Evaluation of the lipase from Bacillus thermocatenulatus as an enantioselective biocatalyst

Article abstract of DOI:10.1016/j.tetasy.2003.10.010

The lipase from Bacillus thermocatenulatus (BTL2) has been immobilized using different techniques, and used in the resolution of different chiral substrates in hydrolytic reactions. This enzyme acts as an interesting biocatalyst for these types of reactio

Full text of DOI:10.1016/j.tetasy.2003.10.010

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 3679–3687
Evaluation of the lipase from Bacillus thermocatenulatus as an
enantioselective biocatalyst
Jose M. Palomo,a Gloria Ferna´ndez-Lorente,a Maria L. Ru´a,b Jose´ M. Guisa´na,* and
Roberto Ferna´ndez-Lafuentea,*
aDepartmento de Biocatalisis, Instituto de Catalisis. CSIC, Campus Autonoma University, 28049 Madrid, Spain
bArea de Bioqu´ımica y Biolog´ıa Molecular, Facultade de Ciencias de Ourense, Universidade de Vigo, As Lagoas s/n, 32004,
Ourense, Spain
Received 14 July 2003; revised 2 September 2003; accepted 16 October 2003
Abstract—The lipase from Bacillus thermocatenulatus (BTL2) has been immobilized using different techniques, and used in the
resolution of different chiral substrates in hydrolytic reactions. This enzyme acts as an interesting biocatalyst for these types of
reactions, whose catalytic properties can be further improved via the use of different immobilization techniques and reaction
conditions. In this way, once the best immobilization techniques and reaction conditions has been chosen, the enzyme can be used
to resolve 2-O-butyryl-2-phenylacetic acid ( )-5 with high enantiomeric excess of the release product (ee>99%, E>100) in the
hydrolysis catalyzed by octadecyl-Sepabeads-BTL2 preparation (at 4°C pH 7 or 9) or glyoxyl-BTL2 preparation (at 4°C pH 7),
yielding (R)-mandelic acid. The immobilized preparations of BTL2 presented high sensitivity to the experimental conditions [e.g.,
BTL2 immobilized on glyoxyl-agarose improve E value from 11 (37°C) to >100 (4°C)]. In the resolution of 2-hydroxy-4-phenyl-
butyric acid ethyl ester ( )-12 the use of octadecyl-Sepabeads-BTL2 in the presence of 20% acetonitrile gave 2-hydroxy-4-phenyl
ethyl butyrate (S)-12 by hydrolyzing the racemic ester to 60% (ee of remaining ester over 90%).
© 2003 Elsevier Ltd. All rights reserved.
Bacillus,14,15 e.g. lipases from B. thermocatenulatus. The
1. Introduction
thermophile B. thermocatenulatus produces two lipases,
BTL1 and BTL2.16a The gene for the BTL2 lipase has
Lipases (glycerol-ester-hydrolase, EC 3.1.1.3) are
enzymes which catalyze the hydrolysis of fatty acid
been cloned and expressed in Escherichia coli.16b The
BTL2 lipase is also a 43 kDa protein (predicted from
the DNA sequence) and shows high stability at medium
temperatures (50°C), alkaline pH (9.0–11.0) and in
organic solvents (2-propanol, acetone, methanol).
These properties suggest that it can be used as a sturdy
biocatalyst for organic synthesis. In the literature, data
about its selectivity towards several chiral substrates
showed17 that this hydrolase gives moderate enantiose-
lectivity toward primary alcohols, in spite of its high
enantioselectivity with aryl-secondary alcohols. Thus,
taking into account these advances, we herein report
the selectivity of this interesting lipase, in resolutions of
racemic mixtures of different compounds, key interme-
diates in the synthesis of drugs, in aqueous media,
which until now had not been studied with this lipase,
e.g. ( )-2-hydroxyphenylacetic acid methyl ester ( )-4
or O-butyryl-2-phenylacetic acid ( )-5, direct precur-
sors of the (R)-isomer of mandelic acid 3, a key inter-
mediate in the synthesis of the styryllactone
(+)-goniodiol [(1%R,2%S,5R)-5-(1%,2%-dihydroxy-2-phenyl-
ester bonds in triacylglycerol to give fatty acids, diac-
ylglycerols, monoacylglycerols and glycerol. These
enzymes are widely used in organic chemistry because
of their high specificity, recognizing very different sub-
strates, sometimes with high regio and enantioselectiv-
ity; as a consequence they are used in the resolution of
many racemic compounds (acids, alcohols, amines,
etc.).1–9 However, many lipases are only moderately
stable at high temperature, drastic pHs, etc. can reduce
their usefulness in some interesting reactions. This can
be solved by using lipases from thermophilic microor-
ganisms, whose resistance to drastic conditions has
been developed by nature.10–13
At present, the majority of the thermophilic lipases that
has been purified and characterized are obtained from
* Corresponding author. Tel.: +34 91 585 48 09; fax: +34 91 585 47
60; e-mail: jmguisan@icp.csic.es; rfl@icp.csic.es
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2003.10.010

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J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 3679–3687
the synthesis of (S)-Enalapril, (S)-1-[N-[1-ethoxycar-
bonyl-3-phenylpropyl]- -alanyl]- -proline (S)-14, a pro-
drug that presents an important relevance in the control
of hypertension and in the treatment of cardiac
deficiency (Scheme 4).
ethyl)-pent-2-eno-5-lactone)] (+)-1, which exhibits a
potent and selective cytotoxicity against human lung
carcinoma with no significant toxicity against brine
shrimp18 (Scheme 1); ( )-trans-4-(4%-fluorophenyl)-6-
oxopiperidin-3-ethyl carboxylate ( )-trans-8 an inter-
mediate in the synthesis of (−)-Paroxetine, used in the
treatment of depression, obsessive compulsive disorder
and panic disorder19 (Scheme 2); ( )-6-(5-Chloropy-
ridin - 2 - yl) - 7 - oxo - 5 - (vinyloxycarboyloxy) - 5,6 - dihy-
dropyrrolo[3,4b]pyrazine ( )-10, which is an interesting
intermediate in the synthesis of (+)-Zopiclone (4-
L
L
Special interest has also been shown regarding the
likely modulation of the properties of the lipase by
using different enzyme immobilized preparations that
have been used successfully with mesophilic lipases.21–26
This modulation is based on the alteration of the
dramatic conformational changes in the enzyme struc-
ture that lipases suffer during catalysis27–30 due to the
different orientation of the enzyme on the support, the
degree of rigidification or the alteration of the enzyme
environment.
methyl-1-piperazinecarboxylic
acid
6-(5-chloro-2-
pyridinyl)-6,7-dihydro-7-oxo-5H-pyrrolo-[3,4b]pyrazin-
5-yl ester) (+)-920, a hypnotic agent (Scheme 3). 2-
Hydroxy-4-phenylbutyric acid ethyl ester ( )-12 was
also used, because it is a very important intermediate in
Scheme 1. Retrosynthetic pathway of (+)-Goniodiol.
Scheme 2. Retrosynthetic pathway of (−)-Paroxetine.
Scheme 3. Synthetic pathway of (S)-(+)-Zopiclone.

J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 3679–3687
3681
Scheme 4. Retrosynthetic pathway of (S)-Enalapril.
Table 1. Specific activity of different immobilized preparations of BTL2 in the hydrolysis of different compounds at pH 7.
Specific activity: (mmol/h mgprot). The relative error was estimated by 3%
Immobilized preparation
Ethyl butyrate
p-NPP
( )-4
( )-5
( )-8
( )-10
( )-12
Octadecyl-BTL2
Glyoxyl-BTL2
PEI-BTL2
0.76
0.1676
0.266
0.24
0.137
0.14
0.0217
5.48×10−3
nd
0.038
–
–
–
–
–
–
2.38×10−3
0.0236
nd
6.52×10−3
5.08×10−3
2. Results
2.2. Enantioselective resolution of ( )-4 and ( )-5, key
intermediates in the synthesis of (+)-Goniodiol, cata-
lyzed by immobilized BTL2
2.1. The screening of activity of BTL2 on different
compounds precursors of drugs
Table 2 shows the results obtained in the resolution of
( )-4 catalyzed by different BTL2 preparations at pH 7
and 25°C. The glyoxyl-BTL2 preparation presented an
enantiomeric excess (ee) of 64% at 10% conversion,
giving the highest enantiomeric ratio (E=4.5), while the
octadecyl-sepabeads-BTL2 only showed a low ee of
16% (E=1.03) at 11% of conversion.
The catalytic activity of BTL2 against different esters at
pH 7 and 25°C was analyzed (Table 1). The lipase,
immobilized on different supports catalyzed the hydro-
lysis of ethyl butyrate and pNPP and gave better activ-
ities when using the chiral substrates ( )-4, ( )-12 (with
the stereogenic centre on the acyl-donor) and ( )-5
(with the stereogenic centre on the acyl chain). How-
ever, the lipase could not hydrolyze compounds ( )-8
and ( )-11, even though they have been successfully
tested with other enzymes.29–33
Table 2. Hydrolytic resolution of ( )-4 catalyzed by BTL2
immobilized at pH 7 and 25°C. The relative error was
estimated by 4%
Immobilized preparation
Conversion
(%)
SP
ee
(%)
E
For all enzyme preparations, the highest activity was
found when using ethyl butyrate as the substrate whilst
the lowest was found against compound 4. The octade-
cyl-Sepabeads-BTL2 preparation presented the highest
activity against most of the compounds, being three
times lower with pNPP and 20 times lower with com-
pound 4 with respect to the activity with ethyl butyrate.
Furthermore, the octadecyl-Sepabeads-BTL2 prepara-
tion was three times more active than PEI-BTL2 prepa-
ration and five times more active with respect to the
glyoxyl-BTL2 preparation against ethyl butyrate. How-
ever, when using pNPP as the substrate, the activity of
the octadecyl-Sepabeads-BTL2 was only a two fold-fac-
tor higher than the other immobilized preparations
which presented similar catalytic activity between them.
With substrates ( )-4 and ( )-5, the octadecyl-Sepa-
beads-BTL2 preparation was around five times more
active than the other immobilized preparations, which
presented similar activities. However, when using com-
pound 12 the glyoxyl-BTL2 was ten times more active
than octadecyl-Sepabeads-BTL2 in the standard condi-
tions analyzed.
Octadecyl-Sepabeads-BTL2
Glyoxyl-BTL2
11
10
R
R
16
64
1.03
4.5
SP: stereochemical preference; ee=enantiomeric excess of the
product; E=value of enantioselectivity.
Table 3 shows the analysis of the enantioselectivity of
different immobilized preparations of BTL2 at pH 7
and different temperatures with substrate ( )-5 (Scheme
5). At 25°C, the glyoxyl-BTL2 immobilized preparation
was the most enantioselective (E=24) towards the (R)-
enantiomer. Interestingly, the PEI-BTL2 preparation
presented a lower E value (9), but hydrolyzed the
(S)-enantiomer quicker.
At 4°C and pH 7, the octadecyl-Sepabeads-BTL2 and
glyoxyl-BTL2 preparations showed a very high enan-
tioselectivity (E>100), hydrolyzing only the (R)-enan-
tiomer, while the PEI-BTL2 preparation decreased the
E value (2) but still preferring the opposite isomer.
Therefore, it would be interesting to study the selectiv-
ity of this thermophilic lipase as a biocatalyst in the
hydrolytic resolution of compounds ( )-4, ( )-5 and
( )-12.
At 37°C and pH 7, the octadecyl-Sepabeads-BTL2
behaved as at 25°C, while the other immobilized prepa-
rations decreased their selectivity or even disappearing
as in the case of the PEI-BTL2 preparation.

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J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 3679–3687
Table 3. Enantioselective resolution of ( )-5 catalyzed by different BTL2 preparations at pH 7 and different temperatures
Immobilized Preparation
Sp
4°C
25°C
37°C
ee
E
ee
E
ee
E
Octadecyl-BTL2
Glyoxyl-BTL2
PEI-BTL2
R
R
S
>99
>99
37
>100
>100
82
92
80
15
24
9
88
84
0
16
11
1
2
SP: stereochemical preference; ee=enantiomeric excess of product at 15% of conversion; E=enantiomeric ratio.
Scheme 5. Enzymatic resolution of compound ( )-5 catalyzed by BTL2 immobilized preparation.
enantioselective between all the immobilized prepara-
tions of BTL2, being 13-fold more enantioselective than
the glyoxyl-BTL2. The PEI-BTL2, maintained the
enantiopreference towards the (S)-enantiomer with an
E value of 2 (Table 5). When the temperature was
decreased to 4°C, an improvement in the E value for all
BTL2 immobilized preparations was observed, in which
the octadecyl-Sepabeads-BTL2 only hydrolyzed the
(R)-enantiomer (E>100); PEI-BTL2 immobilized prepa-
ration increased the E value almost five times (E=9.3),
hydrolyzing the opposite enantiomer faster.
Moreover, slight changes in the pH of the reaction
provoked differences in the selectivity of the different
immobilized preparations of BTL2. Thus, when the pH
of the reaction solution was decreased from 7 to 5, a
decrease in the enantioselectivity of all the immobilized
preparations of BTL2 was observed (Table 4). In this
case, at 25°C the octadecyl-Sepabeads-BTL2 was the
most enantioselective preparation (E=6.5), hydrolyzing
the (R)-isomer quicker while PEI-BTL2 was the lowest
(E=1.5), but curiously maintaining the preference
towards the (S)-enantiomer. At 4°C, an increase in the
E value in all cases was observed, especially in the
octadecyl-Sepabeads-BTL2 preparation (E value from
6.5 to 17).
Therefore, it seems that using some immobilization
techniques and different experimental conditions it is
possible to resolve the racemic mixture of compound 5
with very high enantioselectivity (E>100), with an ee>
99% from 8% until 50% of conversion (Fig. 1).
At pH 9, the octadecyl-Sepabeads-BTL2 presented a
high enantioselectivity (E=56), being again the most
Table 4. Enantioselectivity of immobilized preparations of BTL2-catalyzed hydrolysis of ( )-5 at pH 5
Immobilized preparation
Preferred enantiomer
25°C
4°C
ee
E
ee
E
Octadecyl-Sepabeads-BTL2
Glyoxyl-BTL2
PEI-BTL2
R
R
S
72
46
21
6.5
2.7
1.5
89
49
58
17
2.9
3.8
Ee=enantiomeric excess of the product at 15% of conversion; E=value of enantioselectivity.
Table 5. Enantioselectivity of immobilized preparations of BTL2-catalyzed hydrolysis of ( )-5 at pH 9
Immobilized preparation
Preferred Enantiomer
25°C
E
4°C
ee
ee
E
Octadecyl-Sepabeads-BTL2
Glyoxyl-BTL2
PEI-BTL2
R
R
S
96
62
33
56
4.3
2
>99
70
81
>100
6.4
9.3
Ee=enantiomeric excess of the product at 15% of conversion; E=value of enantioselectivity.

J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 3679–3687
3683
(Table 6). At 25°C, the glyoxyl-BTL2 preparation pre-
sented the highest activity against ( )-12, twenty six
times higher than octadedyl-Sepabeads-BTL2 one.
When the temperature was decreased, the immobilized
preparations suffered a decrease in activity, more dras-
tic than when the enzyme was immobilized on octade-
cyl-Sepabeads support (up to a 6-fold factor).
The hydrolytic resolution of ( )-12 catalyzed by differ-
ent immobilized preparations of BTL2 was performed
(Scheme 6). First, the enantioselectivity at pH 7.5 and
different temperatures in aqueous media was studied
(Table 7). At 25°C, significant differences for the differ-
ent immobilized preparations were not observed, with
enantiomeric excesses of the release product between 37
and 49% at 15% of conversion (Table 7), being
obtained and the (R)-enantiomer hydrolyzed faster.
However, at 4°C, it was possible to see differences in
the E value, with the octadecyl-Sepabeads-BTL2 prepa-
ration being the most enantioselective (E=4.7), up to
more than 2 times higher E value that glyoxyl-BTL2
preparation. Thus, the octadecyl-Sepabeads-BTL2 was
chosen as the best biocatalyst to perform the resolution.
Figure 1. Graphic of evolution of enantiomeric excess (ee)
versus conversion in the hydrolysis of ( )-5 catalyzed by
octadecyl-Sepabeads-BTL2 immobilized preparation. Experi-
ments were performed using concentration of substrate of 0.5
mM at pH 7 and 4°C. ee of remaining substrate (S)-5
(squares), ee of released acid (R)-3 (rhombus).
Table 6. Specific activity of different BTL2 immobilized
preparations in the hydrolysis of ( )-12 at different tem-
peratures. Specific activity: (mmol/h mgprot).
As a result, the effect of the co-solvent on the enan-
tioselectivity of this biocatalyst in the hydrolysis of
( )-12 in aqueous media in order to improve the selec-
tivity of this lipase was studied. Three different solvents
were selected to perform the study (Table 8).
Immobilized preparation
4°C
25°C
Octadecyl-Sepabeads-BTL2
Glyoxyl-BTL2
Glutaraldehyde-BTL2
0.0262
1.85
0.52
0.143
3.77
1.42
In the presence of a co-solvent, the activity of the
octadecyl-Sepabeads-BTL2 was slightly increased and
the enantioselectivity of the biocatalyst improved, being
slightly higher in the presence of 10% acetonitrile (E=
8). Table 9 shows the results obtained using increasing
concentrations of acetonitrile.
2.3. Enzymatic resolution of compound ( )-12 as pre-
cursor of (S)-Enalapril
The enzymatic activity of different immobilized prepa-
rations of BTL2 was also assayed in the hydrolysis
reaction of compound ( )-12 at different temperatures
The octadecyl-Sepabeads-BTL2 preparation gave the
highest E value in 20% of acetonitrile (E=13). The
organic co-solvent did not provoke this increase in the
Scheme 6. Enantioselective hydrolysis of compound ( )-12 catalyzed by BTL2 immobilized preparation.
Table 7. Enantioselective resolution of ( )-12 catalyzed by immobilized BTL2 at different temperatures. Experiments were
performed as described in Section 4
Immobilized Preparation
Preferred enantiomer
4°C
25°C
ee
E
ee
E
Octadecyl-Sepabeads-BTL2
Glutaraldehyde-BTL2
Glyoxyl-BTL2
R
R
R
62
54
32
4.7
3.6
2.1
49
49
37
3
3
2.2
Ee: enantiomeric excess of the product at 15% of conversion (%); E=value of enantioselectivity.

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J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 3679–3687
Table 8. Effect of the presence of co-solvent on the enan-
tioselectivity of octadecyl-Sepabeads-BTL2 preparation.
Experiments were performed with 10% of co-solvent and 2
mM of substrate at pH 7.5
Solvent
Conversion
(%)
Specific
Activity
Ee (%)
E (R/S)
Dioxane
Acetonitrile 15
Diglyme 15
16
0.2
0.21
0.23
73
75
72
7.3
8
6.8
Specific activity: (mmol/h mgprot); ee=enantiomeric excess of the
product; E=value of enantioselectivity.
selectivity of the enzyme immobilized on glutaralde-
hyde-agarose support or glyoxyl-agarose support (E
value remaining around 2).
Figure 2. Graphic of evolution of enantiomeric excess (ee)
versus conversion in the hydrolysis of ( )-12 catalyzed by
octadecyl-Sepabeads-BTL2 immobilized preparation. Experi-
ments were performed using concentration of substrate of 2
mM with 20% (v/v) acetonitrile pH 7.5, 25°C, as described in
Section 4. Ee of remaining substrate (S)-12 (squares), ee of
released acid (R)-13 (rhombus).
In this way, by hydrolyzing the racemic ester with this
immobilized preparation (octadecyl-Sepabeads-BTL2),
a drastic decrease in the enzyme activity at hydrolysis
yields over 60% has been observed showing the great
enantioselectivity of this enzyme for the (R)-enan-
tiomer. Thus, the combined use of this enzyme and
reaction conditions permitted to have quite pure (S)-12
at 60% (ee of remaining ester over 90%) (Fig. 2).
Therefore, this enzyme has been able to resolve com-
pound ( )-5 with high enantiomeric excess (ee>99%,
E>100) in the hydrolysis reaction catalyzed by octade-
cyl-Sepabeads-BTL2 preparation (at 4°C pH 7 or 9)
and glyoxyl-BTL2 preparation (at 4°C pH 7), yielding
only the (R)-enantiomer of mandelic acid, a key inter-
mediate in the synthesis of (+)-Goniodiol, an interesting
drug which has been resulted using different chemical
routes.33–36
3. Conclusion
This enzyme has been able to catalyze the hydrolysis
reaction of esters of very different natures, in some
instances with good enantioselectivity. This enantiose-
lectivity may be strongly modulated by the immobiliza-
tion protocol used and by the reaction conditions. In
this way, different methods of immobilization gave very
different results in the resolution of compound ( )-5,
even observing a selective inversion of the enzyme after
the ionic exchange on agarose support coated with
polyethyleneimine.
With this resolution, the use of immobilized BTL2 and
reaction conditions permitted to have quite pure (S)-12
by hydrolyzing the racemic ester to 60% (ee of remain-
ing ester over 90%), comparable with some resolutions
reported in the literature.37
4. Experimental
4.1. General
Furthermore, the obtained results suggest the impor-
tant influence of slight changes in the experimental
conditions on the selectivity of this lipase. Thus, in the
resolution of compound ( )-5 it was possible to
improve the E value from 11 (at 37°C) to E value of
>100 (at 4°C) for glyoxyl-BTL2 preparation, whereas in
the resolution of compound ( )-12 interesting results
for the E value using octadecyl-Sepabeads-BTL2 immo-
bilized preparation were obtained, from 3 (at 25°C pH
7) to E value of 13 (by adding with 20% acetonitrile),
only by modification of the experimental conditions.
Lipase from Bacillus thermocatenulatus (BTL2) cloned
in E. coli was produced as previously described.16b
Glyoxyl-agarose 6BCL and 10 BCL was kindly donated
by the company Hispanagar SA (Burgos, Spain).
Octadecyl-Sepabeads was generously donated by Resin-
dion Srl (Mitsubishi Chem. Coorp.) (Milan, Italy).
Octyl-agarose 4BCL was purchased from Pharmacia
Table 9. Effect of the co-solvent concentration on the enantioselectivity of different BTL2 preparations at 25°C and pH 7.5.
The percentages show the concentration of acetonitrile used (v/v)
Immobilized preparation
10%
20%
30%
ee
E (R/S)
ee
E (R/S)
ee
E (R/S)
Octadecyl-Sepabeads-BTL2
Glutaraldehyde-BTL2
Glyoxyl-BTL2
76
20
–
8.8
1.5
1
84
10
32
13
1.2
2
81
–
18
11
1
1.4
Ee=enantiomeric excess of the product at 15% of conversion; E=value of enantioselectivity.

J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 3679–3687
3685
Biotech (Uppsala, Sweden). Polyethyleneimine (PEI)
(Mr 25000), Triton X-100, p-nitrophenyl propionate
(pNPP), butyric acid ethyl ester were from Sigma.
Glyoxyl-agarose,38 and PEI-agarose39,40 were prepared
as previously described. ( )-a-Hydroxy-phenylacetic
acid methyl ester ( )-4 were purchased from Sigma.
2-O-butyryl-2-phenylacetic acid ( )-5 was synthesized
as previously described.41 ( )-trans-4-(4%-fluorophenyl)-
6-oxopiperidin-3-ethyl carboxylate ( )-8 and 2-
hydroxy-4-phenylbutyric acid ethyl ester ( )-12 were
kindly donated by Vita Invest S.A. (Barcelona, Spain),
( )-6-(5-Chloropyridin-2-yl)-7-oxo-5-(vinyloxycarboyl-
oxy)-5,6-dihydropyrrolo[3,4b] pyrazine, ( )-10 was
donated by Vicente Gotor (University of Oviedo,
Spain). Other reagents and solvents used were of ana-
lytical or HPLC grade.
(ii) covalent immobilization on amine–agarose beads
activated with glutaraldehyde44 at pH 7 and 25°C.
(iii) multipoint covalent immobilization on glyoxyl-
agarose beads (through areas with the highest density
of lysine (Lys) groups)45 at pH 10.5 and 25°C.
(iv) interfacial adsorption on a hydrophobic support,
Sepabeads resin with the surface covered by octade-
cyl groups.46 To immobilize the purified lipase on
octadecyl-Sepabeads support, the Triton was diluted
500-fold with 5 mM sodium phosphate buffer at pH
7.
A schematic representation of the different immobiliza-
tion protocols is shown in Scheme 7. Enzyme load was
1 mg pure lipase /mL of support (that is approx. 1–2%
of the maximum load) and 23 mg/mL (with compound
12) (maximum capacity of octadecyl-Sepabeads) and in
all cases more than 95% of the lipase became immobi-
lized on all different supports offered. Protein concen-
tration was determined by the Bradford method.43
4.2. Enzymatic activity assay determination
This assay was performed by measuring the increase in
absorbance at 348 nm produced by the release of
p-nitrophenol (pNP) in the hydrolysis of 0.4 mM pNPP
in 25 mM sodium phosphate buffer at pH 7 and 25°C.
To initialize the reaction, 0.05 mL of lipase solution or
suspension was added to 2.5 mL of substrate solution.
One international unit of pNPP activity was defined as
the amount of enzyme that is necessary to hydrolyze 1
mmol of pNPP per minute (IU) under the conditions
described above.
4.5. Enzymatic hydrolysis of esters
The activities of different immobilized preparations of
lipase from B. thermocatenulatus were analyzed in the
hydrolysis reaction of different esters. Ethyl butyrate
was dissolved in a 100 mL solution of 25 mM sodium
phosphate buffer at pH 7 to 10 mM of substrate
concentration and 0.1 g of biocatalyst were added.
pNPP was dissolved in a 10 mL solution of 25 mM
sodium phosphate buffer at pH 7 to 0.4 mM of sub-
strate concentration and 0.1 mL of biocatalyst were
added.
4.3. Purification of the enzyme
To purify the lipase from any other contaminant
proteins (e.g. esterases), the enzyme preparation was
incubated in the presence of octyl-agarose at low ionic
strength, following a previously described procedure.42
Periodically, activity of suspensions and supernatants
was assayed by using the pNPP assay. After immobi-
lization, adsorbed lipase preparation was abundantly
washed with distilled water. To desorbe the enzyme, the
adsorbed lipase was washed with Triton X-100 0.2% in
5 mM sodium phosphate buffer at pH 7 and 25°C.
Substrate ( )-4 was dissolved in a 3 mL solution of 10
mM sodium phosphate buffer to 2 mM of compound
concentration at 25°C and pH 7 under different condi-
tions (pH, T) and 0.6 g of immobilized preparation
were added.
Substrate ( )-5 was dissolved in a 3 mL solution of 10
mM sodium phosphate buffer to 0.5 mM of compound
concentration under different conditions (pH, T) and
0.5 g of immobilized preparation were added.
Substrate ( )-8 was dissolved in a 3 mL solution of 10
mM sodium phosphate buffer to 2 mM of compound
concentration at 25°C and pH 7 under different condi-
tions (pH, T) and 0.1 g of immobilized preparation
were added.
Following these protocols, a quantitative immobiliza-
tion of lipase activity was observed and the SDS-PAGE
analysis of the adsorbed protein preparation only
showed a single band with a molecular weight corre-
sponding to that of the native lipases. Final yield was
near to 100%. Protein concentration was measured
using Bradford’s method.43 The calibration curve was
obtained with bovine serum albumin (BSA) for deter-
mining protein concentrations in the range of 0.1 to 1.5
mg/mL.
Substrate ( )-10 was dissolved in a 3 mL solution of 10
mM sodium phosphate buffer to 2 mM of compound
concentration at 25°C, pH 7 and 50% dioxane and 0.3
g of immobilized preparation were added.
Substrate ( )-12 was dissolved in a 3 mL solution of 10
mM sodium phosphate buffer to 2 mM of compound
concentration at 25°C and pH 7.5 under different con-
ditions (pH, T) and 0.1 g of immobilized preparation
were added.
4.4. Immobilization of lipases on different supports
Different immobilized preparations were prepared fol-
lowing the procedures previously described:
(i) ionically adsorbed lipase on solid supports coated
with PEI39 (ionic microenvironment surrounding
large areas of the protein) at pH 9 and 25°C.
During the reaction, the pH value was maintained
constant using a pH-stat Mettler Toledo DL50 graphic

3686
J. M. Palomo et al. / Tetrahedron: Asymmetry 14 (2003) 3679–3687
Scheme 7. Different immobilized preparations from B. thermocatenulatus lipase.
and the enzymatic activity was defined as mmol of
substrate hydrolyzed per minute per mg of immobilized
protein. The degree of hydrolysis was analyzed by
reverse-phase HPLC (Spectra Physic SP 100 coupled
with an UV detector Spectra Physic SP 8450). For these
assays a Kromasil C18 (25×0.4 cm) column was used,
mobile phase acetonitrile–10 mM ammonium phos-
phate buffer at pH 2.95 (35:65, v/v) for compound 5,
(30:70, v/v) for compounds 4 and 8, (40:60,v/v) for
compound 12, (50:50, v/v) form compound 10, at 1.5
mL/min (or 1 mL/min to 8) and UV detection was
performed at 254 nm (compounds 4 and 5) and 270 nm
(compounds 10 and 8).
authors thank CAM for a PhD Fellowship for Dr.
Palomo. We thank Hispanagar SA for the gift of
glyoxyl-agarose, Resindion Srl by donation of octade-
cyl-Sepabeads and Dr. Vicente Gotor and Vita Invest
to supply some compounds. Angel Berenguer by the
interesting suggestions.
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