Immobilization of β-galactosidase onto Sepharose and stabilization in room temperature ionic liquids

Article abstract of DOI:10.1016/j.molliq.2009.11.003

The hydrolysis of o-nitrophenyl-β-d-galactopyranoside (ONPG) by β-galactosidase immobilized on Sepharose CL-4B was investigated in five different ionic liquids (ILs), 1-butyl-3-methylimidazolium X^-; [X = CF₃SO₃^-, BF₄^-, PF₆^-, CH₃SO₄^-and N(CN)₂^-]. Michaelis-Menten kinetic studies were conducted in phosphate buffer and in the five ionic liquids. For the immobilized enzyme in the ILs, the K_m values were lower (0.36-1.2 mmol ONPG) while the V_max values were higher (0.04-0.008 min^{- 1}) compared to those in aqueous phosphate buffer suggesting a marked increase in the efficiency of the immobilized enzyme in the ionic liquid. For the free enzyme in the ionic liquids, the K_m values, in general, were larger (0.45-4.96 mmol ONPG) than those of the immobilized enzyme in the ionic liquid. A postulated mechanism for the hydrolysis is suggested, involving interception of the intermediate oxonium ion species by the counter ion of the ionic liquid, thereby enabling the hydrolysis to occur at a faster rate.

Full text of DOI:10.1016/j.molliq.2009.11.003

Journal of Molecular Liquids 152 (2010) 19–27
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Immobilization of β-galactosidase onto Sepharose and stabilization in room
temperature ionic liquids
Natasha R. Singh ⁎, Dyer Narinesingh, Gurdial Singh
Department of Chemistry, The University of The West Indies, St Augustine, Trinidad and Tobago
a r t i c l e i n f o
a b s t r a c t
Available online 13 November 2009
The hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPG) by β-galactosidase immobilized on Sepharose
®
−
−
CL-4B was investigated in five different ionic liquids (ILs), 1-butyl-3-methylimidazolium X ; [X=CF
3
SO
3
,
BF , PF , CH SO4 and N(CN) 2− ]. Michaelis–Menten kinetic studies were conducted in phosphate buffer and in
−
−
−
Keywords:
4
6
3
β-galactosidase
Ionic liquids
Immobilization
Sepharose
m
the five ionic liquids. For the immobilized enzyme in the ILs, the K values were lower (0.36–1.2 mmol ONPG)
while the Vmax values were higher (0.04–0.008 min ) compared to those in aqueous phosphate buffer
suggesting a marked increase in the efficiency of the immobilized enzyme in the ionic liquid. For the free
−1
m
enzyme in the ionic liquids, the K values, in general, were larger (0.45–4.96 mmol ONPG) than those of the
immobilized enzyme in the ionic liquid. A postulated mechanism for the hydrolysis is suggested, involving
interception of the intermediate oxonium ion species by the counter ion of the ionic liquid, thereby enabling
the hydrolysis to occur at a faster rate.
©
2009 Elsevier B.V. All rights reserved.
1
. Introduction
Ionic liquids have been referred to as potentially “green solvents”
and other than the obvious benefit to the environment when com-
pared with noxious organic solvents, a new world of non-aqueous
enzymology can be realized. Advantages of using ionic liquids over the
use of normal organic solvents as reaction medium for biocatalysis also
include their high ability of dissolving a wide variety of substrates,
especially those highly polar ones, and their widely tunable solvent
properties through appropriate modification of the cations and anions
[5d].
Ionic liquids as solvents are capable of dissolving both polar and
non-polar compounds and are ideally suited for the dissolution of
carbohydrates [6]. Consequently IL's are ideal candidates for the study
of the chemistry of carbohydrates and of enzymes.
Ionic liquids (IL's) are salts consisting of ions, which exist in the
liquid state at ambient temperatures or in general, have melting points
under 100 °C. They typically consist of organic nitrogen-containing het-
erocyclic cations and inorganic anions [1]. There has been a plethora
of publications in the scientific literature during the last ten years con-
cerning the use of ionic liquids as solvents in chemical and biochemi-
cal reactions. This increased interest has sparked the imagination of
workers in the field and encouraged researchers to employ room tem-
perature ionic salts in a myriad of reactions. New emerging data has
demonstrated the versatility in applicability of these solvents [1].
Room temperature ionic liquids have thus far offered new and
exciting possibilities for the use and application in enzyme catalyzed
reactions [2]. Apart from the ability of ionic liquids to replace tradi-
tional organic solvents in many systems, they are capable of improving
the enantioselectivity in addition to increasing enzyme stability. To
date, reactions involving lipases have benefited most from the use of
ionic liquids [2].
As part of our program for the synthesis of carbohydrates we com-
menced an investigation of the chemistry of the enzyme β-galactosidase
[7–9] immobilized on, Sepharose CL-4B. For our study, β-galactosidase
was used (this catalyzes the hydrolysis of lactose to the parent sugars
D-galactose and D-glucose) from Aspergillus oryaze.
m
The general kinetic parameters (K and Vmax) for the enzyme
The use of ionic liquids with other enzymes and in whole-cell pro-
cesses has also been described [3,4]. In some cases, remarkable results
with respect to yield, (enantio)selectivity or enzyme stability were
observed [5].
were elucidated in traditional working phosphate buffer and in the
five ionic liquids. Although the majority of enzymes reported to be
active in ionic liquids are lipases, other biocatalysts have been inves-
tigated in ionic liquids with improved results compared to water
[16]. To our knowledge there has only been one report thus far on
the activity of β-galactosidase [5] (free enzyme) in ionic liquids,
and no reports thus far have documented the activity of immobi-
lized β-galactosidase in ionic liquids.
⁎
Corresponding author. Tel.: +868 6622002; fax: +868 6453771.
E-mail address: natasha.ramroopsingh@utt.edu.tt (N.R. Singh).
0167-7322/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molliq.2009.11.003

20
N.R. Singh et al. / Journal of Molecular Liquids 152 (2010) 19–27
The five different ionic liquids used in this study were chosen
The amount of protein bound to the gel was determined, using the
Hatree–Lowry method, from the differences in protein concentration
between the enzyme solution offered for coupling and the super-
natant and wash enzyme solutions after coupling.
based upon a difference in the nucleophilic power of the counterions
of these IL's. The anions present in the five different ionic liquids are
listed below in order of increasing nucleophilicity:
2.3. Determination of initial rates for each reaction system under inves-
tigation [15b]
To nine clean dried tubes was added 0.1, 0.2, 0.3, 0.5, 0.7, 0.8, 1.0,
1
.5 and 2.0 mL of ONPG solution (taken from a 1.6 mM stock solution
of ONPG dissolved in phosphate buffer or ionic liquid respectively)
was added. Subsequently each tube was diluted to a final volume of
2.5 ml, with phosphate buffer or ionic liquid. A fixed volume (0.75 mL)
of each solution was subsequently placed into a cuvette and the
hydrolysis reaction triggered by addition of 0.75 ml stock enzyme
solution (0.004 mg) of the free enzyme into the cuvette. The change
in absorbance over time was measured at appropriate time intervals
and graphs of absorbance at 420 nm vs. time (minutes) were plotted
for each concentration of ONPG in the phosphate buffer and in each
of the five ionic liquids, and initial rates determined from the linear
initial portion of each graph. In the case of the immobilized enzyme,
the hydrolysis reaction was triggered by addition of 0.2 mL of the gel
This investigation sought to determine the factors affecting the rate
of the hydrolysis of ONPG and the variation of medium and environ-
ment around the immobilized enzyme and substrate. Our studies add
to the growing knowledge of applications of ionic liquids to organic
reactions, and serves to demonstrate, using a simple system, enhanced
performance, which can be applied to larger, more complex and
important systems, especially in the realm of carbohydrate chemistry.
2
. Experimental
2
.1. Synthesis of [bmim][dca]
(
conc.=0.02 mg enzyme/mL gel) to each tube, and the absorbance
An acidic solution of sodium dicyanamide, 3.562 g (40 mmol), in
0 ml of 2 M HCl, was added to 11.032 g of Ag CO3. This resulted in the
2
formation of a Ag[dca] precipitate. The resultant solid was suction
filtered, and repeatedly washed five times with 150 ml of de-ionized
water.
of the supernatant was measured at appropriate time intervals, and
similar graphs as described for the free enzyme were plotted.
It should be noted that the sample withdrawn from the reaction
tube for determination of absorbance at the various time intervals were
quickly returned to the tube after measurement. There would have
been losses in transfer, but these were minimized as far as possible.
5
To 0.174 g (11 mmol) of Ag[dca] was added to a solution of [bmim]
[Br] (11 mmol) in 30 ml water and the resulting suspension was
stirred overnight. The mixture was filtered and the water removed
m
2.4. K and Vmax: studies of immobilized and free β-galactosidase in
under vacuum to afford the crude title compound. The crude material
aqueous medium and ionic liquid media
was dissolved in DCM (100 ml) and dried (MgSO
removed under vacuum and gave an approximate yield of 96% for
bmim][dca].
4 .
) The DCM was
From the initial rates graphs generated in Section 2.3 above, plots
of the inverse of these initial rates vs. the reciprocal of the correspond-
ing substrate concentrations for each system were investigated (free
and immobilized β-galactosidase) in the phosphate buffer and each of
[
2
.2. Immobilization of β-galactosidase onto Sepharose and test for activity
in phosphate buffer and ionic liquids [14,15a]
the five ionic liquids were generated. Kinetic parameters of K
m
and
V
max values were then determined for β-Galactosidase using ONPG as
2
.2.1. Preparation of Sepharose for immobilization of β-galactosidase
.2.1.1. Sodium periodate oxidation of Sepharose CL-4B. Sepharose CL-4B
substrate in each of these systems investigated.
2
2.5. Kinetic measurements and calculations
(
25 ml) was washed thoroughly using 2 L distilled water to remove
preservatives and storage buffer. A solution of sodium periodate
2.675 g) in 31.25 ml of water was added to the wet gel cake and
All experiments were carried out at 25 °C, unless otherwise stated.
Eppendorf pipettes were used to measure all volumes of substrate and
solvent. A standard quartz cuvette (1 cm in width) was used in a
Unicam 8625 UV/VIS Spectrometer to measure absorbance readings
at 420 nm. All kinetic data was analyzed by means of the Sigma Plot 9.0
Program (©2004 SYSTAT Software Inc.), a Jenway 3310 pH Meter was
used for all pH measurements and all statistical analyses were per-
formed using Minitab 14 software. Water content of each ionic liquid
was determined by weight difference of a given mass after drying for
16 h at 150 °C. HPLC analysis was performed using the Perkin Elmer
Series 200 HPLC along with the SUPELCOGEL CA 59305-U HPLC carbo-
hydrate column and SUPELGUARD CA/C611 59306-U HPLC guard column.
2-Nitrophenol (ONP), 1-methylimidazole, 1-bromobutane, sodium
tetrafluoroborate, Sepharose CL-4B (Cross-linked 4% beaded agarose),
O-nitrophenyl β-D-galactopyranoside (ONPG), β-galactosidase
(10.5 U/mg solid, E.C. 3.2.1.23 from Aspergillus oryzae), 1-butyl-3-
methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium
hexafluorophosphate, 1-butyl-3-methylimidazolium MeSO4, 1-butyl-
3-methylimidazolium bromide, silver nitrate, sodium carbonate, sodi-
um dicyanamide, and trifluoromethane sulfonic acid were obtained
from Sigma Chemical Co. (St. Louis, MO.). All other materials used were
of the analytical reagent grade and were obtained from BDH (Poole,
U.K.).
(
mixed with a paddle stirrer in a RB-flask wrapped in foil in order
to exclude light. Stirring was continued for 2 h at room temperature.
The gel was poured into 1 L of water and suction filtered. The result-
ing cake was washed with a further 1 L of water, under suction and
stored in 20% aqueous ethanol (100 ml) at 4 °C.
2
.2.1.2. Immobilization of β-galactosidase onto oxidized Sepharose CL-4B.
The periodate-activated Sepharose CL-4B (50 ml, settled gel) was
washed on a sintered glass funnel with 100 ml water followed by
1
00 ml 0.1 M sodium phosphate buffer, pH 7.0, containing 1 mM
MgCl . The gel was then suction dried to a wet cake. The resultant wet
2
cake was added to a 55 ml solution of β-D-galactosidase, (0.02 mg/ml)
in sodium phosphate buffer. To the resultant mixture sodium cyano-
borohydride (30 mg, 0.047 mmol) was added and the reaction mix-
ture gently mixed on a shaker at 22 °C for 24 h.
After this period the gel was washed successively with 50 ml phos-
phate buffer, 100 ml of 1.0 M NaCl and 100 ml water and afforded the
immobilized β-D-galactosidase. The resultant system was stored in
5
0% aqueous glycerol containing 0.02% sodium azide and 1 mM MgCl
2
at 4 °C. Preparations of immobilized β-D-galactosidase may be stored
up to 6 months without detectable decrease in activity.

N.R. Singh et al. / Journal of Molecular Liquids 152 (2010) 19–27
21
3
. Results and discussion
around the protein molecule are preserved by the “inclusion” of the
aqueous solution of the enzyme into the IL network, resulting in a
clear enhancement of the enzyme stability.
The hydrolysis reaction of β-galactosidase was monitored by mea-
suring the absorbance at 420 nm of ONP produced. Each concentra-
tion of ONPG utilized in kinetic experiments was allowed to undergo
hydrolysis using either immobilized or free β-galactosidase enzyme in
This may explain the increased rate of the catalysis in ionic liquids
compared to a buffered system. For all the IL's used in this study, the
reactions including the immobilized enzyme, demonstrated an increase
(
a fixed volume of) either phosphate buffer or in one of the five ionic
3 3
in the rate by factors of ca. 3 and 10 (excluding [bmim][CF SO ]). This
liquids utilized in this study. The change in absorbance was noted over
time, and the initial rates calculated, in order to determine a suitable
time frame within which to carry out all kinetic experiments.1
Kinetic parameters elucidated implied that the hydrolysis reaction
followed the Michaelis–Menten behavior. Good linearity was observed
for each Lineweaver–Burke plot, with regression values of 0.97 or
suggests that other factors come into play that affects the rate of these
enzyme catalyzed reactions in ionic liquids. The variation in the rates
amongst the immobilized enzyme systems in the IL's can be attributed
to (a) nucleophilicity of the counter anion comprising the IL, (b) hydro-
phobicity and polarity of the IL and (c) viscosity of the IL.
The five anions present in the five different ionic liquids are listed
−
−
−
higher. In this study, K
m
value was a measure of the dissociation of the
3 3 6 4
below in order of increasing nucleophilicity: CF SO bPF bBF b
−
−
enzyme–substrate complex. The smaller this value, the faster was the
rate of dissociation of the complex. Vmax indicated the maximum rate
of reaction, which occurred when the enzyme was completely satu-
rated with substrate.
CH
3
SO
Our findings lead us to conclude that as the nucleophilic character
of the anion increases, so does the rate (K value decreases), resulting
in the slowest rate for the IL [bmim][CF SO ] and is the fastest when IL
[bmim][N(CN) ] is employed as the solvent for the reaction. However,
[bmim][CH SO ], does not follow this trend, as a higher K value is
4 2
bN(CN) .
m
3
3
The data, (Table 4), obtained for the immobilized enzyme in the
2
ionic liquids, in general, showed that the K
m
values are lower and the
3
4
m
V
max values are higher, compared to the activity in aqueous phosphate
obtained when it is employed as the reaction medium. Consideration
of the dielectric constants of IL's [10] leads one to conclude that they
can be classified as solvents of moderate polarity, having static dielec-
tric constants ε of ca. 11.4 compared to 78.4 for water [11].
A postulated mechanism involving the anion in the hydrolysis can
occur where the intermediate oxonium ion that is formed is inter-
cepted by the anion of the ionic liquid enabling the hydrolysis to occur
at a faster rate, and with greater efficiency in the presence of the IL and
immobilized enzyme. As a consequence of this not one, but two path-
ways present themselves to the reaction, in the form of the glutamic
acid residue (proton donor) of the enzyme and the anion component
(nucleophile) of the ionic liquid.
To explain the different rates demonstrated in the IL, [bmim]
4
[MeSO ], the factors of viscosity and hydrophobicity must be con-
sidered. Chiappe et al. [12] have reported hydrolysis reactions rates
(in the case of epoxide hydrolase and trans-β-methylstyrene oxide as
substrate), that are actually lower in more hydrophilic ionic liquids. For
buffer thus demonstrating a marked increase in the stability and
efficiency of the immobilized enzyme in the ionic liquid.
m
For the free enzyme in the ionic liquids, the K values were also
generally lower compared to their values in the buffer.
From these findings we concluded that the immobilized enzyme
had higher catalytic efficiency than the free enzyme in the ionic liquids.
Normally, the activity of an enzyme decreases upon immobilization —
Tables 5 and 6 compares the free as well as the immobilized enzyme
in this study with reported kinetic parameters in the literature. The
findings in this study indicate that the observed K
m
values actually
decreased on immobilization, showing an increase in the activity. For
the hydrolysis reaction to occur a certain amount of water, regardless
of how little, is required. This may be explained by the fact that in the
free enzyme system in ionic liquids, the water content present is prac-
tically zero. In contrast, the solid support utilized, Sepharose, is a
highly hydrophilic polymer that contains approximately 10% of the
weight of the gel as water, which passes onto the enzyme micro-
environment on immobilization. This water is never completely re-
moved as its total removal (by vacuum filtration) causes deformation
of the polymer beads. β-Galactosidase catalyzes the hydrolysis of
β-1,4-glycosidic bonds using a two step, double displacement mech-
anism involving the formation and breakdown of a covalent glycosyl–
enzyme intermediate via an oxocarbenium ion-like transition states
example, [bmim][PF
lysis reaction actually proceeded faster in this IL than in [bmim][BF
6
], despite being polar, is hydrophobic, and hydro-
], the
4
more hydrophilic IL. This feature is opposite to what is observed for most
common organic solvents. Therefore, in these IL's, at constant water con-
tent, the increase in hydrophobicity may determine an increase in water
activity around the protein, favoring the enzyme action by enhancement
of the concentration of free water molecules [12]. Additionally, the in-
crease in viscosity might confer a mass-transfer limiting parameter that,
in principle, could reduce the enzymatic activity [13].
[7]. The hydrolysis of β(1–3) and β(1–4) galactosyl bonds in oligo-
saccharides takes place through general acid catalysis wherein Glu-
2
00 is the proton donor and Glu-299 the nucleophile [9].
Kragl has reported [5] a β-1,4-cleavage of lactose utilizing a
The pH did not vary drastically across the spectrum of ionic liquids
used in this study, and they actually all lie between the range of 6.6 to
7.2. The phosphate buffer used was of pH 7.3, therefore all media
employed were in the optimal pH for the functioning of the enzyme
(6.0–8.0).
β-galactosidase, isolated from Bacillus cirulans. The galactose generated
by this process was subsequently used in the synthesis of N-acetyl-
lactosamine in aqueous [mmim][MeSO ]. These authors found that the
4
reactivity in pure IL was low, but completely recovered on dilution with
water.
The ionic liquid offers an environment that increases the avail-
ability of the active site of the enzyme to the substrate — alternatively,
the turnover is greater in our system.
The introduction of other molecules within the bonding frame-
work of the ionic liquid e.g. enzymes, occurs with a disruption of the
bonding network causing inclusion-type compounds to be formed.
The presence of these bonded structures with polar and non-polar
regions may be responsible for the stabilization of enzymes supported
in ionic liquids that can maintain their functionality under very ex-
treme denaturative conditions. Thus, both the solvophobic interac-
tions essential to maintain the native structure and the water shell
Our findings lead us to conclude that [bmim][dca] is the ionic
liquid of choice to perform the hydrolysis of ONPG using immobilized
β-galactosidase. In this case the high nucleophilicity of the dicyana-
mide anion complemented the hydrophilic nature and low viscos-
ity, of the IL as well as its high ability to dissolve the substrate, [6]
is responsible for the observed outcome. All of these factors, namely
nucleophilicity, viscosity, polarity and hydrophobicity must be care-
fully balanced for a particular enzyme given the residues present in
the active site which are responsible for catalysis. For the case where
4
[bmim][MeSO ] is used as the solvent, the delicate balance of the many
solvent parameters is not achieved demonstrating a more complex
interaction between the physical properties of the ionic liquid, the
substrate and the enzyme system under investigation.
In order to gauge the stability of the immobilized enzyme used
in this study, storage stability studies were carried out independently.
1
Initial rates graphs are presented at the end of the text.

22
N.R. Singh et al. / Journal of Molecular Liquids 152 (2010) 19–27
For the immobilized β-galactosidase, there was no significant differ-
ence in the observed activity when stored in the IL 1-butyl-3-methyli-
midazolium tetrafluoroborate at 4 °C or in the IL at room temperature,
over a six month period. For both, there was a 10% decrease in the acti-
vity over this time period. However, when stored in a pH 7.0 phosphate
buffer at 4 °C, there was a marked decline in the activity after fifteen
weeks and a total loss of 40% activity after the six month period.
Stability studies of various enzymes documented in the literature
with respect to variations in the water content of the storage medium
gave similar results. In a study by Yoshioka et al. [29], it was suggested
that as the water content increases, the mobility of water around the
enzyme (β-galactosidase) increases, resulting in enhanced enzyme
inactivation. In another report by Yoshioka et al. [30], the effects of
excipients on the protein stability during lyophilization as well as the
storage stability of lyophilized bilirubin oxidase and β-galactosidase
formulations were investigated. Storage stability was improved by
absorption of a small amount of water for all the formulations studied.
Absorption of a larger amount of water, however, decreased the stor-
age stability of the formulations. However, the storage stability of for-
mulations containing dextran did not decrease noticeably with
increasing water. This was so, since formulations containing dextran
had a higher glass transition temperature than formulations containing
PVA, PAA or PHEA when a large amount of water is absorbed.
The rational use of ionic liquids in chemical and engineering
processes will involve a comprehensive appreciation of what makes
these liquids superior to commonly used organic and aqueous sol-
vents. Studies into physiochemical behavior are in the embryonic
stages, and hence the researcher, before delving into applications,
should look toward building a data-base of knowledge on the local
steric interactions amongst ions which govern a given ionic liquid,
which is suited for a particular application. Industrially, applications
can include enzymatic synthesis of methanol, conversion of natural gas
to single cell protein for animal feed and convergent syntheses of
pharmaceuticals.
4. Conclusion
The anion of the ionic liquid, [bmim][dca] is able to act as a
nucleophilic catalyst for the hydrolysis of ONPG. The IL also provides
a solvent system which is able to favorably affect the functioning of
the β-galactosidase enzyme, by virtue of its superior physiochemical
properties.
Acknowledgment
We thank the UWI for a postgraduate research scholarship (NRS).
Appendix A
m
A.1. K and Vmax studies of immobilized β-galactosidase in aqueous medium and in five ionic liquids
Table 1
Contents of all tubes used in the kinetic runs in terms of substrate and derivation of the 1/[S] terms.
Tube #
Vol ONPG (1.6 mM)/mL
Vol working buffer or IL/mL
mM substrate (ONPG)
1/[S]
1
2
3
4
5
6
7
8
9
0.1
0.2
0.3
0.5
0.7
0.8
1.0
1.5
2.0
2.2
2.1
2.0
1.8
1.6
1.5
1.3
0.8
0.3
0.064
0.128
0.192
0.32
0.448
0.512
0.64
15.63
7.81
5.21
3.13
2.23
1.95
1.56
1.04
0.78
0.96
1.28
N.B.— Fixed amount of gel used in each tube=0.2 mL, which contains 0.004 mg enzyme.
Table 2
Results of analysis in phosphate buffer and in the five ionic liquids for product formation over time using immobilized enzyme.
TUBE #
1
2
3
4
5
6
7
8
9
Immob. enzyme in buffer
Abs @420 nm
0.045
1.8411
1.5343
0.091
3.7096
3.0914
0.117
4.8051
4.0043
0.186
7.605
6.3375
0.292
11.96
9.9671
1003.3
0.031
0.334
13.68
11.404
876.9
0.039
0.388
15.88
13.24
755.2
0.045
0.483
19.766
16.427
607.1
0.058
0.491
20.081
16.734
597.6
0.072
v
V)×10−
/[V]
4
(
1
6517.5
0.007
3234.8
0.011
2497.3
0.017
1577.9
0.025
Immob. enzyme in [bmim] [PF
6
]
Abs @420 nm
v
V)×10−
/[V]
5.331
4.4426
2250.943
0.007
4.9244
4.103
2436.874
0.008
8.8834
7.4029
1350.873
0.011
7.818
6.515
1534.871
0.013
13.294
11.079
902.64
0.017
12.04
10.03
20.026
16.689
599.215
0.028
20.004
16.67
25.037
20.864
479.291
0.037
26.16
21.8
30.796
25.664
389.657
0.0423
29.95
24.96
400.564
0.029
35.715
29.763
335.992
0.050
35.1
29.25
45.678
38.065
262.71
0.063
44.3
36.94
57.115
47.495
210.104
0.085
60.48
50.4
4
(
1
Immob. enzyme in [bmim] [BF
4
]
Abs.@420 nm
v
V)×10−
/[V]
4
(
1
996.712
0.017
599.940
0.021
458.76
0.025
341.834
0.034
270.745
0.045
198.451
0.066
Immob. enzyme in [bmim] [dca]
Abs.@420 nm
v
V)×10−
/[V]
7.8346
6.5289
1531.65
0.002
12.731
10.609
942.553
0.005
16.648
13.873
720.776
0.007
20.566
17.138
583.486
0.012
24.483
20.402
490.128
0.014
28.401
23.667
422.524
0.015
33.297
27.748
360.388
0.019
44.07
64.636
53.863
185.655
0.026
4
(
1
36.725
272.293
0.022
Immob. enzyme in [bmim] [CF
3
SO
3
]
Abs.@420 nm
v
V)×10−
/[V]
1.8196
1.5163
6594.8
0.006
3.95
5.245
9.4801
7.9001
1265.8
0.025
10.785
8.9876
1112.64
0.029
12.007
10.005
999.452
0.037
15.181
12.651
790.436
0.045
17.538
14.615
684.215
0.051
20.339
16.949
589.987
0.064
4
(
1
3.2917
3037.92
0.011
4.3711
2287.73
0.017
Immob. enzyme in [bmim] [MeSO
4
]
Abs.@420 nm
v
V)×10−
/[V]
4.7816
3.985
2509.41
8.7663
7.305
1368.93
13.548
11.289
885.818
19.923
16.603
602.301
23.111
19.259
519.238
29.486
24.572
406.967
35.862
29.885
334.616
40.643
33.869
295.255
51.004
42.503
235.278
4
(
1

N.R. Singh et al. / Journal of Molecular Liquids 152 (2010) 19–27
23
Table 3
Results of analysis in phosphate buffer and in five ionic liquids for product formation over time with free enzyme.
TUBE #
1
2
3
4
5
6
7
8
9
Free enzyme in buffer
Abs @420 nm
0.017
0.707
0.589
0.032
1.315
1.096
0.046
1.865
1.55
0.079
3.222
2.685
0.112
4.599
3.83
0.125
5.116
4.263
0.157
6.44
5.368
0.213
8.702
7.252
0.273
11.18
9.31
−
7
v (×10
)
−
4
(V)×10
1
/[V]
16978.92
0.006
9125.77
0.012
6432.9
0.015
3723.8
0.024
2609.3
0.034
2345.7
0.039
1862.8
0.048
1378.9
0.057
1073.6
0.083
Free enzyme in [bmim] [PF
6
]
Abs @420 nm
v
(
1
4.585
9.1701
7.6418
1308.59
0.006
4.061
3.384
2954.994
0.008
11.4626
9.5522
1046.88
18.3402
15.284
654.27
0.011
7.78
6.482
25.9819
21.6516
461.859
0.017
12.06
10.05
29.8028
24.8357
402.646
0.021
14.62
12.2
36.6804
30.567
327.1501
0.024
16.96
14.13
43.558
36.2983
275.495
0.032
22.4
18.7
63.426
52.855
189.19
0.038
27.0
22.5
V)×10−
/[V]
4
3.8208
2617.25
0.003
1.897
1.581
6324.673
0.005
Free enzyme in [bmim] [BF
4
]
Abs.@420 nm
0.008
5.94
4.95
v
(
1
V)×10−
/[V]
4
2019.534
0.011
1542.811
0.014
995.136
0.017
820.633
0.021
707.453
0.024
534.967
0.029
443.98
0.034
Free enzyme in [bmim] [dca]
Abs.@420 nm
v
(
1
4.7203
3.9336
2542.19
0.003
7.8346
6.5289
1531.65
0.006
10.772
8.97724
1113.92
0.009
13.710
11.4255
875.228
0.014
16.64
20.566
17.1384
583.485
0.021
23.504
19.5867
510.55
0.025
28.400
23.6673
422.524
0.029
33.297
27.747
360.38
0.034
V)×10−
/[V]
4
13.8739
720.776
0.017
Free enzyme in [bmim] [CF
3
SO
3
]
Abs.@420 nm
v
(
1
2.2925
1.91041
5234.47
0.005
4.585
6.8775
5.7313
1744.8
0.014
10.6984
8.9153
1121.67
0.023
12.9909
10.8258
923.719
0.035
16.0476
13.373
747.775
0.040
19.1043
15.9203
628.129
0.047
22.161
18.4675
541.492
0.059
25.981
21.651
461.85
0.082
V)×10−
/[V]
4
3.8208
2617.25
0.009
Free enzyme in [bmim] [MeSO
4
]
Abs.@420 nm
v
(
1
3.9846
3.321
3011.14
7.1724
5.977
1673.08
11.157
9.298
1075.5
18.329
15.274
654.7
27.893
23.244
430.219
31.878
26.565
376.435
37.456
31.213
320.379
47.019
39.183
255.213
65.349
54.458
183.27
V)×10−
/[V]
4
Fig. 1. Graphs of 1/[V] vs. 1/[S] for immobilized enzyme in phosphate buffer and in five different ionic liquids.
Fig. 2. Graphs of 1/[V] vs. 1/[S] for free enzyme in phosphate buffer and in five different ionic liquids.

24
N.R. Singh et al. / Journal of Molecular Liquids 152 (2010) 19–27
A.2. Initial rates graphs for ONPG hydrolysis using free or immobilized enzyme in ionic liquids and in phosphate buffer
Fig. 3. Initial rates graphs for ONPG hydrolysis using immobilized enzyme in ionic liquids and in phosphate buffer.

N.R. Singh et al. / Journal of Molecular Liquids 152 (2010) 19–27
25
Fig. 4. Initial rates graphs for ONPG hydrolysis using free enzyme in ionic liquids and in phosphate buffer.

26
N.R. Singh et al. / Journal of Molecular Liquids 152 (2010) 19–27
A.3. Summary
Table 4
The Michaelis–Menten kinetic parameters derived from Lineweaver–Burke plots for free and immobilized β-galactosidase in the five IL's and in phosphate buffer.
Free enzyme
Solvent
Immobilized enzyme
Solvent
M–M Equation
K
m
(± 0.07)
V
max (± 0.001)
M–M Equation
K
m
(± 0.07)
Vmax (± 0.001)
Phosphate buffer
y=50.358x+6.6328
R =0.9867
7.592
1.959
1.415
0.786
0.615
0.384
0.151
0.004
0.007
0.005
0.004
0.003
Phosphate buffer
y=30.079x+9.1553
R =0.9983
3.285
0.664
0.559
0.365
0.347
0.239
0.109
0.004
0.005
0.002
0.002
0.002
2
2
[
[
[
[
[
bmim][CHF
3
SO
3
]
y=527.16x+268.97
[bmim][MeSO
4
]
y=157.62x+237.51
2
2
R =0.9887
R =0.989
bmim][MeSO
4
]
y=201.51x+142.44
[bmim][PF
6
]
y=117.83x+210.86
2
2
R =0.9975
R =0.9986
bmim][BF
bmim][PF
4
]
y=156.39x+198.95
[bmim][BF
4
]
y=199.33x+546.2
2
2
R =0.9873
R =0.9983
6
]
y=152.54x+247.95
[bmim][CHF
3
SO
3
]
y=193.87x+559.32
2
2
R =0.9886
R =0.9951
bmim][dca]
y=138.74x+361.71
[bmim][dca]
y=138.04x+576.39
2
2
R =0.9837
R =0.9973
N.B — Unit of K
Unit of Vmax =min
m
=millimoles ONPG.
−
1
.
Temperature of reaction systems=27 °C.
Table 5
m
Comparison of K values of immobilized β-galactosidase on various supports.
Immobilization matrix
K
m
/mM ONPG
Reference
Sepharose® CL-4B using [bmim][dca] as reaction medium
Cross-linked onto graphite using gluteraldehyde
Polyacrylamide beads
0.239
8.7
This study
[17]
[18]
4.2, 5.4 and 30 (enzyme isolated from L. bulgaris, E. coli and K. lactis respectively)
Diethyl aminoethylcellulose
Cross-linked poly(vinyl alcohol)
Chitosan
0.047
7.1
5.2
[19]]
[20]
[20]
Table 6
Comparison of the K
m
values of free β-galactosidase in the literature.
Source of enzyme (purified from)
m
K /mM ONPG
Reference
Aspergillus niger
Lactobacillus bulgaricus
E. coli
D201N-β-gal from E. coli
Streptococcus cremoris
Streptococcus thermophilus
Einset seedless grapes
Thermophilic Rhizomucor sp
Bacteroides polypragmatus
0.384
0.365
2.42
0.36
0.384
0.25
0.5
This study
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
0.785
0.43
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