Improved activity of horseradish peroxidase (HRP) in 'specifically designed' ionic liquid

Article abstract of DOI:10.1016/j.tetlet.2007.06.022

The ionic liquid (IL), tetrakis (2-hydroxyethyl) ammonium triflouromethanesulfonate is rationally designed for horseradish peroxidase (HRP) on the basis of its stability and activity in the presence of an excipient, tris(hydroxymethyl)aminoethane (TRIS) in different ILs. The activity of HRP in this tailor made IL is at least 30-240-fold higher than that in conventional ILs. Also, the activity is more than 10 times greater than that in methanol, a common organic solvent used for HRP.

Full text of DOI:10.1016/j.tetlet.2007.06.022

Tetrahedron Letters 48 (2007) 5635–5639
Improved activity of horseradish peroxidase (HRP) in
‘specifically designed’ ionic liquid
Dibyendu Das, Antara Dasgupta and Prasanta Kumar Das*
Department of Biological Chemistry and Centre for Advanced Materials, Indian Association for the Cultivation of Science,
Jadavpur, Kolkata 700032, India
Received 24 April 2007; revised 29 May 2007; accepted 7 June 2007
Available online 9 June 2007
Abstract—The ionic liquid (IL), tetrakis (2-hydroxyethyl) ammonium triflouromethanesulfonate is rationally designed for horse-
radish peroxidase (HRP) on the basis of its stability and activity in the presence of an excipient, tris(hydroxymethyl)amino-
ethane (TRIS) in different ILs. The activity of HRP in this tailor made IL is at least 30–240-fold higher than that in
conventional ILs. Also, the activity is more than 10 times greater than that in methanol, a common organic solvent used for HRP.
Ó 2007 Elsevier Ltd. All rights reserved.
Research in the arena of ionic liquids (ILs)1–3 has wit-
nessed significant progress mainly due to their eco-
friendly nature and designer flexibility. The application
of these green solvents in the field of biocatalysis is also
increasing rapidly. The merits of ILs in enzyme catalysis
can be exploited if the enzyme exhibits improved effi-
ciency within these green solvents. Significant enhance-
ment in the stability and activity of enzymes in ILs
could increase their versatility in biotransformations.
Although preliminary reports on the hydrolytic activity
of lipase and chymotrypsin in ILs are available,4–14 liter-
ature on oxido-reductase enzymes has rapidly increased
in recent years.15–22 However, the efficiency of biocata-
lysts in conventional imidazolium based ILs is not sub-
stantial enough to substitute organic solvents. To this
end, Walker and Bruce were the first to describe the de-
sign of an ionic liquid in which the cation contained a
hydroxyalkyl group to stabilize the dissolved enzyme
(alcohol dehydrogenase).23,24 On the basis of this con-
cept, herein, we report the design of ILs containing mul-
tiple hydroxyethyl groups for dissolving horseradish
peroxidsase (HRP) while maintaining high activity by
improving the compatibility of the enzyme in ILs. The
stability and activity of HRP was investigated in differ-
ent ILs (1–6, Fig. 1). To improve HRP activity further,
the hydrophilic lyoprotectant tris(hydroxymethyl)amino-
ethane (TRIS) was applied as excipient25 in the various
ILs. The enhanced activity in the presence of TRIS
led us to design novel ILs (7–8, Fig. 1), tailor made
for the enzyme. The activity of HRP in 8 increased
ꢀ30–240-fold compared to that observed in other ILs.
Stereoselective oxidation of thioanisole by HRP in the
newly developed IL was also demonstrated.
The ILs ([bmim][Br], 2–4) were prepared following
microwave assisted synthesis and subsequent ion-
exchange of the bromide ions by hydroxide ions which
were neutralized by the respective acids for 2–4. ILs 5
and 6 were synthesized directly from [bmim][Br] by stir-
ring with ammonium tetrafluoroborate and hexafluoro-
phosphoric acid, respectively. IL 7 was synthesized by
quaternization of triethanolamine with 2-chloroethanol
while 8 was prepared by subsequent replacement of
the chloride anion of 7 with hydroxide which was neu-
tralized by triflic acid. Detailed synthetic procedures
with analytical data are given in the Supplementary
data.
To improve the activity/stability of HRP in ILs, it was
essential to study the dependence of HRP on the compo-
nents of the ILs. Initially, the stability of HRP was
investigated in three ILs, namely [bmim][Cl] (1),
[bmim][CF3CO2] (2) and [bmim][alanine] (3). While 1
and 2 are conventional ILs, 3 is a relatively new IL,
whose role in biocatalysis has not yet been probed.
HRP loses half of its initial activity within 24 h at 5%
water content in 1 and 2 (Fig. 2).26a Rapid deactivation
Keywords: Biocatalysis; Excipients; Ionic liquids; Peroxidase.
*
Corresponding author. Fax: +91 33 24732805; e-mail: bcpkd@
iacs.res.in
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.06.022

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D. Das et al. / Tetrahedron Letters 48 (2007) 5635–5639
Figure 1. Structures of the ionic liquids.
inhibiting effect of the carboxylate anion, which binds
to the sixth coordination position of Fe(III) in HRP,
and thereby blocks further access to the substrate.28,29
At high water (low IL) content, the enzyme active site
becomes sufficiently hydrated to restore its natural con-
formation. Inhibition by the counterion is also reduced,
resulting in greater stability of HRP. However, when the
enzyme is exclusively dispersed in 3 at 0% water (lyoph-
ilized HRP), it takes 24 h for complete deactivation. In
the presence of water, the active site of HRP is possibly
more accessible to the inhibiting counterions of 2 and 3,
leading to its deactivation. To ascertain the influence of
the anionic component of the IL, the stability of HRP
was investigated (Fig. 3c) in [bmim][CF3SO3] (4), where
Figure 2. Stability of HRP in the ILs with 5% water. The concentra-
tion of HRP in the cuvette was 1 lg/mL. [Guaiacol] = 0.3 mM,
[H2O2] = 0.1 mM. The experimental error in these measurements was
typically within 8–12% in triplicate measurements.
À
CF3SO3 is an ineffective inhibitor of HRP. The results
were encouraging as the enzyme was stable for up to 30
h irrespective of the water content in 4 and was stable
for longer time even at 0% water.
of HRP in 1 and 2 led us to introduce 3,27 to determine
whether the presence of an amino acid component in the
IL would improve the activity and stability of the en-
zyme. However, IL 3 was found to be quite detrimental
to the enzyme, causing irreversible deactivation. HRP
becomes inactive within an hour of incubation in 3
(Fig. 3b) and also the stability was lower than that
found in 1 (see Supplementary data) and 2 (Fig. 3a).
The deactivation rate increases with decreasing the
water content from 70% to 5% for all the three ILs
(Fig. 3a and b for 2 and 3, respectively, see Supplemen-
tary data for 1) while the order of deactivation was
3 > 2 > 1, particularly at lower water content. The high
deactivation of HRP in 2 and 3 could be due to the
In another common IL, [bmim][BF4] (5), the stability of
HRP was found to be comparable to that in 4 at high
water content. Between 30% and 50% deactivation of
HRP was observed in 5 at 640% water content
(Fig. 3d). In concurrence with earlier reports,15,30 the
stability of HRP was again hampered in the medium
where 5 was the major component, presumably due to
binding of fluoride anions released from tetrafluoro-
borate to the heme iron. The stability of HRP was also
investigated in a hydrophobic IL, [bmim][PF6] (6),
where the lyophilized enzyme was quite stable for up
to 24 h. However, further study in the presence of water
could not be carried out due to the immiscibility of the
enzyme solution with 6. On the basis of the preceding
Figure 3. (a)–(d) Stability of HRP with varying water content in 2–5, respectively. The concentration of HRP in the cuvette was 1 lg/mL.
[Guaiacol] = 0.3 mM, [H2O2] = 0.1 mM. The experimental error in these measurements was typically within 8–12% in triplicate measurements.

D. Das et al. / Tetrahedron Letters 48 (2007) 5635–5639
5637
observations, 4 was the best IL in terms of the stability
of HRP.
enzyme by holding water in proximity of the active site
and thereby increase the efficiency of the biocatalyst.25
Excipients have been previously used for enhancing
enzyme activity in organic solvents.25Their use in ILs
has however, not been reported previously. In this case,
the hydrophilic lyoprotectant, TRIS was used as an
excipient and succeeded in enhancing HRP activity in
2 and 3 up to an order of magnitude depending on the
water content (Fig. 5).26c Moreover, in the presence of
TRIS, the minimum amount of water necessary for
HRP to be active was reduced by 10% in both these
ILs. Thus, stability and activity studies in imidazolium
based ILs have illustrated the crucial role of counterions
on the efficiency of HRP.
Apart from the stability of HRP, it is important to
determine its activity directly within these green solvents
in order to be able to replace organic solvents.26b In con-
currence with the stability study, 3 was found to be the
most inferior medium for HRP as more than 92% water
was necessary to show minimum visible activity (Fig. 4).
HRP also showed poor efficiency in 2 where the presence
of ꢀ70–80% water was required for measurable activity.
As discussed earlier, the presence of COOÀ in both these
ILs presumably deactivates peroxidase. In the case of 1,
the minimum amount of water essential for HRP activ-
ity was 20%. As expected (from the stability study), in all
cases, the activity of HRP increased with water content
and importantly, HRP needed only 5% water to remain
active in 4 and 5. Its efficiency was also markedly higher
than in other ILs in comparable solution compositions.
Interestingly, biocatalysis in ILs with structural varia-
tion of the cationic component is rarely re-
ported,23,24,31,32 and to our knowledge, no such work
has been published on HRP. The activity study in the
presence of excipient33 motivated us to design an IL pos-
sessing four hydroxyethyl moieties (7, Fig. 1), whilst
keeping a structural resemblance to TRIS. We expected
that such a similarity in structure would help the IL (7)
to act as an activator for the enzyme. HRP showed a
3-fold increase in activity in 7 compared to the best
imidazolium based IL, 4 at 40% water content (Fig. 6,
inset). However, the activity could not be measured
below 40% water content in 7 due to solidification of
the reaction medium. At this point, the ClÀ counterion
To improve the activity of HRP in 2 and 3, an excipient
was added as an activity stimulator. In general, hydro-
philic excipients maintain the native structure of the
À
of 7 was replaced by CF3SO3 to minimize the opera-
tional difficulty and also to have the most compatible
counterion for HRP (as evidenced in the imidazolium
based ILs). The resulting IL, 8 (tetrakis(2-hydroxy-
ethyl)ammonium triflouromethanesulfonate) was not-
ably better than its predecessor (7), as activity was
observed even at 0.8% water content and 8 also showed
ꢀ6-fold higher activity compared to 4 at 40% water
content.
In similar solution compositions (at 5% water), the
activity of HRP in 8 was ꢀ30 and 50-fold higher than
that in the best performing imidazolium based ILs, 4
and 5, respectively (Fig. 6). Also, the observed activity
was ten times greater than that in methanol, a common
Figure 4. Initial rate of oxidation of guaiacol by HRP in different ILs
with varying water content. [HRP] = 50 lg/mL. [Guaiacol] = 0.3 mM,
[H2O2] = 0.1 mM. The experimental error in these measurements was
typically within 8–12% in triplicate measurements.
Figure 5. (a, b) Initial rate of oxidation of guaiacol by HRP in 2 (a) and 3 (b) in the presence and absence of TRIS (100 mM) with varying water
content. [TRIS] = 0.1 M, [HRP] = 50 lg/mL [Guaiacol] = 0.3 mM, [H2O2] = 0.1 mM.

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D. Das et al. / Tetrahedron Letters 48 (2007) 5635–5639
Figure 6. Initial rate of oxidation of guaiacol by HRP in different ILs and methanol at 5% water content. Inset shows the activity profiles of HRP in
4, 7 and 8 at varying percentages of water. [HRP] = 50 lg/mL [Guaiacol] = 0.3 mM, [H2O2] = 0.1 mM. The experimental error in these
measurements was typically within 8–12% in triplicate measurements.
organic solvent for HRP. Under varying water content,
HRP showed ꢀ60–240 times higher activity in 8 as com-
pared to that found in 1 and was beyond comparison
with respect to 2 and 3. Although the activity of HRP
was significantly improved in 8 compared to other ILs
and methanol, it was 15–20 times lower than that in
water. Spectroscopic studies are in progress to under-
stand the effects of the components of the IL on the
enzyme.
dation of thioanisole was observed in IL 8, which was
developed on the basis of enzyme stability and activity
in different ILs. Hence, the present approach provides
a prelude to the development of novel green solvents
which are tailor made for enzyme catalysis.
Acknowledgements
D.D. and A.D.G. acknowledge CSIR, India, for their
Research Fellowships and P.K.D. is thankful to the
Department of Science and Technology, India, for
financial assistance through a Ramanna Fellowship
(No. SR/S1/RFPC-04/2006).
Improving the stereoselectivity of an enzyme towards a
given substrate is also a very challenging objective in
the field of biocatalysis. The prospect of ionic liquids
as reaction media in this field has been studied re-
cently.19,21 At this time, we questioned whether the pres-
ent ILs could also boost the stereoselectivity of HRP in
asymmetric sulfoxidation. Accordingly, we investigated
three ILs 3, 4 and 8, with varying percents of water as
reaction media for carrying out the sulfoxidation of
thioanisole (0.5 mM) by HRP (15 mg/mL) using 1 mM
H2O2 as the oxidant. It was found that no sulfoxide
was formed using 3 and 4 in the presence of varying per-
cents of water (Table 1); whereas in 8 with 10%, 30%
and 50% v/v content of water, HRP showed >99% ee
with conversions ranging from 16% to 40% on reaction
for 2 h (details of the experimental procedure are given
in the Supplementary data). In concurrence with the
observed trend in guaiacol oxidation by HRP, here also
peroxidase showed improved activity as well as stereo-
selectivity in designed IL, 8.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2007.06.022.
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Table 1. Asymmetric oxidation of phenyl methylsulfide by HRP at
room temperature in ILsa
Ionic liquid
Water added
to IL (%)
Conversion
(%)
(R) ee
(%)
3
4
8
50
50
10
30
50
0
0
—
—
>99
>99
>99
15.8
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40.34
a Concentrations in the reaction mixtures: [H2O2] = 1 mM, [HRP] =
15 mg/mL, [phenyl methylsulfide] = 0.5 mM.

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was diluted (100–500 lL) with buffer and an aliquot
(3.75 lL) was added to a cuvette (1.5 mL) to monitor the
initial rate of oxidation (V) of guaiacol spectrophotomet-
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required % water (buffer). Subsequently, the enzyme,
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oxidation of guaiacol. [HRP] = 50 lg/mL; (c) Aqueous
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containing 100 mM TRIS at pH = 7 was used. The
addition of TRIS increased the pH to ꢁ9. Hydrochloric
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