Effect of imidazolium ionic liquids on the hydrolytic activity of lipase

Article abstract of DOI:10.1016/S1872-2067(11)60521-4

The effect of 1-alkyl-3-methylimidazolium ionic liquids (ILs) on the hydrolysis activityof Candida rugosa lipase (CRL) toward triacylglycerol was investigated. The critical micelle concentrations (CMC) of ILs with Br ^-, Cl^-, and [BF₄]^- anions and the solubility of ILs with [PF₆]^- anions were determined in phosphate buffer. Results suggested that the content of the ILs, not kosmotropicity, highly influenced the effects of anions and cations of ILs on CRL activity. As the length of alkyl chain of the cation [C_nMIM] ⁺ increased, lower IL content was required to achieve high enzyme activity. Once the concentrations of the ILs with Br^-, Cl ^-, and [BF₄]^- anions exceeded the CMC value, enzyme activity was suppressed. The positive promotion effect of anions on enzyme activity was in the order of Br^- Cl^- [BF ₄]^- [PF₆]^-. The effect of ionic liquid on enzyme activity was highly dependent on the pH and temperature of the system, with the optimum pH being 7.000. Under optimal conditions of pH 7.000, 30 °C, and 47.6 mmol/L of [C₈MIM]Br, the highest relative activity of CRL (1734%) was achieved, with a specific activity of 54.4 U/mg protein.

Full text of DOI:10.1016/S1872-2067(11)60521-4

Chinese Journal of Catalysis 34 (2013) 769–780
ava i l a b l e a t w w w. s c i e n c ed i r e c t . c o m
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e /c h n j c
Article
Effect of imidazolium ionic liquids on the hydrolytic activity of lipase
LI Naa,*, DU Weiyana, HUANG Zhuonanb, ZHAO Weia, WANG Shoujianga
a Key Laboratory of Thermo‐Fluid Science and Engineering, Ministry of Education, School of Chemical Engineering and Technology, Xi’an Jiaotong
University, Xi’an 710049, Shaanxi, China
b Department of Chemistry and Engineering, Baoji University of Arts and Science, Baoji 721007, Shaanxi, China
A R T I C L E I N F O
A B S T R A C T
Article history:
The effect of 1‐alkyl‐3‐methylimidazolium ionic liquids (ILs) on the hydrolysis activity of Candida
rugosa lipase (CRL) toward triacylglycerol was investigated. The critical micelle concentrations
(CMC) of ILs with Br–, Cl–, and [BF4]– anions and the solubility of ILs with [PF6]– anions were deter‐
mined in phosphate buffer. Results suggested that the content of the ILs, not kosmotropicity, highly
influenced the effects of anions and cations of ILs on CRL activity. As the length of alkyl chain of the
cation [CnMIM]+ increased, lower IL content was required to achieve high enzyme activity. Once the
concentrations of the ILs with Br–, Cl–, and [BF4]– anions exceeded the CMC value, enzyme activity
was suppressed. The positive promotion effect of anions on enzyme activity was in the order of Br–
> Cl– > [BF4]– > [PF6]–. The effect of ionic liquid on enzyme activity was highly dependent on the pH
and temperature of the system, with the optimum pH being 7.000. Under optimal conditions of pH
7.000, 30 °C, and 47.6 mmol/L of [C8MIM]Br, the highest relative activity of CRL (1734%) was
achieved, with a specific activity of 54.4 U/mg protein.
Received 20 November 2012
Accepted 24 December 2012
Published 20 April 2013
Keywords:
Imidazolium ionic liquid
Lipase
Hydrolysis
Enzyme activity
Critical micellar concentration
Hofmeister
© 2013, Dalian Institute of Chemical Physics, Chinese Academy of Sciences.
Published by Elsevier B.V. All rights reserved.
1. Introduction
ported, the miscibility of an IL is mainly influenced by the abil‐
ity of the anions to form hydrogen bonds, and slightly by the
Because of the lack of vapor pressure, thermal stability,
non‐flammability, and widely tunable physicochemical proper‐
ties, ionic liquids (ILs) are great reaction media and efficient
additives for enzymatic reactions. They provide an alternative
reaction media for volatile organic solvents and are advanta‐
geous for the use of enzymes as they can obtain higher activity,
selectivity, and stability [1–3]. Ionic liquids usually contain a
bulky cation and a small anion, and are available in a number of
varieties. The structure of the cation and anion both determine
the polarity, hydrophobicity or hydrophilicity, and ionization of
the ILs. These properties influence the micro structure of the
reaction system and the catalytic activity of any enzyme em‐
ployed. Thus, appropriate ILs need to be screened for each spe‐
cific enzyme‐medium‐substrate reaction system [4–6]. As re‐
length and number of alkyl chains linked to the imidazolium
ring. With the same anion, the hydrophobicity of an IL increas‐
es as the length of the alkyl chain of the cation increases [7–9].
Some researchers found that enzyme activity was mainly in‐
fluenced by anions rather than cations [10–12]. Yigitoglu et al.
[13] considered that the enzyme activity increased as the
length of alkyl chain increased. In contrast, Ulbert et al. [2] and
Zhao [4] indicated that enzyme activity and stability increased
as the length of the alkyl chain decreased. In addition, the con‐
tent of the IL affects the ionization and aggregation structure of
one or more ILs in the system as well as the properties of ionic
strength, pH, viscosity, and interfacial properties of the reaction
system as a whole [14]. Zhao et al. [15] found that an appropri‐
ate concentration of ILs promoted lipase activity for amino acid
*Corresponding author. Tel: +86‐29‐82664554; E‐mail: lina@mail.xjtu.edu.cn
This work was supported by the National Basic Research Program of China (973 Program, 2009CB2199006).
DOI: 10.1016/S1872‐2067(11)60521‐4 | http://www.sciencedirect.com/science/journal/18722067 | Chin. J. Catal., Vol. 34, No. 4, April 2013

LI Na et al. / Chinese Journal of Catalysis 34 (2013) 769–780
ester hydrolysis. However, most research has only been carried
and acetone were removed through filtration and rotary evap‐
oration, respectively. The residual was washed 4–6 times using
methylene chloride. The ionic liquid was dissolved in deionized
water until no white precipitate appeared with the addition of
silver nitrate. After methylene chloride removal, [CnMIM]BF4
was obtained with a yield of 62.5%–73.2%.
out with specific concentrations of ILs, thereby limiting a sys‐
tematic and general study of their influences on enzyme activi‐
ties. Thus, investigating the effects of structures of different
cations and anions on enzyme activity within a wide range of IL
content is required.
Lipase has high specific activity, enantioselectivity, and
thermal stability. Few studies have been conducted on the in‐
fluence of IL content and structure on hydrolysis activity in
IL‐water systems under different pH and temperature condi‐
tions. Our previous study [16] showed that lipase hydrolytic
activity can be efficiently and accurately analyzed using an au‐
tomatic pH‐stat potentiometric titration method, which was
carried out based on changes in micro electrode potential in‐
duction. This automatic titration system has advantages of pre‐
cision, stability, and reproducibility. Thus, using this method to
investigate the enzyme activity with various ILs under different
conditions can help to understand the influences of the ionic
liquid on the mechanism of enzymatic catalytic hydrolysis and
determine proper ionic liquid and reaction conditions.
Imidazolium ionic liquid, widely used during biocatalytic
processes, can improve enzyme activity, selectivity, and stabil‐
ity [17–19]. In the present study, constant potential automatic
titration was applied to investigate the effects of cations / ani‐
ons and concentrations of 19 ILs with 1‐alkyl‐3‐ methylimidaz‐
olium on hydrolysis activity. The cations were [CnMIM]+ (n = 2,
4, 6, 8, 10, 12) with different lengths of the alkyl chain, and the
anions were Br–, Cl–, [BF4]–, and [PF6]–. The hydrolysis activity
of Candida rugosa lipase (CRL) in different ionic liq‐
uid‐phosphate buffer solutions was analyzed and the optimum
pH and temperature of the systems were determined. Critical
micelle concentrations and solubility of ILs were determined
using conductance. The relationships among the concentra‐
tions of ILs, ionization, and enzyme activity are discussed.
Synthesis of [C4MIM]Cl [20]. A total of 18.51 g of chlorobu‐
tane was added into 16.42 g of N‐methylimidazole slowly with
stirring. The mixture was put into a microwave synthesis reac‐
tion device, and was stirred and refluxed under 70 °C for 6 h.
The sticky products were washed three times using ethyl ace‐
tate, after which the ethyl acetate was removed under 90 °C
using a rotary evaporator to obtain the product [BMIM]Cl with
a yield and purity of 94.6% and 98.9%, respectively.
Synthesis of [CnMIM]PF6 (n = 2, 4, 6, 8, 10, 12) [22].
[CnMIM]Br and KPF6 at a molar ratio of 1:1.1 were each dis‐
solved in deionized water, and then mixed together and reacted
at 80 °C for 3 h in a microwave synthesis reaction device. After
removal of the upper aqueous phase, the residue was washed
using deionized water repeatedly until no precipitate appeared
with the addition of silver nitrate in the upper aqueous phase.
[CnMIM]PF6 was obtained with a yield of 39.2%–66.1% after
the removal of water by evaporation under 90 °C.
2.2. Determination of CRL hydrolysis activity
A 7 mg/ml crude enzyme solution was obtained by dissolv‐
ing CRL powder in 50 mmol/L phosphate buffer solution (pH
7.000), which was centrifuged at 10 °C, 1000 r/min for 15 min.
The protein content of the supernatant was measured using the
bicinchoninic acid method [23]. A total of 40 ml phosphate
buffer (pH 7.000), 2 ml of olive oil, and a certain amount of
ionic liquids were mixed together and were preheated in a wa‐
ter bath at 30 °C for 30 min. After pH adjustment to 7.000 with
stirring at 1200 r/min, 2 ml of enzyme supernatant solution
was added to start the titration. The consumption of the sodi‐
um hydroxide standard solution after 15 min of titration was V
ml for the systems containing ILs and V0 ml for the control [16].
One unit of enzyme activity was defined as the production of
1 µmol of fatty acid for every 1 min under the determination
conditions, which is calculated by
2. Experimental
2.1 Synthesis of ionic liquid
Synthesis of [CnMIM]Br (n = 2, 4, 6, 8, 10, 12) [20]. To a set
quantity of N‐methylimidazole in a 100 ml round‐bottomed
flask, alkyl bromide was slowly added with stirring at a molar
ratio of 1:1. The following alkyl bromides were used: ethyl
bromide, n‐butyl bromide, 1‐bromohexane, bromooctane,
1‐bromodecane, and bromododecane. The mixture was put
into a microwave synthesis reaction device, and was stirred
and refluxed at 70 °C for 6 h. The sticky products were washed
three times using ethyl acetate, after which the ethyl acetate
was removed under 90 °C using a rotary evaporator to obtain
the products of [CnMIM]Br with yields and purities of
92.1%–94.8% and 98.6%–99.2%, respectively.
(V V )  C
1000
NaOH


0
(1)
U 
15
where CNaOH represents the concentration of sodium hydroxide
(mol/L).
The relative enzyme activity is calculated by
Relative activity (%) 
(2)
Enzyme activity in the system with ILs
100%
Enzyme activity in system without ILs
The specific enzyme activity (U/mg protein) is calculated by
Enzyme activity
(3)
Specific enzyme activity 
Synthesis of [CnMIM]BF4 (n = 2, 4, 6, 8, 10, 12) [21]: To a set
Pr otein content
quantity of NaBF4 dissolved in acetone in
a 250 ml
Each CRL hydrolysis activity test was carried out 2–3 times
with a relative error in the range of 0.19%–3.67%.
round‐bottomed flask, [CnMIM]Br dissolved in acetone was
slowly added with stirring at a molar ratio of 1:1. The mixture
was put into a microwave synthesis reaction device, and was
stirred and refluxed under 55 °C for 6 h. The precipitate NaBr
2.3 Electrical conductivity measurement of the ILs‐water
system

LI Na et al. / Chinese Journal of Catalysis 34 (2013) 769–780
Electrical conductivity (EC) of the phosphate buffer solution
and 505%, respectively. The effect of the ILs with the Br– anion
was higher than that of ILs with [BF4]– anion. It was noted that
as the carbon atomic number of the alkyl chains of the cations
increased, the optimum concentrations of ILs decreased. Once
the concentrations of ILs with longer alkyl chains of the cations
were over the optimum values, the CRL activity decreased
much faster, which led to a narrower concentration range of ILs
suitable for promoting CRL activity. The relative enzyme activ‐
ity would drop to 100% as the increase in IL concentrations
reached a certain value (called the maximum concentration
hereafter). Thus, the IL content had an important influence on
enzyme activity. In the literature, without considering the effect
of IL concentrations, conflicting conclusions about the effect of
the length of the alkyl chain on enzyme activity have been
made. As shown in Fig. 1, only when the number of alkyl chain
carbon atoms of the cation was less than 10 and the IL concen‐
trations were less than the optimum values did the influence of
ILs on enzyme activity show a Hofmeister effect. This phe‐
nomenon was consistent with the conclusion made by Bi et al.
[3], who found that only when the concentrations of I– based
imidazolium ILs were low and the number of alkyl chain car‐
bon atoms of the alkyl chain was less than 10 did the promo‐
tion effect of ILs on black plum glycosidase (catalyzes the syn‐
thesis of salidroside) increase with alkyl chain length, which is
consistent with the Hofmeister effect. When the length of the
alkyl chain was higher than 10, the promotion effect of ILs on
enzyme activity decreased.
(pH 7.000) before and after the addition of ILs was measured
using a conventional conductometer DDS‐11AW (Shanghai
Bante) at a constant temperature of 30 °C and 1200 r/min. The
value of the sample without ILs was used as the blank.
3. Results and discussion
3.1 Effect of the structure of cations and concentrations of ILs
with Br– and [BF4]– anions on CRL hydrolysis activity
Imidazolium ILs with Br– and [BF4]– anions had good misci‐
bility with water. The effect of the cation ([CnMIM]+(n = 2, 4, 6,
8, 10, 12)) structures and IL concentrations on CRL hydrolysis
activity is presented in Fig. 1. When the number of cationic
alkyl chain carbon atoms was less than 12, the ILs increased
CRL activity. As the concentrations of the ILs increased, the
positive effect increased at first and then decreased. Once the
concentrations of ILs exceeded a certain value, they actually
suppressed CRL activity. If the number of cationic alkyl chain
carbon atoms reached 12, both [C12MIM]Br and [C12MIM]BF4
thoroughly suppressed CRL activity. The maximum positive
promotion effect of cations on enzyme activity was achieved as
the concentrations of ILs reached the optimal values. The
maximum positive promotion effect of cations on enzyme ac‐
tivity was in the order of [C8MIM]+ > [C6MIM]+ > [C4MIM]+ >
[C2MIM]+ > [C10MIM]+ for ILs with the Br– anion, which had the
highest relative activities of 1734%, 1729%, 1709%, 1579%,
As seen in Fig. 1, the effect of the structure of the cations and
concentrations of IL on the enzyme hydrolysis activity might be
related to IL properties of ionization and aggregation. Within
the concentration range presented in Fig. 1, the conductivity of
the solution increased as the concentrations of IL increased
(Fig. 2). However, the growth rate slowed down gradually. Ac‐
cording to the study by Sirieix‐Plénet et al. [24], when the con‐
centrations of the IL were less than the critical micelle concen‐
tration (CMC), the molar conductivity of the solution increased
linearly with the square root value of the concentration. Once
the concentration of the IL was higher than the CMC, no linear
relationship was observed. Based on this observation, CMC can
be determined. Though some researchers have determined the
CMC of ILs in pure water and found that the addition of an in‐
organic salt affected the CMC [25,26], few CMC measurement
studies have been conducted in phosphate buffer solution. Ac‐
cording to the results illustrated in Fig. 2, the CMC of some of
the ILs could be obtained (Table 1). The CMC of the ILs in pure
water at 25 °C, as well as the optimum concentrations of ILs
and the maximum concentrations of ILs that led to the highest
relative enzyme activity and 100% relative enzyme activity,
respectively, are all presented in Table 1.
2000
[C2MIM]Br
[C4MIM]Br
[C6MIM]Br
[C8MIM]Br
[C10MIM]Br
[C12MIM]Br
(a)
1800
1600
1400
1200
1000
800
600
400
200
0
500
400
300
200
100
0
0
10 20 30 40 50
0
150 300 450 600 750 900 1050 1200 1350
Ionic liquid concentration (mmol/L)
2000
1800
1600
1400
1200
1000
800
500
400
300
200
100
0
(b)
0
10 20 30 40 50
[C2MIM]BF4
[C4MIM]BF4
[C6MIM]BF4
[C8MIM]BF4
[C10MIM]BF4
[C12MIM]BF4
The concentration ranges of [C2MIM]Br and [C4MIM]Br,
which increased enzyme activity, were both less than their
reported CMCs. The CMCs of the other ILs with longer alkyl
chain of the cation decreased as the carbon atomic number of
[CnMIM]+ decreased, which was coincident with the decreasing
ability of the cations to form hydrogen bonds and the increas‐
ing hydrophobicity of the ILs, as reported in the literature [9]. It
was obvious that the optimum concentrations of the ILs were
600
400
200
0
0
150 300 450 600 750 900 1050 1200
Ionic liquid concentration (mmol/L)
Fig. 1. Effect of concentrations and cation structure of Br– (a) and [BF4]–
(b) based imidazolium ionic liquids on CRL hydrolytic activity.

LI Na et al. / Chinese Journal of Catalysis 34 (2013) 769–780
Table 1
[C2MIM]Br
[C4MIM]Br
[C6MIM]Br
[C8MIM]Br
[C10MIM]Br
[C12MIM]Br
(a)
Optimum concentration and maximum concentration of Br– and [BF4]–
40
35
30
25
20
15
10
5
based imidazolium ionic liquids and their CMCs in phosphate buffer
solution.
Optimum
Maximum
CMCc
Reported CMCd
(mmol/L)
ILs
concentrationa concentrationb (mmol
(mmol/L)
778.7
208.6
78.4
47.6
4.6
—
466.9
208.6
(mmol/L)
—
/L)
—
—
5
[C2MIM]Br
[C4MIM]Br
[C6MIM]Br
[C8MIM]Br
[C10MIM]Br
[C12MIM]Br
[C2MIM]BF4
[C4MIM]BF4
1900 [29]
700 [29], 970 [30]
4
3
2
1
0
—
379.5
81.3
17.2
—
385.5 400 [29],770 [30]
89.4 150 [29], 160 [30]
17.5 30 [29], 39 [30]
0.8
—
0
20 40 60 80 100
9 [30]
—
0
—
0
150 300
450 600 750 900 1050
732.8
704.5
912 [31], 829
[31], 846 [32]
Ionic liquid concentration (mmol/L)
50
45
40
35
30
25
20
15
10
5
0.8
0.6
0.4
0.2
0.0
[C2MIM]BF4
[C4MIM]BF4
[C6MIM]BF4
[C8MIM]BF4
[C10MIM]BF4
[C12MIM]BF4
[C6MIM]BF4
[C8MIM]BF4
[C10MIM]BF4
78.0
18.3
1.7
244.4
75.5
9.4
242.5 282 [31], 312 [31]
78.2 89 [31], 86 [31]
7.5 27 [31], 23 [31]
1.2 8.7 [31], 8.3 [31]
(b)
[C12MIM]BF4
—
—
a
Concentrations of ILs with highest CRL activity in phosphate buffer
solution (pH 7.000) at 30 °C.
0 3 6 9 121518
b Concentrations of ILs over which the beneficial effect on activity was
eliminated.
c CMC of ILs in phosphate buffer solution (pH 7.000) at 30 °C.
d CMC of ILs in pure water at 25 °C.
celles and the amount of enzyme embedded in the micelles
increases as the surfactant content increases [28]. Thus, it can
be deduced that as the concentrations of ILs increased to their
CMCs, the amount of enzymes embedded in the micelles in‐
creased, which led to structural disruption of the enzyme and
hence a decrease in hydrolysis rate.
0
0
150 300 450 600 750 900 1050 1200
Ionic liquid concentration (mmol/L)
Fig. 2. Effect of concentration and cation structure of Br– (a) and [BF4]–
(b) based imidazolium ionic liquids on the conductivity of phosphate
buffer solution.
3.2 Effect of the structure of cations and concentrations of ILs
with [PF6]– anion on CRL hydrolysis activity
all smaller than the CMCs. When the number of alkyl chain
carbon atoms reached 6, 8, or 10, their maximum concentra‐
tions were almost the same as their CMCs. Moreover, when the
concentration of [C12MIM]Br was 0.83 mmol/L (just over the
CMC of 0.79 mmol/L), the CRL relative enzyme activity was
83%. When the concentration of [C12MIM]BF4 was 1.3 mmol/L
(just over the CMC of 1.2 mmol/L), the CRL relative enzyme
activity was 94%. These results suggested that the aggregation
behavior of ILs in the system significantly influenced the en‐
zyme activity. When the concentrations of ILs were less than
the CMC, the ILs increased the enzyme activity. Once the con‐
centrations were equal to their CMCs, this effect disappeared. If
the concentrations were higher than the CMCs, they inhibited
enzyme activity. Very few studies on the relationship between
the aggregation properties of ILs and enzyme activity have
been conducted. Geng et al. [27] systematically studied the
secondary structure of bovine serum albumin (BSA) in a
[C14MIM]Br– water system using circular dichroism. When the
concentrations of imidazolium ILs were less than their CMCs,
the ILs electrostatically interacted with BSA, which stabilized
the secondary structure of BSA. However, when the concentra‐
tions reached their CMCs, the hydrophobicities of the ILs in‐
creased and they strongly adsorbed to BSA, disrupting its sec‐
ondary structure. Once the concentrations were higher than the
CMCs, BSA denaturation occurred. It is known that activity is
reduced after enzymes become embedded in surfactant mi‐
The ILs with [PF6]– anion were strongly hydrophobic. The
effect of the concentrations and the length of the cation alkyl
chain on CRL activity in the ILs‐water system are illustrated in
Fig. 3. All the ILs had a beneficial effect on the CRL activity,
which reached the highest level under optimum concentra‐
tions. When the number of carbon atoms of the alkyl chain was
less than 10, the beneficial effect on enzyme activity was in the
order of [C8MIM]+ > [C6MIM]+ > [C4MIM]+ > [C2MIM]+. As the
number of carbon atoms of the cation alkyl chain increased, the
optimum concentrations of ILs decreased. Once the concentra‐
tions of ILs with longer cation alkyl chains were over the opti‐
mum values, the CRL activities rapidly decreased. This phe‐
nomenon coincided with the ILs containing Br– and [BF4]– ani‐
ons. However, the length of the alkyl chain had a significant
influence on the highest enzyme activity. In addition, ILs with
alkyl chains containing 10 and 12 carbon atoms still had strong
beneficial effects on enzyme activity. Two reasons can explain
this phenomenon: 1) the phase interface formed between the
hydrophobic ILs and water favored interfacial lipase catalytic
activity, and 2) because of the low solubility of ILs in the buffer
solution, the system is less toxic toward the enzyme. Previous
research has proven that compared with hydrophilic ILs‐water
one‐phase systems, hydrophobic ILs‐water multiphase systems
are less toxic for the biocatalyst.

LI Na et al. / Chinese Journal of Catalysis 34 (2013) 769–780
2000
1800
1600
1400
1200
1000
800
Table 2
Solubility and optimum content of [PF6]– based imidazolium ionic liq‐
uids in phosphate buffer.
[C2MIM]PF6
[C4MIM]PF6
[C6MIM]PF6
[C8MIM]PF6
[C10MIM]PF6
[C12MIM]PF6
Optimum
Solubilityb
(mmol/L)
Reported solubilityc
(mmol/L)
ILs
concentrationa
(mmol/L)
313.9
[C2MIM]PF6
[C4MIM]PF6
[C6MIM]PF6
[C8MIM]PF6
[C10MIM]PF6
[C12MIM]PF6
133.9
65.1
28.8
10.7
6.5
184 [9], 201.2 [37]
71.7 [9], 86.5 [37]
112.0
62.7
49.1
22.6
26.0 [37]
8.1 [37]
—
600
400
47.0
< 1.3
—
a Concentrations of ILs with highest CRL activity in phosphate buffer
solution (pH 7.000) at 30 °C.
b Solubility of ILs in phosphate buffer solution (pH 7.000) at 30 °C.
c Solubility of [C2MIM]PF6 in pure water at 35 °C; solubility of other ILs
in pure water at 30 °C.
200
0
0
100 200 300 400 500 600 700
Ionic liquid concentration (mmol/L)
Fig. 3. Effect of concentration and cation structure of [PF6]– based im‐
idazolium ionic liquids on hydrolytic activity of CRL.
ity, which might contribute to this phenomenon. However, once
the concentrations of the ILs increased to certain values, the
enzyme activity decreased. Some researchers support the idea
that imidazolium ILs can dissolve CRL to a certain extent, lead‐
ing to a decrease in enzyme activity [36]. If the concentration of
the IL was too high, a larger proportion of the total enzyme
amount might have dissolved, further resulting in a lower reac‐
tion rate. It was also observed that some oil was transferred
into the phase interface between the IL and water. Further, the
increase in the amount of IL led to a higher system viscosity.
Taken together, this might hinder the mass transfer of the sub‐
strate and enzyme. Consequently, the lipase catalytic reaction
rate decreased.
When the content of an IL in the buffer solution was higher
than its solubility, phase splitting occurred between the IL and
the buffer solution. Consequently, as the concentration of IL
with [PF6]– anion increased, the EC of the buffer solutions in‐
creased, and finally reached a constant high value (Fig. 4). With
the same concentration of IL, the EC of the buffer solution in‐
creased as the length of the alkyl chain decreased. It has been
reported that because of its large size, [PF6]– cannot effectively
bond to the micelle surface, resulting in phase splitting before
the formation of the micelle. Thus, it can be considered that an
IL will be fully ionized in water when its concentration is less
than its solubility [35]. Based on the relationship between mo‐
lar conductivity and the square root of concentration, the solu‐
bility of the ILs in buffer solution was determined (Table 2).
The solubility of [CnMIM]PF6 in buffer solution decreased as the
length of the alkyl chain of the cation increased, exhibiting the
same pattern as IL hydrophobicity. In addition, the optimum
concentrations of the ILs were higher than their solubilities in
buffer solution, which indicated that when the concentrations
of ILs were over their solubility in a certain range, their benefi‐
cial effects on enzyme activity still increased as their concen‐
trations increased. The phase interface formed between the ILs
and the buffer solution favored interfacial lipase catalytic activ‐
3.3 Effect of the structure of anions and concentrations of ILs
on CRL hydrolysis activity
The effect of the concentrations of ILs with different anions,
such as Br–, Cl–, [BF4]–, and [PF6]–, and [C4MIM]+ cation on CRL
activity, is shown in Fig. 5. Obviously, the anions contributed to
the enzyme activity in the order of Br– > Cl– > [BF4]– > [PF6]–,
which was not exactly the same as the previously reported
kosmotropicity sequence [4]. According to the Hofmeister ef‐
fect, the kosmotropicity sequence of anions is Cl– > Br– > [BF4]–
> [PF6]–. Bi et al. [3] reported that the beneficial effect of
[C4MIM]Cl was weaker than that of [C4MIM]Br. In addition, for
ILs with [PF6]– as an anion, because of the significant influence
of the alkyl chain of the cation on the maximum enzyme activi‐
ty, as the length of alkyl chain increased the effect of [PF6]– on
the enzyme activity did not fully conform to this order. This
also suggests that the effect of hydrophobic ILs on enzyme ac‐
tivity cannot be explained using the Hofmeister effect, which is
coincident with prior findings [30].
As shown in Fig. 5, the optimum concentrations of ILs with
Br–, Cl–, [BF4]–, and [PF6]– anions were 208.6, 209.4, 208.6, and
110.6 mmol/L, respectively. This indicates that the optimum
concentrations of hydrophilic ILs are very similar. The ILs (Br–
and [BF4]–) with the same cation alkyl chain length had similar
optimum concentrations (Fig. 1). This indicated that it was the
length of the alkyl chain of the cation but not the anion that
affected the optimum concentration of hydrophilic ILs. It was
14
12
[C2MIM]PF6
[C4MIM]PF6
[C6MIM]PF6
[C8MIM]PF6
[C10MIM]PF6
10
8
[C12MIM]PF6
6
4
2
0
0
100
200
300
400
500
Ionic liquid concentration (mmol/L)
Fig. 4. Effect of concentration and cation structure of [PF6]– based im‐
idazolium ionic liquids on the conductivity of phosphate buffer solution.

LI Na et al. / Chinese Journal of Catalysis 34 (2013) 769–780
2000
1800
1600
1400
1200
1000
800
55
[C4MIM]Cl
[C2MIM]Br
[C4MIM]Br
[C6MIM]Br
[C4MIM]BF4
[C4MIM]PF6
[C6MIM]PF6
without ionic liquid
(a)
50
45
40
35
30
25
20
15
10
5
[C4MIM]Br
[C4MIM]Cl
[C4MIM]BF4
[C4MIM]PF6
600
400
200
0
0
100
200
300
400
500
600
0
25 30 35 40 45 50 55 60
Temperature (oC)
Ionic liquid concentration (mmol/L)
Fig. 5. Effect of concentration and anion type of [C4MIM]+ based imid‐
azolium ionic liquids on the hydrolytic activity of CRL.
50
45
40
35
30
25
20
15
10
5
(b)
unlikely that it was the anion and not the length of the alkyl
chain of the cation that affected the activity of CRL.
[C4MIM]Cl
[C2MIM]Br
[C4MIM]Br
[C6MIM]Br
[C4MIM]BF4
[C4MIM]PF6
[C6MIM]PF6
without ionic liquid
The effect of anions on the EC of the buffer solution is shown
in Fig. 6. The beneficial effect of the anions was in the order of
Cl– > Br– > [BF4]– > [PF6]–, coinciding with anion size and mass.
It was reported that anions of ILs with larger diameters and
masses have lower migration speed, which results in a low EC
[38]. According to the relationship between molar conductivity
and the square root of IL concentration, as the investigated
concentrations of [C4MIM]Cl, [C4MIM]Br, and [C4MIM]BF4 were
less than their CMCs, the enzyme activity should be enhanced.
In addition, when the alkyl chain had 2, 6, 8, 10, and 12 carbons,
the effect of the anions Br– and [BF4]– on EC was also in the
order of Br– > [BF4]–.
0
6.0
6.5
7.0
7.5
pH
8.0
8.5
9.0
Fig. 7. Effects of temperature (a) and pH (b) of the IL‐phosphate buffer
solution system on CRL hydrolytic activity.
mum temperature for the systems without an IL, with the addi‐
tion of a hydrophilic IL with Br–, Cl–, and [BF4]– anions, and with
the addition of a hydrophobic IL with a [PF6]– anion, were 37,
30, and 37 °C, respectively (Fig. 7(a)). This indicated that the
highest enzyme activity could be reached at a lower tempera‐
ture in hydrophilic ILs‐water systems compared with that
without ILs, while the optimum temperature was the same as
that of a system without an IL in a heterogeneous system of a
hydrophobic [C4MIM]PF6 IL combination and water.
3.4 Effect of temperature and pH on CRL hydrolysis activity in
ILs‐water system
High temperature can help achieve a high enzymatic reac‐
tion rate but may accelerate enzyme denaturation. With opti‐
mum IL concentrations, the effect of temperature and pH on
CRL hydrolysis activity in the ILs‐water system was investigat‐
ed. As shown in Fig. 7, as temperature and pH increased, the
relative activity first increased and then decreased. The opti‐
The pH value of the reaction system may affect the dissocia‐
tion state and conformational space of an enzyme’s active cen‐
ter, thereby affecting enzyme activity. The optimum pH for the
reaction decreased from 7.200 to 7.000 after the addition of a
variety of ILs at 30 °C (Fig. 7(b)). When the pH value was lower
than 6.0, the enzyme activity decreased to 30%–40% of the
highest value. When the pH value was higher than 8.0, no en‐
zyme activity was observed. These results suggest that CRL was
sensitive to high pH. It might be that the negatively charged
enzyme in the alkaline environment interacted with IL cations
more severely than in the acid environment as the isoelectric
points of CRL are 4.4(A) and 4.9(B).
The beneficial effect of different ILs on enzyme activity
changed as the temperature and pH were varied (Fig. 7). Gen‐
erally speaking, the enzyme activity in systems with the addi‐
tion of ILs having the same anion but different cations changed
similarly with each other as temperature and pH changed. In
25
[C4MIM]Br
[C4MIM]Cl
[C4MIM]BF4
[C4MIM]PF6
20
15
10
5
0
0
100
200
300
400
500
600
Ionic liquid concentration (mmol/L)
Fig. 6. Effect of concentration and anion type of [C4MIM]+ based imid‐
azolium ionic liquids on the conductivity of ionic liquid‐phosphate
buffer solution.

LI Na et al. / Chinese Journal of Catalysis 34 (2013) 769–780
contrast, the enzyme activity of systems with the addition of ILs
Acknowledgments
having the same cation but different anions changed quite dif‐
ferently as temperature and pH changed. This suggests that the
effect of temperature and pH on CRL hydrolysis activity was
highly dependent on the types of anions of the ILs.
The authors acknowledge Prof. Bolun Yang’s research team
at Xi’an Jiaotong University for their provision of materials for
the synthesis of ILs.
As shown in Fig. 7, under the optimum conditions of pH 7.0,
30 °C and 47.6 mmol/L [C8MIM]Br, the highest relative enzyme
activity and specific activity reached 1734% and 54.4 U/mg
protein, respectively. Dominguez de Maria et al. [39] measured
different CRL activities (Sigma, Roche, and Fluka) using an au‐
tomatic pH‐stat potentiometric titration method. Under the
conditions of pH 7.0 and 30 °C, CRL activity was in the range of
3.8–14.0 U/mg protein. The construction of a micro‐emulsion
system efficiently increased the activity of all the sources of
enzymes with the specific enzyme activity reaching 19.8 U/mg
protein. By comparison, much higher enzyme activity has been
reported herein through the addition of appropriate ILs. In
addition, as reported previously [16], the addition of PVA, AOT
or lecithin increased the maximum relative activity of CRL to
255%, which was still much lower than the enzyme activity
obtained with IL addition.
References
[1] Moniruzzaman M, Nakashima K, Kamiya N. Goto M. Biochem Eng J,
2010, 48: 295
[2] Ulbert O, Fráter T, Bélafi‐Bakó K, Gubicza L. J Mol Catal B, 2004,
31: 39
[3] Bi Y H, Wang Ch Y, Mao Y Y, Zheng Sh Y, Zhang H J, Shi H. Chin J
Catal, 2012, 33: 1161
[4] Zhao H. J Mol Catal B, 2005, 37: 16
[5] Stock F, Hoffmann J, Ranke J, Störmann R, Ondruschka B, Jastorff
B. Green Chem, 2004, 6: 286
[6] Du W Y, Li N, Wang Sh J. Chemistry, 2012, 75: 780
[7] Seddon K R, Stark A, Torres M J. Pure Appl Chem, 2000, 72: 2275
[8] Stark A, Seddon K R. Ionic Liquids, in Kirk‐Othmer Encyclopedia of
Chemical Technology. New York: Wiley, 2007. 859
[9] Freire M G, Neves C M S S, Carvalho P J, Gardas R L, Fernandes A M,
Marrucho I M, Santos L M N B F, Coutinho J A P. J Phys Chem B,
2007, 111: 13082
4. Conclusions
[10] Kaar J L, Jesionowski A M, Berberich J A, Moulton R, Russell A J. J
Am Chem Soc, 2003, 125: 4125
The effect of 1‐alkyl‐3‐methylimidazolium ionic liquids (ILs)
on the activity of Candida rugosa lipase (CRL) for the hydrolysis
of triacylglycerol was investigated. The CMC and solubility of
the ILs were determined in phosphate buffer. The results sug‐
gested that the effect of the ILs on CRL activity was highly de‐
pendent upon their concentration, alkyl chain length of the
cation and type of anion employed. The strongest beneficial
effect on enzyme activity was achieved at the optimum concen‐
trations of ILs. The maximum beneficial effect of the anions was
in the order of Cl– > Br– > [BF4]– > [PF6]–. The lengthier the alkyl
chain of the cation [CnMIM]+, the lower the optimum content of
ILs required to achieve high enzyme activity. When the length
of the alkyl chain of [CnMIM]+ in the ILs with Br– or [BF4]– was
less than or equal to 10 and their concentrations in phosphate
buffer solution were smaller than their CMCs, the ILs increased
enzyme activity. Once the concentrations of the ILs with Br– or
[BF4]– anions exceeded their CMC value, enzyme activity was
suppressed. Their optimum concentrations were much smaller
than their CMCs. Only when the number of carbon atoms of the
alkyl chain of [CnMIM]+ of ILs with Br– or [BF4]– was in the
range of 2–8 and the concentrations were smaller than the
optimum values, the beneficial effect of the alkyl chain of the
cations on enzyme activity coincident with the Hofmeister ef‐
fect. The effect of [CnMIM]+ from ILs with [PF6]– anion showed
no relationship with the Hofmeister effect. The effect of ILs on
enzyme activity was highly dependent on the pH and tempera‐
ture of the system and an optimum pH of 7.000 was deter‐
mined. The optimum temperatures for maximal enzyme activi‐
ty for hydrophilic and hydrophobic ILs were 30 and 37 °C, re‐
spectively. Under the optimal conditions, the highest relative
activity of CRL with the addition of [C8MIM]Br reached 1734%.
This study provides insight into the structure‐activity relation‐
ships of ionic liquids that influence enzyme activity.
[11] Zhao H, Olubajo O, Song Z Y, Sims A L, Person T E, Lawal R A,
Holley L A. Bioorg Chem, 2006, 34: 15
[12] Lou W Y, Zong M H, Liu Y Y, Wang J F. J Biotechnol, 2006, 125: 64
[13] Yigitoglu M, Temocin Z. J Mol Catal B, 2010, 66: 130
[14] Sun Sh, Zhai Y J, Sun Y, Zhang Y P, Liu H, Wang X H, Yu A M, Zhang
H Q. Chem J Chin Univ, 2010, 31: 468
[15] Zhao H, Luo R G, Malhotra S V. Biotechnol Progr, 2003, 19: 1016
[16] Zhao W, Huang Zh N, Li N. Chin J Anal Chem, 2011, 39: 351
[17] Shan H X, Lu Y, Li Z J, Li M, Cai Y, Sun X L, Zhang Y Zh. Chin J Catal,
2010, 31: 289
[18] Nicole W, Christian W, Vaclav C, Andreas P, Eva H, Rainer R, Frank
B. Chem Biol Chem, 2008, 9: 1493
[19] Debnath S, Das D, Dutta S, Das P K. Langmuir, 2010, 26: 4080
[20] Park S, Kazlauskas R J. J Org Chem, 2001, 66: 8395
[21] Lozano P, De Diego T, Guegan J P, Vaultier M, Iborra J L. Biotechnol
Bioeng, 2001, 75: 563
[22] Huddleston J G, Rogers R D. Chem Commun, 1998: 1765
[23] Shang Y, Li N, Qin X H, Lu B W, Fu Q. Chin J Anal Chem, 2009, 37:
1173
[24] Sirieix‐Plénet J, Gaillon L, Letellier P. Talanta, 2004, 63: 979
[25] Dong B, Li N, Zheng L Q, Yu L, Inoue T. Langmuir, 2007, 23: 4178
[26] Miskolczy Z, Sebők‐Nagy K, Biczók L, Göktürk S. Chem Phys Lett,
2004, 400: 296
[27] Geng F, Zheng L Q, Yu L, Li G Z, Tung C H. Process Biochem, 2010,
45: 306
[28] Borgstrom B, Donner J. J Lipid Res, 1976, 17: 491
[29] Goodchild I, Collier L, Millar S L, Prokeš I, Lord J C D, Butts C P,
BowersJ, Webster J R P, Heenan R K. J Colloid Interf Sci, 2007, 307:
455
[30] Wang J J, Wang H Y, Zhang Sh L, Zhang H Ch, Zhao Y. J Phys Chem B,
2007, 111: 6181
[31] Fu S Zh, Chen Q D, Shen X H. Acta Phys‐Chim Sin, 2011, 27: 1913
[32] Katayanagi H, Nishikawa K, Shimozaki H, Miki K, Westh P, Koga Y.
J Phys Chem B, 2004, 108: 19451
[33] Rehmann L, Ivanova E, Ferguson J L, Gunaratne H Q N, Seddon K R,

LI Na et al. / Chinese Journal of Catalysis 34 (2013) 769–780
Graphical Abstract
Chin. J. Catal., 2013, 34: 769–780 doi: 10.1016/S1872‐2067(11)60521‐4
Effect of imidazolium ionic liquids on the hydrolytic activity of lipase
LI Na*, DU Weiyan, HUANG Zhuonan, ZHAO Wei, WANG Shoujiang
Xi’an Jiaotong University; Baoji University of Arts and Science
Lipase hydrolytic activity was strongly affected by the concentrations, cation structure and anion type of the ILs. The beneficial effect of
the IL on lipase activity was eliminated as the IL approached its CMC.
Stephens G M. Green Chem, 2012, 14: 725
[34] Gorke J, Srienc F, Kazlauskas R. Biotechnol Bioprocess Eng, 2010,
15: 40
[35] Blesic M, Marques M H, Plechkova N V, Seddon K R, Rebelo L P N,
Lopes A. Green Chem, 2007, 9: 481
[37] Wong D S H, Chen J P, Chang J M, Chou C H. Fluid Phase Equilibr,
2002, 194‐197: 1089
[38] Wang F H, Li Ch X, Meng H, Wang Z G. J Beijing Univ Chem Technol ,
2006, 33(6): 17
[39] Dominguez de Maria P, Martinez‐Alzamora F, Moreno S P, Valero
F, Rua M L, Sánchez‐Montero J M, Sinisterra J V, Alcantara A R.
Enzyme Microb Technol, 2002, 31: 283
[36] Xin J Y, Zhao Y J, Zheng Y, Xia Ch G, Li Sh B. Chin J Catal, 2006, 27:
263