1
24
Y. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 123–127
and the effect of the reaction medium on the transition state of the
reaction. But now, using Fourier transform-infrared (FT-IR) spec-
troscopy, it is very easy to know the changes of the non-covalent
forces (hydrogen bonding, ionic, hydrophobic and van der Waals
interactions) that maintain the native secondary and tertiary struc-
tures of lipases [15,16]. In our previous works, we reported the
effect of conventional organic solvents and ILs on Burkholderia cepa-
cia (BCL) lipase activity and its conformation structure using FT-IR
and circular dichroism (CD) [14,17]. To our best knowledge, the
effect of ILs pretreatment with different cation and anion structures
on CRL activity and its conformation has rarely been well studied.
And the understanding of the effects of sub- and super-critical CO2
pretreatment on CRL catalytic activity and conformation has still
few reports so far.
Therefore, the objectives of this work are: (1) to investigate the
esterification activity of CRL pretreated with 5 conventional organic
solvents with different log P from 0.8 to 4.5; (2) to evaluate the effect
of 17 different ILs, and under sub-/super-critical CO2 pretreatment
on the CRL esterification activity; (3) to elucidate the secondary
structure variance of CRL by FT-IR in the cases of above mentioned
pretreatments.
Each treatment was immersed and stirred in a rotary shaker with
the rate of 200 rpm at 37 C for 10 min. Then, the ILs was removed off
with water washing and the treated CRL was used for esterification
activity and its conformation assays. CRL without ILs pretreatment
was used as the control.
◦
2.3. CRL pretreatment with organic solvents
In order to evaluate the effects of log P of organic solvents on
the CRL activity and its conformation, five conventional organic sol-
vents with different log P (from 0.8 to 4.7) were tested, including
tert-butanol (log P = 0.8), tert-amyl alcohol (log P = 1.3), petroleum
ether (log P = 2.0), n-hexane (log P = 3.5) and isooctane (log P = 4.7).
The procedure of CRL treatment with organic solvents was modi-
fied according to that in our previous work [17]. Generally, 40 mg
CRL was placed in the different test tubes and then 0.5 mL of various
organic solvents were added to the tubes to swell the enzyme pow-
der. Each treatment was immersed and stirred in a rotary shaker
with the rate of 200 rpm at 37 ◦C for 10 min. Then, the organic
solvent was removed off and the treated CRL was used for esteri-
fication activity and its conformation assays. CRL without organic
solvents pretreatment was used as the control.
2
. Materials and methods
2.4. CRL pretreatment with sub- and super-critical CO2
2.1. Materials
The effect of SC-CO2 on the CRL activity and its conformation
was also investigated in this work. The SC-CO equipment consisted
C. rugosa lipase (CRL) (lyophilized powder) with spe-
2
mainly of a liquid CO2 reservoir, thermostatic bath with the preci-
cific lipase activity ≥ 700 U/mg-protein was bought from
◦
sion of ± 1 C, stainless steel vessel (autoclave) with the internal
Sigma Co. (St, Louis, MO, USA). Seventeen ILs of 1-ethyl-3-
volume of 1 L, high pressure liquid pump, and pressure gauge with
the precision of ± 0.1 MPa. The equipment scheme is shown in Fig. 1.
The procedure of CRL treatment with SC-CO2 was described as
follows: First of all, 200 mg CRL was placed in the catalyst bas-
ket and put into the autoclave, then CO2 was supplied by pump
into the autoclave, and then the whole system was heated until
methylimidazolium tetrafluoroborate ([Emim][BF ]), 1-butyl-3-
4
methylimidazolium tetrafluoroborate ([Bmim][BF ]), 1-butyl-
4
3
1
[
1
-methylimidazolium
hexafluorophosphate
([Bmim][PF ]),
6
-butyl-3-methylimidazolium
trifluoromethanesulfonate
Bmim][TfO], N-octyl-3-pyridine tetrafluoroborate ([OmPy][BF ]),
4
-hexyl-3-methylimidazolium methanesulfonate ([Hmim][CH3
◦
the desired pressure (6 MPa and 15 MPa) and temperature (40 C)
SO ]), 1-ethyl-3-methylimidazolium trifluoromethanesulfonate
3
◦
were achieved. Each treatment was set at 40 C for 20 min. After
(
[Emim][TfO]),
1-octyl-3-
methylimidazolium
tetrafluo-
depressurizing, the sub- and supercritical treated CRL was used
for esterification activity and its conformation assays. CRL without
pretreatment with SC-CO2 was used as the control.
roborate ([Omim][BF ]),
1-hexyl-3-methylimidazolium
4
hexafluorophosphate
methylimidazolium
([Hmim][PF ]),
hexafluorophosphate
1-ethyl-3-
6
([Emim][PF ]),
6
1
-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide
(
[Bmim][Tf N]),
1-butyl-3-methylimidazolium
methanesul-
2.5. Assay of CRL esterification activity
2
fonate ([Bmim][CH SO ]), 1-butyl-3-methylimidazolium chloride
3
3
(
1
[Bmim][Cl]), 1-ethyl-3-methylimidazolium chloride ([Emim][Cl]),
-butyl-3-methylimidazolium hydroxide ([Bmim][OH]), 1-octyl-3-
The determination procedure of CRL esterification activity of
lauric acid and dodecanol was modified according to the method
in our previous work [17]. Specifically, 40 mg CRL were added into
a screw capped vial, which contained 0.40 g lauric acid (0.2 M) and
0.37 g dodecanol (0.2 M) in a mixture solution of 10 mL isooctane
methylimidazolium chloride ([Omim][Cl]), and 1-octyl-3-
methylimidazolium hexafluorophosphate ([Omim][PF ]) with
6
purifity ≥ 99.9% were purchased from Shanghai Cheng Jie Chemical
Co. Ltd. (Shanghai, China). All ILs were dried to remove the water
content before experiments. Organic solvents of tert-amyl alcohol,
tert-butanol, petroleum ether, n-hexane and isooctane were of
analytical grade and purchased from Sinopharm Chemical Reagent
Co. Ltd. (Shanghai, China). Liquid CO with the purification of 99.9%
2
was purchased from Shanxi Kewei chemical company (Shanxi,
China).
2.2. CRL pretreatment with ILs
To elucidate the effect of cation and anion structures of ILs on
the CRL esterification activity and its conformation, seventeen ILs
with five cations and seven anions structures were employed in
this work. The procedure of CRL treatment with ILs was modified
according to the method by Pan et al. [14]. Generally, 40 mg CRL
was placed in the seventeen different test tubes and then 0.5 mL of
various ILs were added to the tubes to swell the enzyme powder.
Fig. 1. The scheme of supercritical CO2 equipment (1: CO2 reservior; 2: thermostatic
bath; 3: high pressure liquid pump; 4: reactant loop; 5: stainless vessel (autoclave);
6: high pressure gauge; 7: catalyst basket; 8: 6-way sample valve; 9: sample vessel).