Novozym
435®, the acid produced is extracted to the water phase.
In the bulk solution, the acid will be titrated with KOH,
driving the reaction towards completion. This was confi-
rmed by a separate experiment in which swollen beads
(containing 4-methyloctanoic acid) were titrated. The
total amount of acid that sorbed into the beads was
completely removed by titration. In cases like this, the
enantiomeric excess of the product (acid) should be used
to determine the enantiomeric ratio.19
4.3. Apparatus
Microscope: Olympus automatic exposure photo micro-
graphic system model PM-10AK, Olympus C35DA-2
photo camera (4×10), Magic Imaging MTI series 68.
Video: Olympus BH2 microscope, Sony model DXC-
151AP camera.
4.4. Esterification of 4-methyloctanoic acid
Esterification was carried out in 4 mL screw-cap vials
placed in a New Brunswick Scientific Innova™ incubator
at 35°C and 350 rpm for up to 100 h. The vials contained
the substrates 4-methyloctanoic acid and ethanol in
different molar ratios, as well as tetradecane (internal
standard, 0.05 g) and immobilised C. antarctica lipase B
(20–30 mg of CALB). The total volume was approxi-
mately 2 mL. During the course of the reaction 20 mL
samples were taken and diluted to 1 mL with octane,
containing 0.5% (v/v) acetic acid. The amounts used are
given in Table 2.
3. Conclusions
For the Novozym 435® catalysed esterification of 4-
methyloctanoic acid at different substrate concentra-
tions, we found that we could not fit the Chen equation
for equilibrium reactions to our data in a reliable way.
This was caused by the fact that part of the substrate
sorbed into the enzyme beads. Microscopic techniques
showed that the amount of 4-methyloctanoic acid sorbed
into Novozym 435® approximated 7.2 mmol per g lipase.
Since this amount does not take part in the esterification,
a correction must be made to calculate the actual
conversion. This resulted in reliable plots yielding E
values with an acceptable confidence interval. The
highest E value (57) was found for an acid/ethanol ratio
of 1:8 (mol:mol). The effect of substrate sorption was
only found important for esterification reactions; the
transesterification and hydrolysis reactions that were
investigated previously were not affected by sorption
effects.
4.5. GC analysis
Aliquots of 0.1 mL were analysed on a gas chro-
matograph using a GC-8000 series Fisons Instruments
(8160) with a MFC 800 control unit and an A200S CE
Instruments autosampler. The GC was equipped with a
chiral g-DEX™ 120 WCOT SUPELCO capillary column
(ID: 30m×0.25 mm, film: 0.25 mm).32 Hydrogen was used
as the carrier gas (70 kPa). A temperature program was
used to separate both the enantiomers of 4-methyloc-
tanoic acid and 4-methyloctanoic acid ethyl ester (T1=
75°C, t1=53 min, r1=5°C/min, T2=115°C, t2=33 min).
The peaks were detected by an FID (EL 980 Fisons
Instrument) set at 200°C. The split temperature was
225°C. The GC was connected to the computer program
Xchrom/Windows 4.0 version 2.11b for Windows NT
(ThIS Labsystems) for online data acquisition. Tetra-
decane was used as an internal standard in all measure-
ments. The retention times for 4-methyloctanoic acid
ethyl ester were 49.25 and 49.79 min [h=1.05], for
4-methyloctanoic acid 82.77 and 84.10 min [h=1.02]).
The liner of the GC was replaced by a new silylated liner
every week, since injection of fatty acids leads to a
desilylation reaction which gives rise to the adsorption
of the fatty acids to the glass. Liners were silylated in a
solution of dichlorodimethylsilane (5%) in toluene,
rinsed with methanol and dried. A piece of silylated glass
wool was placed into the liner to protect the main column
from contamination.
Novozym 435® (immobilised C. antarctica lipase B) is
one of the enzymes that are most often used in organic
chemistry.31 However, for other enzymes that are immo-
bilised on organic polymers, sorption of the substrate or
product should also be taken into account when perform-
ing kinetic measurements in solventless reaction systems.
4. Experimental
4.1. Chemicals
4-Methyloctanoic acid (4-MOA) was purchased from
Oxford Chemicals. Glacial acetic acid, toluene and
octane were obtained from Acros. Tetradecane was
purchased from Aldrich, ethanol was obtained from
Merck and dichlorodimethylsilane was supplied by
Sigma. All solvents were of analytical quality.

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4.6. Characterisation of CALB beads
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3. Wescott, C. R.; Klibanov, A. M. Biochim. Biophys. Acta
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5. Faber, K. In Biotransformations in Organic Chemistry, 4th
ed.; Faber, K., Springer Verlag: Berlin, Heidelberg, 2000;
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4.6.1. Weight. The mean weight of one enzyme bead
was determined by weighing 5 series of 10 beads and
taking the average. One bead of Novozym 435® (C.
antarctica lipase B, CALB) did have an average weight
of 5.4×10−2 1.0×10−2 mg and so 1 g of Novozym 435®
thus contained 18500 2900 beads.
6. Engel, K.-H. Tetrahedron: Asymmetry 1991, 2, 165–168.
7. Berglund, P.; Vo¨rde, C.; Ho¨gberg, H.-E. Biocatalysis 1994,
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1999, 10, 1821–1826.
4.6.2. Diameter and volume. Approximately 300 ‘dry’
CALB beads, 300 CALB beads soaked in 4-methyloc-
tanoic acid and 300 CALB beads soaked in 4-methyloc-
tanoic acid ethyl ester were scanned. For each bead the
diameter was determined using the program RESULTS
from Applied Imaging, version 5.1, 1991.
The Sauter mean diameter was calculated using:
11. Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am.
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12. Chen, C.-S.; Wu, S.-H.; Girdaukas, G.; Sih, C. J. J. Am.
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A. Recl. Trav. Chim. Pays-Bas 1991, 110, 247–254.
14. Rakels, J. L. L.; Romein, B.; Straathof, A. J. J.; Heijnen,
J. J. Biotechnol. Bioeng. 1994, 43, 411–422.
%ni·d3i
d32=
(1)
%ni·d2i
The mean volume was calculated using:
n
1
4
15. Rakels, J. L. L.; Straathof, A. J. J.; Heijnen, J. J. Enzyme
V=
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k=1 3
y·r3k
(2)
n
Microb. Technol. 1993, 15, 1051–1056.
16. Straathof, A. J. J.; Jongejan, J. A. Enzyme Microb Technol.
1997, 21, 559–571.
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de Sousa Teixeira, A.; Franssen, M. C. R.; van der Padt,
A.; Jongejan, J. A.; de Groot, Ae. Biocatal. Biotransform.
1998, 16, 145–162.
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Biocatal. Biotransform. 2001, 19, 181–189.
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van der Padt, A.; Franssen, M. C. R.; de Groot, Ae.; van’t
Riet, K. Biotechnol. Bioeng. 2001, 76, 193–199.
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with n=390 (dry CALB beads), 305 (CALB beads
saturated with 4-methyloctanoic acid) and 381 (CALB
beads saturated with 4-methyloctanoic acid ethyl ester).
4.6.3. Rate of swelling. The swelling of enzyme beads
was recorded on videotape. To one bead, viewed under
the microscope, a drop of 4-methyloctanoic acid was
added. The swelling was recorded in time. The volume
was calculated in time using Eq. (2).
4.7. Determination of E values
For the esterification of 4-methyloctanoic acid with
ethanol, the enantiomeric ratio was determined solely
using enantiomeric excess data of the substrate and the
extent of conversion. This was necessary because of the
poor separation of the 4-methyloctanoic acid ethyl ester
enantiomers on the GC. Plotting the ees versus the
conversion in the computer program SIMFIT13 gave
the enantiomeric ratio.
To determine the peak areas of the 4-methyloctanoic
acid enantiomers at t0, a sample was taken from the
mixture in the absence of lipase. In case of substrate
sorption, the initial amount of 4-methyloctanoic acid
(mol/g) was different from the one determined by GC
at t0. Therefore, the conversions that were calculated
from the GC data at time t and t0 were corrected for
4-methyloctanoic acid (mol/g) sorbed into the enzyme
beads. Plotting the ees versus the conversion in the
SIMFIT program13 gave new E values.
27. Berglund, P.; Holmquist, M.; Hult, K.; Ho¨gberg, H.-E.
Biotechnol. Lett. 1995, 17, 55–60.
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