Modulation of immobilized lipase enantioselectivity via chemical amination

Article abstract of DOI:10.1002/adsc.200600555

The aspartic and glutamic carboxylic groups of the surface of three different immobilized lipases (those from Candida antarctica (form B) (CAL-B), Thermomyces lanuginose (TLL) and Pseudomonas fluorescens (PFL) have been modified with ethylenediamine (after activation of the carboxylic function with carbodiimide). The exchange of groups with a negative charge for positively charged groups permitted us to greatly alter the activity, specificity and stereoselectivity of the lipase. Thus, in some cases, the activity of the lipases increased after the chemical modification while in other cases the activity was strongly reduced. Moreover, the effect of the experimental conditions on the activity was also strongly altered. Remarkably, the enantioselectivity of the enzymes in the hydrolysis of different mandel ic acid derivatives was strongly modulated. For example, amination of the CNBr-CAL-B preparation greatly increased the enantioselectivity of the enzyme in the hydrolysis of (±)-2-hydroxyphenylacetic acid methyl ester, from an E value of 2 without modification up to E>100, affording (R)-mandelic acid in high purity (ee>99% at 50% conversion) at pH 7 and 4°C. Thus, the chemical modification of lipases seems to be a very powerful tool to improve their performance as enantioselective biocatalvsts.

Full text of DOI:10.1002/adsc.200600555

FULL PAPERS
DOI: 10.1002/adsc.200600555
Modulation of Immobilized Lipase Enantioselectivity via
Chemical Amination
Jose M. Palomo,a Gloria Fernµndez-Lorente,a Jose M. Guisµn,a,*
and Roberto Fernµndez-Lafuentea,*
a
Departamento de Biocatµlisis, Instituto de Catµlisis (CSIC), Campus UAM, Cantoblanco, 28049 Madrid, Spain
Phone: (+34)-91-585-4809; Fax: (+34)-61-585-4760; e-mail: rfl@icp.csic.es or jmguisan@icp.csic.es
Received: October 28, 2006; Revised: February 27, 2007
Abstract: The aspartic and glutamic carboxylic ic acid derivatives was strongly modulated. For ex-
groups of the surface of three different immobilized ample, amination of the CNBr-CAL-B preparation
lipases (those from Candida antarctica (form B) greatly increased the enantioselectivity of the
(CAL-B), Thermomyces lanuginose (TLL) and Pseu- enzyme in the hydrolysis of (Æ)-2-hydroxyphenyl-
domonas fluorescens (PFL) have been modified with acetic acid methyl ester, from an E value of 2 with-
ethylenediamine (after activation of the carboxylic out modification up to E>100, affording (R)-man-
function with carbodiimide). The exchange of groups delic acid in high purity (ee>99% at 50% conver-
with a negative charge for positively charged groups sion) at pH 7 and 48C. Thus, the chemical modifica-
permitted us to greatly alter the activity, specificity tion of lipases seems to be a very powerful tool to
and stereoselectivity of the lipase. Thus, in some improve their performance as enantioselective bio-
cases, the activity of the lipases increased after the catalysts.
chemical modification while in other cases the activi-
ty was strongly reduced. Moreover, the effect of the Keywords: amination of protein surface; chemical
experimental conditions on the activity was also modification of immobilized lipases; improved activi-
strongly altered. Remarkably, the enantioselectivity ty; improved enantioselectivity; lipase modulation;
of the enzymes in the hydrolysis of different mandel- mandelic acid
Introduction
lipase becomes adsorbed to it and this equilibrium
shifts towards the open form (interfacial activation).[3]
The alteration of this equilibrium or the shape of
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3)
are the most used enzymes in organic chemistry be- the open form of the lipase could change the catalytic
cause they can recognize a wide diversity of substrates properties of the lipase, such as its selectivity. For ex-
with a high regio- and enantioselectivity. In this way, ample, this has been already accomplished by using
they have been successfully employed as catalysts for different immobilization protocols.[4] Here, taking into
kinetic resolutions of racemates (e.g., racemic secon- account that the movement of the lid involves differ-
dary alcohols, racemic carboxylic acids, etc) and in ent interactions among residues placed on the protein
asymmetric reactions.[1]
surface, (e.g., electrostatic interactions),[2] the altera-
Lipases may exist in two different structural tion of this movement by chemical modification of
forms.[2] One of them, in which the active site of the the protein surface has been proposed. The modifica-
lipase is secluded from the reaction medium by a heli- tion of carboxylic groups of the superficial Asp and
cal oligopeptide chain called “lid”, is considered an Glut residues by chemical amination[5] may produce
inactive (closed) form. The other one, which presents an alteration in the interaction between the lid and
the lid displaced and the active site exposed to the re- the rest of the protein (e.g., forming new ionic bridges
action medium, is considered to be the lipase in an or promoting repulsion between groups formerly in-
active (open) form. In homogeneous aqueous media, volved in ionic bridges).
the lipase molecule exists as an equilibrium between
Furthermore, it has been shown that small changes
these two structures. This equilibrium is, however, in the reaction conditions could exert an especially in-
shifted towards the closed form. Upon interaction tense effect on the properties of the lipases.[4,6] This
with a hydrophobic surface, such as a lipid droplet or should be a consequence of the change in the global
other hydrophobic surface, the open form of the interactions of the open-closed forms of lipases, alter-
Adv. Synth. Catal. 2007, 349, 1119 – 1127
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Jose M. Palomo et al.
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ing the exact shape of the open structure of the Results and Discussion
lipase. This effect of the experimental conditions
could also be altered on the chemically modified li- Analysis of the Lipase Surface
pases.
In this work, we have tried to study the prospects The surface of the three lipases was studied to deter-
of the chemical modification of lipases, previously co- mine the presence of Asp or Glu residues on areas
valently immobilized in a very mild fashion on a near the active centre, and the suitability of the pro-
CNBr-agarose support,[7] through amination by ethyl- posed strategy to alter the lipaseꢁs performance (Fig-
enediamine for the modulation of the properties of ures 1–3).
the lipase, especially its enantioselectivity.
The use of lipases that have been immobilized
under dissociation conditions (e.g., in the presence of
detergents) permitted us to avoid undesired lipase-
lipase interactions in the study of properties of the
lipase (e.g., specific lipase-lipase aggregation).[8] The
immobilization of the lipases on CNBr-agarose at 48C
and pH 7 in the presence of 0.1% detergent permit-
ted only a much reduced enzyme-support multi-inter-
action, keeping the lipaseꢁs properties almost unal-
tered and assuring the immobilization of lipaseꢁs indi-
vidual molecules. Moreover, the use of immobilized
proteins prevents any inactivation of the lipases due
to aggregation.[5]
To check the feasibility of this technique, we have
used the kinetic resolution of two derivatives of a-hy-
droxyphenylacetic acid as a model reaction, (Æ)-a-hy-
droxyphenylacetic acid methyl ester (1) (asymmetric
centre in the acyl donor site) and (Æ)-2-O-butyryl-2-
phenylacetic acid (2) (asymmetric centre in the acyl
acceptor site). (R)-a-Hydroxyphenylacetic acid (man-
delic acid) (3) is an important precursor of cephalo-
sporin antibiotics such as Cefamandole and Cefoni-
cid.[9] Moreover, it is a key intermediate in the synthe-
sis of (+)-goniodiol, a potent and selective cytotoxic
agent against human lung carcinoma[10] (Scheme 1)
Figure 1. 3D surface model structure of CAL-B. a) lid face,
and other interesting products.[11]
b) lateral face. Asp or Glu residues (green), lid oligopeptide
(blue), catalytic serine (red); c) Representation of the active
site surrounded of the CAL-B marking the key amino acid.
The 3D structure was obtained from the Protein Data Bank
(PDB) using Pymol vs. 0.99. The pdb code for CAL-B is
TCA.
Three widely used lipases from different sources
were selected as model enzymes, those from Candida
antarctica (form B) (CAL-B),[12] Thermomyces lanugi-
nose (TLL),[13] and Pseudomonas fluorescens
(PFL).[14]
Scheme 1. Synthesis of biologically active compounds from (R)-mandelic acid.
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The open structure of the CAL-B (Figure 1) shows
several Asp and Glu groups that are placed in impor-
tant areas of the protein. Going into detail, it may be
remarked that Asp 145 – the amino acid placed in the
lid – can establish hydrogen bonds with Ser 150 and
Thr 158 in both lipase conformations (Figure 1c).
Moreover, Asp 223 and Glut 188 are two negatively
charged groups very near to the active centre, so in
this case the chemical amination will change this neg-
atively charged group into a larger and positively
charged group, perhaps influencing the movements of
this area of the protein during catalysis.
Figure 2 shows the different sides of the 3D-struc-
ture of TLL. We can observe that this enzyme pres-
ents a very high number of Asp/Glut residues on its
surface, even presenting some of them in the “lid” or
in areas near to the active centre.
Figure 3. 3D surface model structure of PFL. a) lid face, b)
lateral face. Asp or Glu residues (green), lid oligopeptide
(blue), catalytic serine (red).The 3D structure was obtained
from the Protein Data Bank (PDB) using Pymol vs. 0.99.
The pdb code for PFL is 2LIP.
Table 1. Effect of the amination in the enzymatic activity of
different lipases immobilized on CNBr in the hydrolysis of
pNPB at pH 7. Other specifications as described in methods.
Finally, PFL presents Asp and Glu residues close to
the lid but not so many in the surroundings of the
active site (Figure 3).
Lipase
Modification
Relative activity[a] [%]
Thus, it may be expected that the three different li-
pases have enough Asp and Glu groups in critical po-
sitions to be good models for the potential application
of this strategy to modulate lipases via chemical modi-
fication of these groups.
CALB
CALB
CALB
TLL
TLL
TLL
PFL
PFL
PFL
NO
100
100
200
100
190
350
100
60
PARTIAL
TOTAL
NO
PARTIAL
TOTAL
NO
PARTIAL
TOTAL
Effect of the Chemical Amination of Immobilized
Lipases on Catalytic Activity
30
[a]
Ratio between the activity of the unmodified enzyme
and the modified enzyme in%.
Table 1 shows the initial activity displayed by differ-
ent lipase preparations in the hydrolysis of p-NPB (an
ester with a good leaving group), at pH 7 and 258C.
In the case of CAL-B and TLL immobilized on agar-
ose activated with CNBr, an increase in the specific
activity was observed after chemical treatment. A
direct correlation between the modification degree
and the activity increase was found, multiplying the
activity by three in the case of TLL.
However the CNBr-PFL preparation underwent a
significant decrease in enzyme activity depending on
the modification degree. Thus the immobilized
enzyme maintained only 30% of the initial activity
after full amination.
Therefore, the effect of this modification on the ac-
tivity of the lipases in the hydrolysis of simple sub-
strates was quite dissimilar when comparing different
enzymes, from a noticeable increase to a significant
decrease.
Figure 2. 3D surface model structure of TTL. a) lid face, b)
lid opposite face c) lateral face. Asp or Glu residues
(green), lid oligopeptide (blue), catalytic serine (red).The
3D structure was obtained from the Protein Data Bank
(PDB) using Pymol vs. 0.99. The pdb code for TLL is
1DTB.
Effect of the Amination on the Enantioselectivityof
the CNBr-CALB Immobilized Preparation Catalyzed
Hydrolysis of (Æ)-1 and (Æ)-2
The specific activities and enantiomeric ratio values
(E) of the CNBr-CALB immobilized preparation with
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Table 2. Effect on the the amination grade in the enantioselectivity of the CNBr-CALB immobilized preparation.
Aminated Biocatalyst
Experimental Conditions
Substrate
Initial Rate[a]
c[b]
E Ratio
Not modified
Partially modified
Fully modified
Not modified
Fully modified
Not modified
Fully modified
Not modified
Fully modified
Not modified
Fully modified
Not modified
Fully modified
pH 7, 258C
1
1
1
1
1
1
1
1
1
2
2
2
2
180
139
90
93
83
54
28
18
15
0.83
0.26
0.11
0.05
16
20
16
15
14
14
15
18
15
15
11
10
12
4.4
8.5
10
8
12
pH 5, 258C
pH 7, 48C
pH 5, 48C
pH 7, 258C
pH 7, 48C
2
>100
45
>100
1
2.6
1
2.5
[a]
the initial rate in UI/h (”10À3).
c=conversion.
[b]
different amination degrees in the kinetic hydrolysis 258C, the modified enzyme showed higher activity to-
of (Æ)-1 and (Æ)-2 at different conditions are shown wards pNPB than the non-modified enzyme while the
in Table 2.
expressed activity was lower with substrates (Æ)-1
In the hydrolytic resolution of (Æ)-1, the effect of and (Æ)-2.
the modification on the enzyme activity was quite dif-
If we divided the enzyme activity corresponding to
ferent to that detected using pNPB. The partial ami- substrates (Æ)-1 or (Æ)-2 by the enzyme activity
nation of the enzyme surface produced a moderate against pNPB, we can calculate the specificity of the
decrease of the enzyme activity (around 25%), and enzyme by the different substrates. To simplify the
the full modification produced a further decrease in comparison among the different preparations, we
the enzyme activity (50%). The decrease in tempera- have given a relative ratio of 1 to the value obtained
ture from 25 to 48C decreased the enzyme activity by using the non-modified enzyme. Thus, fully aminated
around 65% for non- and full aminated enzymes. enzymes give a value of 0.5 and 0.3, respectively, that
Nevertheless, the effect of changing the pH value – is, the enzyme activity is negatively relatively affected
from pH 7 to pH 5 – was very different. The non- by the amination when using less suitable substrates
modified enzyme suffered a decrease in activity up to for the enzyme.
50% and 33% at 258C and 48C, respectively, while
These results in enzyme activity and specificity sug-
the fully aminated enzyme almost maintained its ac- gested that the amination of the CNBr-CALB prepa-
tivity unaltered at 258C and retained more than 50% ration had altered its catalytic properties, but the
of its initial activity if the change in the pH was per- most interesting property that we intend to alter (and
formed at 48C.
if possible to improve) is the enantioselectivity. Thus,
Using substrate (Æ)-2, which presents a negative the enantioselectivity of the different preparations
charge at pH 7 by the carboxyl group (Scheme 3), the was evaluated in the hydrolysis of (Æ)-1 (Table 2).
situation is slightly different. Full chemical modifica- All enzyme preparations favoured the hydrolysis of
tion of the immobilized lipase produced a reduction the R isomer, but the E values depended strongly on
in the activity in 69% at 258C and around 50% at the preparation employed (Scheme 2).
48C.
The unmodified CNBr-CALB preparation present-
Interestingly, the substrate specificity of the enzyme ed a quite low E value at pH 7 and 258C (E=4.4).
changed when comparing the activities of the aminat- The decrease in the pH value to pH 5 improved the
ed and non-aminated enzymes. Thus, at pH 7 and enantiomeric ratio up to 8, although when the reac-
Scheme 2. Enzymatic kinetic resolution of (Æ)-1.
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Modulation of Immobilized Lipase Enantioselectivity
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Scheme 3. Enzymatic kinetic resolution of (Æ)-2.
tion was performed at 48C, the E value of the biocat- drop in the enzyme activity (by a 7-fold factor). This
alyst reached 45. At pH 7, the decrease in tempera- result was unexpected since the lowest modification
ture produced a decrease in the E value from 4.4 to 2. presented the highest effect on lipase activity, suggest-
Partial amination permitted us to increase the E ing that the decrease in activity is not due to the
value to 8.5.
direct effect on relevant groups in the proteinꢁs active
The fully aminated CAL-B immobilized prepara- centre (that obviously will be also modified in the
tion gave better values of enantiomeric ratio than the more aminated enzyme) but due to an alteration of
unmodified enzyme in all cases, permitting us to the enzyme structure, perhaps by altering the exact
obtain an E>100 (ee>99%) at 48C at both pH shape of the open form of the enzyme. In fact, these
values (Table 2).
lipases having only 50% of the carboxylic groups
Interestingly, this fully aminated enzyme, in opposi- modified may be the ones with more “new” interac-
tion to the behaviour of the unmodified CNBr-CALB, tions.
improved the E value at pH 7 when decreasing the T,
from E=10 to >100.
Using substrate (Æ)-2, full modification of external
carboxylic acids in the immobilized lipase produced a
In the hydrolysis of (Æ)-2 (Table 2), the unmodified slight increase in the activity at pH 7 and 258C (but
CNBr-CALB preparation was not selective at all, only by 10%). However, at pH 5 and 258C, the activi-
whereas full chemical amination permitted us to ties of the different immobilized preparations showed
obtain a slight selectivity towards the R isomer an increase, higher in the fully aminated one, by more
(Scheme 3).
than a two-fold factor.
In this case the substrate specificity of the enzyme
has been changed. The full modification of the
enzyme, which improves the activity against pNPB by
a three-fold factor, presented an almost null effect on
the activity against the other substrates at pH 7, with
a slight decrease using (Æ)-1 and slight increase using
Effect of the Amination on the Enantioselectivityof
the CNBr-TLL Immobilized Preparation Catalyzed
Hydrolysis of (Æ)-1 and (Æ)-2
Results are summarized in Table 3. Full modification (Æ)-2.
of the immobilized enzyme promoted a slight de-
When the enantioselectivity factor of the different
crease in the enzyme activity compared to substrate TLL-immobilized preparations was studied in the hy-
(Æ)-1 (by 10%) at the different conditions evaluated, drolysis of (Æ)-1 (Scheme 2), the unmodified CNBr-
while the partial amination produced an important TLL preparation presented a low E value at pH 7
Table 3. Effect on the amination grade in the enantioselectivity of the CNBr-TLL immobilized preparation.
Aminated Biocatalyst
Experimental Conditions
Substrate
Initial Rate[a]
c[b]
E Ratio
Not modified
Partially modified
Fully modified
Not modified
Fully modified
Not modified
Fully modified
Not modified
Fully modified
pH 7, 258C
1
1
1
1
1
2
2
2
2
0.9
0.13
0.8
0.34
0.31
0.40
0.44
0.70
1.75
15
12
13
10
17
18
17
10
13.5
3.5
1.2
6.5
1.9
3.6
3.0
6.0
1.1
1.3
pH 5, 258C
pH 7, 258C
pH 5, 258C
[a]
The initial rate in UI/h (” 10À3).
c=conversion.
[b]
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Table 4. Effect on the amination grade in the Enantioselectivity of the CNBr-PFL immobilized preparation.
Aminated Biocatalyst
Experimental Conditions
Substrate
Initial Rate[a]
c[b]
E Ratio
Not modified
Partially modified
Fully modified
Not modified
Fully modified
Not modified
Fully modified
pH 7, 258C
1
1
1
2
2
2
2
0.22
0.036
0.33
0.41
0.32
0.15
0.11
11
12
17
19
18
12
13
1
1.1
1.7
2.0
3.4
29
12
pH 7, 258C
pH 5, 258C
[a]
The initial rate in UI/h (”10À3).
c=conversion.
[b]
(E=3.5), which was reduced when the reaction was ing a certain selectivity towards the R isomer
performed at pH 5 (E=1.9). The amination on the (Scheme 2). At pH 5, all preparations presented a
TLL immobilized preparation improved the E values very low activity against substrate 1 and could not be
by around a two-fold factor (Table 3). Curiously, a used in this reaction.
partial amination gave a decrease in the E value
(from 3.5 to 1.2).
Using substrate (Æ)-2 (Scheme 3), the aminated
immobilized preparation presented better E value at
Using substrate (Æ)-2 (Scheme 3), the amination pH 7 compared to the unmodified one (E=3.4 versus
produced an improvement in the E value for the TLL 2). When the reaction was performed at pH 5, the
preparation, especially at pH 7 (from
(Table 3).
3
to 6) enantioselectivity of both preparations increased, al-
though the highest E value obtained was found when
using the non-aminated one (E=29) (Table 4).
Effect of the Amination on the Enantioselectivityof
CNBr-PFL Catalyzed Hydrolysis of (Æ)-1 and (Æ)-2
Production of EnantiomericallyPure ( R)-Mandelic
Acid
The specific activities and enantiomeric ratio values
of the CNBr-PFL immobilized preparation with dif- The chemical amination of the CNBr-CAL-B immobi-
ferent amination degrees in the kinetic hydrolysis of lized preparation has permitted us to obtain an enan-
(Æ)-1 and (Æ)-2 at different conditions are shown in tioselective biocatalyst (E>100%) for the prepara-
Table 4.
Full modification of the enzymes promoted a signif-
icant increase in the enzyme activity (by a 1.5-factor)
in the hydrolysis of (Æ)-1, while the partial amination
of the enzyme provoked a very significant reduction
in the enzyme activity (by 85%). Again, this inverse
relation between activity variation and modification
degree suggested that the chemical modification did
not have a direct effect on chemical groups related to
the enzyme activity, but may be altering the percent-
age or the shape of the open form of the lipase. In
contrast, the effect of full modification on the enzyme
activity was negative when using substrate 2, reducing
the activity by around 25% at both pH values.
Following this procedure, the enzyme specificity
was altered again, in this case favouring substrate 1
(relative specificity was 4.5 for the full modified prep-
aration comparing substrate (Æ)-1 and pNPB, and 1
for the non-modified enzyme).
Figure 4. Evolution of enantiomeric excess (ee) of 1 and 3
With respect to the enantioselectivity towards (Æ)-
1 (Table 4), the unmodified CNBr-PFL preparation
did not present a significant E at pH 7, that is im-
versus hydrolysis conversion of (Æ)-1 catalyzed by aminated
BrCN-CAL-B immobilized preparation. Experiments were
performed using 10 mM of substrate at pH 7 and 48C. ee of
proved after full amination of the preparation, detect- [(R)-1] (square), ee of released acid [(R)-3] (rhombus).
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Modulation of Immobilized Lipase Enantioselectivity
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tion of enantiomerically pure (R)-mandelic acid [(R)- Enzymatic Kinetic Assay
3], a precursor of different biologically active com-
This assay was performed by measuring the increase in the
absorbance at 348 nm produced by the release of p-nitro-
phenol in the hydrolysis of 0.4 mM pNPB in 25 mM sodium
phosphate buffer at pH 7 and 258C. To initialize the reac-
tion, 0.05 mL of lipase solution or suspension were added to
2.5 mL of substrate solution. One international unit (IU) of
pNPB activity was defined as the amount of enzyme that is
necessary to hydrolyze 1 mmol of pNPB per minute under
the conditions described above.
pounds (Scheme 1). Figure 4 shows the catalystꢁs per-
formance; the ee value of both the acid and the ester
is around 99% at 50% conversion.
Conclusions
Chemical modification of lipase surfaces may be a
simple and powerful tool to modify their specificity.
Amination has permitted us to improve the enzyme
activity against several substrates and has given a sig-
nificant increment in the E values in some instances.
In this way, the amination of the CNBr-CAL-B prepa-
ration permitted us to greatly modulate its enantiose-
lectivity from an E value of 2 without modification up
to E>100, affording (R)-mandelic acid in high purity
(ee>99% at 50% conversion) at pH 7 and 48C.
Therefore, the results presented in this paper – on
the potential of chemical modification of the lipases
surface – together with the previous results on the
effect of the immobilization of lipases following dif-
ferent strategies,[4] show that the coupled use of physi-
cochemical tools may be a simple and rapid way of
preparing a small library of biocatalysts with very dif-
ferent properties, significantly increasing the probabil-
ities of finding a biocatalyst useful for the resolution
of any racemate. This methodology could be consid-
ered as a very useful tool in the combinatorial bioca-
talysis background.[17]
Immobilization of Lipases on CNBr-Activated
Support
Commercial agarose support activated with CNBr was dis-
solved in an acidic aqueous solution (pH 2–3) for one hour.
After that the solid support is filtered by vacuum. 1 mL of
the purified lipase solution containing a concentration of
10 mg protein/mL was added to 20 mL of 25 mM sodium
phosphate buffer solution at pH 7 containing 0.1% Triton
X-100. After that, 1 g of the CNBr-agarose support was
added. The mixture was then shaken at 48C and 250 rpm for
18 h. After that, the solution was removed by filtration and
the supported lipase was washed several times with distilled
water. In all cases, the immobilization percentage was more
than 95%. The immobilization was followed by the assay
described above.
Chemical Amination of Immobilized Enzymes
One g of immobilized enzyme was added to 50 mL solution
of EDA 1M in buffer at pH 4.75 (1/5 v/v). Solid EDC was
added to the suspension to different final concentrations,
10À2 M (fully aminated biocatalyst) or 10À3 M (partially ami-
nated biocatalyst).[5] After 90 min of gentle stirring at 258C,
the immobilized-modified preparations were filtered and in-
cubated for 4 h with a 0.1M hydroxylamine solution at pH 7
to recover the modified tyrosines.[16] The enzyme prepara-
tions were filtered and washed with 25 mM potassium phos-
phate buffer at pH 7.5 and an excess of distilled water. The
aminated derivatives were stored at 48C. Scheme 4 shows
the reaction. We have checked that the colour developed by
the reaction with picrylsulfonic acid with the derivatives pre-
viously modified with glutaraldehyde (to eliminate endoge-
nous primary amino groups) were doubled in strength when
using 10 mM EDA than when using 1 mM EDA for the 3
immobilized enzymes.
Experimental Section
General Remarks
The lipases from Candida antarctica B (CAL-B) and Ther-
momyces lanuginosa (TTL) were from Novo Nordisk (Den-
mark). The lipase from Pseudomonas fluorescens (PFL) was
from Amano. Cyanogen bromide (CNBr-activated Sephar-
ose 4BCL) was purchased from Pharmacia Biotech (Uppsa-
la,Sweden). p-Nitrophenyl butyrate (pNPB), 1-ethyl-3-(3-di-
methylaminopropyl)-carbodiimide (EDC), ethylenediamine
(EDA) and (Æ)-a-hydroxyphenylacetic acid methyl ester
(mandelic acid methyl ester) (1) were purchased from
Sigma. NMR data were recorded on a Bruker AC-300 (1H
at 250 MHz, 13C at 75.5 MHz) spectrometer at room temper-
ature. MALDI-TOF mass spectra were recorded with a
Voyager-DE Pro BioSpectrometer from PerSeptive Biosys-
tems with 2,5-dihydroxybenzoic acid (DHB) as the matrix.
Synthesis of 2-O-Butyryl-2-phenylacetic Acid [(Æ)-2]
A solution of butyryl chloride (20 mmol) in diethyl ether
(100 mL) was added dropwise to a stirred solution of (Æ)-3
(22 mmol) in diethyl ether (200 mL) with NEt3 (22 mmol).
The mixture was stirred at 258C for 4 h and the reaction
was followed by HPLC. The mixture was extracted with
neutral aqueous solution and, after re-extraction of the
water phase with Et2O, the combined organic solvents were
dried with MgSO4 and the solvent was evaporated. The
crude was washed several times with cold ether (5”2 mL)
Purification of the Lipases
The different lipases were purified prior to use. The used
strategy was the interfacial adsorption on octyl-agarose sup-
port at low ionic strength and a second step of desorption
using 1% Triton X-100 following a previously described
procedure.[15]
1
and dried under vacuum; yield: 60%. H-NMR (250 MHz,
CDCl3): d=7.4 (m, 4H), 5.9 (s,1H, CH), 2.4 (m, 2H, aCH2),
1.7 (septet, 2H, bCH2), 0.95 (m, 3H, CH3); 13C NMR
Adv. Synth. Catal. 2007, 349, 1119 – 1127
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1125

Jose M. Palomo et al.
FULL PAPERS
Scheme 4. Chemical amination of protein carboxyl groups. EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride.
(CDCl3): d=173 (C=O), 170 (C=O), 131.8 (C), 127.8 (CH),
127.3 (CH), 126.1 (CH),76.1 (CH), 34.2 (CH2), 16.8 (CH2),
12.1 (CH3); MS (MALDI-TOF+): m/z=245.7, calcd. for
C12H14O4 [M+Na]+: 245.1.
racemic mixture) at hydrolysis degrees between 15 and
20%.
Acknowledgements
Enzymatic Hydrolysis of Esters
This work has been sponsored by the Spanish CICYT (pro-
jects BIO-2005–8576). We gratefully recognize CSIC by I3P
contracts for Dr Palomo and Dr Fernandez-Lorente
(FEDER founds). The authors would like to thank Dr.
Angel Berenguer Murcia (Universidad de Alicante) for his
help during the writing of this paper.
The activities of the different immobilized lipase prepara-
tions on the hydrolysis reaction of different esters were per-
formed by adding 0.2 g of catalyst in 10 mL of (Æ)-1 (5
mM) and 0.5 g of catalyst in 6 mL of (Æ)-2 (0.5 mM) under
different conditions (pH, T) under mechanic stirring.
During the reaction, the pH value was maintained con-
stant by automatic titration and the enzymatic activity
(mmol of substrate hydrolyzed per hour per mg of immobi-
lized protein) was evaluated from NaOH consumption using
a pH-stat Mettler Toledo DL50 graphic system. The degree
of hydrolysis was confirmed by reverse-phase HPLC (Spec-
tra Physic SP 100 coupled with a UV detector Spectra
Physic SP 8450) on a Kromasil C18 (25”0.4 cm) column
supplied by Analisis Vinicos (Spain). At least triplicate runs
of each assay were made. The elution was isocratic with a
mobile phase of acetonitrile (30%) and 10 mM amonium
phosphate buffer (70%) at pH 2.95 and a flow rate of
1.5 mLminÀ1. The elution was monitored by recording the
absorbance at 254 nm [substrate (Æ)-1] or 225 nm [substrate
(Æ)-2].
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