Effect of ionic liquids as additives in the catalytic properties of different immobilized preparations of Rhizomucor miehei lipase in the hydrolysis of peracetylated lactal

Article abstract of DOI:10.1039/c003829f

The addition of small amount of different ionic liquids modified the activity and regioselectivity of different immobilized preparations of R. miehei lipase catalyzing the hydrolysis of hexa-O-acetyl lactal in aqueous media. ILs with [emim] as cation and different anions were first evaluated affecting in a different manner depending on the immobilized preparation used. The enzymatic activity of RML immobilized on octyl-agarose or CNBr-agarose decreased in the following order: NO₃^-≈ BF₄^- > MeOSO₃^- > PF₆^-, whereas when RML was immobilized on Q-Sepharose, the enzymatic activity decreased in a different order: MeOSO₃^- > PF₆^- > BF₄^- > NO₃^-. Using [bdmim], the activity of octyl-RML and CNBr-RML preparations resulted higher in the presence of PF₆^- than BF₄^-, 6-fold for octyl-RML and 2-fold for CNBr-RML if compared with the enzyme activity without additive_. In both preparations the enzyme was completely regioselective in the presence of the IL hydrolyzing at C-3 position in 99% yield. The modification of the cation in the IL did not affect to the activity of Q-RML with BF₄^- or decreased the activity with PF ₆^-, although affected to the regioselectivity producing another undesired product, a bihydrolized product in 20-25% yield. In this case, the addition of [emim][MeOSO₃] caused the best increment in the activity for this RML biocatalyst, 2-fold with only 8% of bihydrolized production.

Full text of DOI:10.1039/c003829f

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www.rsc.org/greenchem | Green Chemistry
Effect of ionic liquids as additives in the catalytic properties of different
immobilized preparations of Rhizomucor miehei lipase in the hydrolysis of
peracetylated lactal†
Marco Filice, Jose M. Guisan* and Jose M. Palomo*
Received 3rd March 2010, Accepted 19th May 2010
First published as an Advance Article on the web 29th June 2010
DOI: 10.1039/c003829f
The addition of small amount of different ionic liquids
modified the activity and regioselectivity of different im-
mobilized preparations of R. miehei lipase catalyzing the
hydrolysis of hexa-O-acetyl lactal in aqueous media. ILs
with [emim] as cation and different anions were first
evaluated affecting in a different manner depending on
the immobilized preparation used. The enzymatic ac-
tivity of RML immobilized on octyl-agarose or CNBr-
viscosity and easily handled materials with very interesting
properties as solvents. Furthermore the miscibility with water
or some organic solvents (e.g. hexane) can be tuned by selecting
the appropriate cation and anion.1–2
One of the most interesting applications of ILs has been
on biocatalysis.2–5 ILs have been used in the preparation of
biocatalysts by additive in the immobilization process,6 coating
the enzyme,7 as solid phase support for immobilization,8 as
solvent substituent how medium engineering,9 or an additive in
organic solvents or aqueous media with interesting improvement
of enzyme activity and selectivity in several kind of reactions.10
-
-
agarose decreased in the following order: NO3 ª BF4
>
MeOSO3- > PF6 , whereas when RML was immobilized on
-
Q-Sepharose, the enzymatic activity decreased in a different
-
Indeed, some ILs, especially those containing PF6 , BF4- and
-
-
-
-
order: MeOSO3 > PF6 > BF4 > NO3 . Using [bdmim],
-
MeOSO3 anions, were known as positive influences on the
the activity of octyl-RML and CNBr-RML preparations
enzyme stability,11 activity and selectivity.12
-
resulted higher in the presence of PF6- than BF4 , 6-fold for
Particularly, in recent years many examples of the application
of ILs in biotransformations with lipases have been reported.13
This is because of the high versatility of these enzymes
which recognize a broad range of substrates with high regio-
and enantioselectivity.14–15 The interesting and complex catalytic
mechanism of these enzymes and the great flexibility of their
active centre have permitted development of strategies to
modulate their catalytic properties, such as using immobilization
strategies.16–17 Also small changes in the experimental conditions
(e.g. co-solvent addition,18 pH changes,18 addition of salts,19 etc.)
have been observed to introduce interesting modifications of the
activity and selectivity of lipases.
octyl-RML and 2-fold for CNBr-RML if compared with
the enzyme activity without additive. In both preparations
the enzyme was completely regioselective in the presence
of the IL hydrolyzing at C-3 position in 99% yield. The
modification of the cation in the IL did not affect to the
-
activity of Q-RML with BF4 or decreased the activity with
-
PF6 , although affected to the regioselectivity producing
another undesired product, a bihydrolized product in 20–
25% yield. In this case, the addition of [emim][MeOSO3]
caused the best increment in the activity for this RML
biocatalyst, 2-fold with only 8% of bihydrolized production.
Although the lipase catalytic properties have been interpreted
in terms of IL properties including polarity, hydrogen-bond
basicity, anion nucleophilicity, and viscosity,20 hydrophobic ILs
seem to be the better choice as reaction solvent in term of lipase
activity.21
Therefore, in the present manuscript, we have studied the
effect of six different ionic liquids (Scheme 1) as additive on
the activity and regioselectivity of Rhizomucor miehei lipase
immobilized by different strategies. Furthermore, this study has
Introduction
Ionic liquids (ILs) are emerging as ‘green’ alternatives to com-
mon solvents because they have no measurable vapor pressure
and are able to dissolve compounds of varying polarity. They are
simply salts and therefore entirely composed of ions that are liq-
uid below 100 ◦C. Ionic liquids are also chemically diverse owing
to the huge number of possible cation/anion combinations that
can be synthesized. Typical ILs are based on organic cations, e.g.
1,3-dialkylimidazolium, tetraalkylammonium, etc., paired with
a variety of anions that have a strongly delocalized negative
charge (e.g. [BF4], [PF6], etc.), which results in colourless, low
Departamento de Biocata´lisis, Instituto de Cata´lisis (CSIC), c/marie
curie 2, Cantoblanco, Campus UAM, 28049, Madrid, Spain.
E-mail: josempalomo@icp.csic.es, jmguisan@icp.csic.es; Fax: +34-91
585 47 60
† Electronic supplementary information (ESI) available: Experimental
data and MALDI-TOF. See DOI: 10.1039/c003829f
Scheme 1 Ionic liquids tested as additives.
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Table 1 Activity, specificity and regioselectivity of different immobilized preparations of RML in the hydrolysis of 1 in the presence of different
additives
Support
Octyl
ILs
Activitya
Time/h
Cb (%)
2c (%)
Otherd
—
0.58 0.01
2.40 0.02
0.64 0.01
2.50 0.03
1.00 0.01
1.30 0.01
3.30 0.02
24
8
22
8
12
9
7
100
100
100
100
100
100
100
100
95
97
100
100
100
100
[emim][BF4]
[emim][PF6]
[emim][NO3]
[emim][MeOSO3]
[bdmim][BF4]
[bdmim][PF6]
5
7
CNBr
—
0.038 0.001
0.052 0.002
0.038 0.002
0.052 0.003
0.038 0.001
0.040 0.002
0.064 0.002
144
144
144
144
144
144
144
80
88
78
88
81
82
100
80
88
78
88
81
82
100
[emim][BF4]
[emim][PF6]
[emim][NO3]
[emim][MeOSO3]
[bdmim][BF4]
[bdmim][PF6]
Q-Sepharose
—
0.36 0.005
0.11 0.003
0.36 0.004
0.10 0.002
0.77 0.003
0.11 0.004
0.11 0.003
48
72
48
72
26
72
72
100
100
100
100
100
100
100
100
78
78
76
92
80
75
[emim][BF4]
[emim][PF6]
[emim][NO3]
[emim][MeOSO3]
[bdmim][BF4]
[bdmim][PF6]
22
15
24
8
20
25
a The initial rate in mmol mgprot h-1. It was calculated at 10–20% yield. IL without enzyme was tested and no conversion was detected. The
experiments were performed by triplicate. b C = conversion c Yield of monodeprotected product 2. d The compound corresponds to a bi-hydrolyzed
product detected by MALDI-TOF, [M+Na]+: 499.15, found: 499.13.
-1
been focused on the regioselective hydrolysis of peracetylated
lactal 1, a quite interesting building block in biological active
oligosaccharides synthesis.22
worst ones (the best ones with the other RML preparations),
causing a more than 3-fold decrease in the activity. Addition
of [emim][PF6] did not produce any different in the biocatalyst
activity (Table 1).
Secondly, the effect of the anion in ILs was evaluated, by
substitution [emim] for [bdmim] (Table 1).
Results and discussion
Using a more hydrophobic anion, the behavior on the specific
activity varied. The best result in term of activity enhancement
was achieved by using an IL bearing [PF6] – the worst cation in
emim ILs. The addition of [bdmim][PF6] enhanced the activity
of octyl-RML 6-fold in the hydrolysis of 1. Using [bdmim][BF4]
an improvement of 2-fold was observed (Table 1).
The presence of [bdmim][PF6] also gave the best activity
improvement on the CNBr-RML, 1.68-fold compared without
additive or [emim][PF6] (Table 1). With [bdmim][BF4] the
enzyme slightly improved its activity.
Using Q-Sepharose-RML, the addition of these ILs caused a
decrease on the activity (Table 1).
Using NaCl as typical salt as additive, almost no effect on
the activity for the different immobilized RML preparation was
observed (see ESI†).
The specific activity displayed by three different immobilized
preparations of RML in the regioselective hydrolysis of peracety-
lated lactal 1 in aqueous solution containing 5 eq.‡ of different
ILs (Scheme 1) is shown in Table 1. The influence of different
cations or anions in the IL was evaluated and the results were
quite different depending on the immobilization protocol used.
Firstly, the influence of different anions of ILs on the lipase
properties was analyzed. Thus, four ionic liquids – constituted
by [emim] as cation – were evaluated.
In all cases, the addition of the IL improved the catalytic
activity on the lipase immobilized on octyl-agarose beads
-
-
(by interfacial adsorption), although ILs with BF4 or NO3
permitted to enhance the activity in almost 4-fold. The addition
of [emim][MeOSO3] improved the enzymatic activity 2-fold
whereas [emim][PF6] just slightly increase the activity value
(Table 1).
When RML immobilized on CNBr-agarose (by covalent
attachment) was used as biocatalyst, a similar effect was
observed but with less activity increment (Table 1). [emim][BF4]
or [emim] [NO3] increased the activity in 1.4-fold whereas the
rest did not affect to the activity.
The kinetic parameters for the octyl-RML immobilized
preparation were calculated in order to understand the nature
of the effect (Table 2).
Table 2 Kinetic parameters for octyl-RML with 1 as substrate
ILs
Km/mM
Vmax (nmol min-1 mg-1) Kcat/ms-1
Different behavior was observed when these ILs were added in
the reaction catalyzed by RML immobilized on Q-Sepharose (by
anionic exchange).23 [emim][MeOSO3] was the best IL increasing
—
5.02 0.09 4.47 0.1
3.33 0.05 2.51 0.08
0.0051 0.0003
0.0287 0.0009
0.0029 0.0001
[emim][PF6]
[bdmim][PF6] 1.66 0.02 25.5 1.2
-
-
2-fold the enzyme activity. ILs with BF4 or NO3 were the
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The Km value of the immobilized enzyme was lower in the
presence of [bdmim][PF6] (1.66 mM) compared with the value
of the immobilized preparation without ionic liquid (5.02 mM).
The Km value for the biocatalyst in the presence of [emim][PF6]
was 3.33 mM, lower than the value without additive, showing
that the affinity of the enzyme for the substrate increased
in the presence of both ILs. However, the influence of the
presence of IL was different on the Kcat. The turnover number
was almost 6-fold higher with the addition of [bdmim][PF6]
and almost 2-fold lower with the addition of [emim][PF6]
(Table 2). This result seems to indicate that the effect on the
activity of the immobilized preparation in the presence of
[emim][PF6] is exclusively based on a decrease of the kcat whereas
the improvement on the catalytic activity for the octyl-RML
preparation in the presence of [bdmim][PF6] is based on an
influence on both parameters (a better substrate affinity and
an increase of turnover number).
Analyzing the reaction course for octyl-RML (Fig. 1), all ILs
added in this enzymatic process decreased the time for a full
conversion of the product. The reaction without additive was
finished at 24 h whereas using [bdmim][PF6] the substrate was
completely consumed in 7 h. The specificity and regioselectivity
of the biocatalyst was maintained in the presence of most ILs
producing exclusively the deprotection of the acetyl group on
C-3 (Scheme 2, Table 1). Only a 5–7% bi-hydrolyzed product
was observed using [emim][PF6]or [emim][BF4] (Table 1).
Fig. 2 The influence of addition of different ionic liquids on the
hydrolysis of 1 catalyzed by CNBr-RML. Without additive (aster-
isks), [emim][MeOSO3] (rhombus), [emim][NO3] (circles), [emim][PF6]
(empty circles), [emim][BF4] (empty rhombus), [bdmim][BF4] (triangles),
[bdmim] [PF6] (empty triangles).
The specificity and regioselectivity of this biocatalyst was also
maintained after the addition of ILs.
Using Q-Sepharose-RML preparation the effect of the IL on
the reaction course was quite different, especially in the final
yield of product. In this case the addition of ILs caused a
change in the regioselectivity of the enzyme, producing another
substance (a bi-hydrolyzed product analyzed by HPLC and
MALDI-TOF, see ESI) in different yields together the hydrolysis
in C-3 (Table 1). The ratio between 2 and the other peak was
different depending on the ionic liquids. In the presence of
[emim][MeOSO3], 100% conversion is achieved in 26 h with 92%
of 2 whereas with the other ILs the full conversion is achieved
in 72 h and around 70–78% yield of 2 is obtained (Fig. 3).
Fig. 1 The influence of addition of different ionic liquids on the
hydrolysis of 1 catalyzed by octyl-RML. Without additive (aster-
isks), [emim][MeOSO3] (rhombus), [emim][NO3] (circles), [emim][PF6]
(empty circles), [emim][BF4] (empty rhombus), [bdmim][BF4] (triangles),
[bdmim] [PF6] (empty triangles).
Fig. 3 The influence of addition of different ionic liquids on the
hydrolysis of 1 catalyzed by Q-Sepharose-RML. Without additive (aster-
isks), [emim][MeOSO3] (rhombus), [emim][NO3] (circles), [emim][PF6]
(empty circles), [emim][BF4] (empty rhombus), [bdmim][BF4] (triangles),
[bdmim] [PF6] (empty triangles).
The addition of the same equivalents of NaCl affected the
reaction catalyzed by CNBr-RML and Q-Sepharose-RML,
where 30% of undesired product was produced (see ESI).
Scheme 2 Regioselective hydrolysis of per-O-acetylated lactal 1 cat-
alyzed by different RML immobilized preparations in aqueous media
containing ionic liquids.
Conclusion
The profile for the CNBr-RML preparation was different
(Fig. 2), where only a 100% conversion was achieved after 144 h
by the addition of [bdmim][PF6]. Others ILs almost did not
affect on the rate.
In this paper, we have described the influence of small amounts
of different ionic liquids on the catalytic activity and re-
gioselectivity of three different immobilized preparations of
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RML – besides on different manner – catalyzing the hydrolysis
of hexa-O-acetyl lactal 1 in aqueous media.
under mechanical stirring, and the pH value was controlled
by automatic titration. Hydrolysis reactions were followed by
HPLC.
We have observed how the enzymatic activity of the different
RML preparations varied depending on cation or anion con-
stituting the IL. The activity of octyl-RML was rise with all
ILs, the CNBr-RML activity increased with some ILs whereas
it was not affected with others. Two ILs improved the activity
of Q-Sepharose-RML while the rest caused a decrease. The
regioselectivity in most cases remained unaltered, although ILs
changed this property for Q-Sepharose-RML, producing an
undesired bi-hydrolyzed product from 5–25%.
Therefore, the addition of [bdmim][PF6] enhanced 6-fold the
activity of octyl-RML preparation keeping a high regioselectiv-
ity, producing 3-hydroxy product 2 in high overall yield (>95%),
key intermediate in the synthesis of different glycoderivatives.
The origin of the effects observed could be explained by
considering the nature of the immobilized enzyme. The en-
zyme immobilized on octyl-agarose exists on a fixed open
conformation with a high hydrophobic pocket surrounding
the active site. The best results for this lipase immobilized
preparation were found by using the most hydrophobic ionic
liquids, probably because they can penetrate easily changing the
active site environment. The study of the kinetic parameters
demonstrated that the affinity of the substrate was better (lower
Km) and the turnover number (Kcat) was increased. In the case
of the lipase immobilized on an ionic exchanger Q-Sepharose,
the most hydrophilic ILs gave the best results; in this case
considering the most hydrophilic area around the active site.
Also the alteration of ionic charges on the protein and the
influence on the conformational changes, in the same way as
previously described for pH change18 could be also another
artifact.
Analytical method
HPLC analyses were performed using HPLC-spectra P100
(Thermo Separation products). The column was a Kromasil-
C
18 (250 ¥ 4.6 mm and 5 mm) from Analisis V´ınicos (Tomelloso,
Spain). Analyses were run at 25 ◦C using an L-7300 column oven
and UV detector L-7400 at 215 nm. The eluent was an isocratic
mixture of 40% acetonitrile in 10 mM ammonium phosphate
buffer at pH 3.8; flow rate 1.0 mL min-1. The retention time of
the product 1 and 2 were 18.50 min and 11.10 min respectively in
these conditions. Compound 2 has been recently characterized.24
Acknowledgements
This work has been sponsored by the Spanish Ministry of
Science and Innovation (CTQ2009-07568).
Notes and references
‡ 10, 15 and 20 eq. of ionic liquids were also assayed and no significant
differences were observed.
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