Solubilisation of α-chymotrypsin by hydrophobic ion pairing in fluorous systems and supercritical carbon dioxide and demonstration of efficient enzyme recycling

Article abstract of DOI:10.1039/b904761a

Hydrophobic ion-pairing (HIP) with the fluorinated surfactant KDP 4606 (KDP) was used to extract the protein α-chymotrypsin (CMT) into perfluoromethylcyclohexane (PFMC). The diameter of the solubilised CMT-KDP complexes formed in PFMC was determined by dynamic light scattering (DLS) to be 25 nm which suggested the formation of a protein aggregate containing ～100 protein molecules surrounded by KDP 4606 surfactant molecules per particle. The catalytic activity of the protease CMT either solubilised by HIP or as the suspended native enzyme has been investigated in both a fluorous biphasic system (FBS) and a supercritical carbon dioxide (scCO₂) batch reactor. Transesterification of N-acetyl-l-phenylalanine ethyl ester (APEE) with n-butanol or rac-2-butanol was catalysed by the protease in the FBS hexane-PFMC or scCO₂ at 40 °C. Under comparable conditions, the amount of transesterification of the solubilised protease-surfactant (CMT-KDP) complex in PFMC (6-10%) was shown to be significantly higher than that of the suspended protease (1-3%) in either hexane-PFMC or scCO₂. This suggested the formation of a catalytically active CMT-KDP aggregate in PFMC. The CMT-KDP complex which is retained in the fluorous phase on cooling the solution was successfully reused over four cycles with no loss of activity.

Full text of DOI:10.1039/b904761a

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PAPER
www.rsc.org/greenchem | Green Chemistry
Solubilisation of a-chymotrypsin by hydrophobic ion pairing in fluorous
systems and supercritical carbon dioxide and demonstration of efficient
enzyme recycling
Karima Benaissi,a,b Martyn Poliakoff*a and Neil R. Thomas*b
Received 10th March 2009, Accepted 21st August 2009
First published as an Advance Article on the web 29th September 2009
DOI: 10.1039/b904761a
Hydrophobic ion-pairing (HIP) with the fluorinated surfactant KDP 4606 (KDP) was used to
extract the protein a-chymotrypsin (CMT) into perfluoromethylcyclohexane (PFMC). The
diameter of the solubilised CMT-KDP complexes formed in PFMC was determined by dynamic
light scattering (DLS) to be 25 nm which suggested the formation of a protein aggregate
containing ~100 protein molecules surrounded by KDP 4606 surfactant molecules per particle.
The catalytic activity of the protease CMT either solubilised by HIP or as the suspended native
enzyme has been investigated in both a fluorous biphasic system (FBS) and a supercritical carbon
dioxide (scCO2) batch reactor. Transesterification of N-acetyl-L-phenylalanine ethyl ester (APEE)
with n-butanol or rac-2-butanol was catalysed by the protease in the FBS hexane-PFMC or scCO2
at 40 ◦C. Under comparable conditions, the amount of transesterification of the solubilised
protease–surfactant (CMT-KDP) complex in PFMC (6–10%) was shown to be significantly
higher than that of the suspended protease (1–3%) in either hexane–PFMC or scCO2. This
suggested the formation of a catalytically active CMT-KDP aggregate in PFMC. The CMT-KDP
complex which is retained in the fluorous phase on cooling the solution was successfully reused
over four cycles with no loss of activity.
is unpredictable and modifications can result in a considerable
Introduction
loss of enzymatic activity.3,4,6–11
The lipids, esters, acids and alcohols that are widely used as
The technique of hydrophobic ion-pairing (HIP) has pre-
starting materials for the commercial production of important
viously been reported to solubilise proteins in hydrocarbon
pharmaceuticals, cosmetics and food products, are often poorly
solvents by forming ion pairs with anionic organic surfactants
soluble in water. Therefore, biocatalysis in non-aqueous media
such as Aerosol-OT (AOT) (see Fig. 1 for structure).12 In some
to produce these materials has attracted considerable attention
in recent decades.1 Given the move to replace hydrocarbon
cases, including with protease a-chymotrypsin (CMT),1,13–16 an
increase in catalytic activity was observed with these anionic
solvents with more environmentally benign alternatives, one
surfactant–enzyme complexes in the organic solvent.1,13–19 The
area of significant research activity is the identification of new
use of HIP to solubilise enzymes using anionic fluorinated
solvents and reaction systems that are compatible with the use
surfactants such as KDP 4606 (see Fig. 1 for structure) and
of enzymes.1
Krytox 157 FSL developed from these studies.10,13,19 In the case
Different strategies, such as the suspension of the enzyme in
of CMT, the enzyme–CO2-philic surfactant complex formed has
an organic solvent,2 lipid-coated enzymes,3 microencapsulation
been shown to be active and dispersible in CO2.10,13
of enzymes in reverse-micelles,4 immobilisation of enzymes on
a microgel-matrices or within membranes,5 covalent attachment
of polymers (pegylation) to the enzyme,6 stabilisation of enzymes
R
forming cross-linked enzyme crystals (CLECsꢀ)7 or cross-linked
R
enzyme aggregates (CLEAsꢀ),7,8 aimed at increasing the stability
and active site accessibility of enzymes have been employed
to enhance enzymatic activity in organic media. However, the
effect on the catalytic activity of these modified enzyme forms
Fig. 1 Structures of the anionic surfactants sodium bis(2-ethylhexyl)
aClean Technology Group, School of Chemistry, University of
Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom.
E-mail: martyn.poliakoff@nottingham.ac.uk
sulfosuccinate (Aerosol-OT, AOT) and KDP 4606 (DuPontꢀR ).
In this paper we describe the use of both a fluorous biphasic
system (FBS) and supercritical carbon dioxide (scCO2) as
solvents for a transesterification catalysed by CMT.
bCentre for Biomolecular Sciences, School of Chemistry, University of
Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom.
E-mail: neil.thomas@nottingham.ac.uk
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Only a few examples of biocatalysis under FBS conditions
have been reported.9,10,13 Candida rugosa lipase (CRL) sus-
pended in hexane was used to catalyse the separation of rac-
2-methylpentanoic acid via fluorinated tagging of one of the two
enantiomers using a highly fluorinated alcohol.9 This promising
approach was shown to allow easy separation and recovery of
both product and catalyst. This approach was recently developed
further through the use of enzymes solubilised in fluorous
solvents by HIP to allow a homogeneous reaction to be achieved
in a fluorous biphasic system,10 the enzyme was retained in the
fluorous phase allowing easy recycling.
5 mM to 70 mM in PFMC (1 mL) were stirred with 1 ml of
sodium phosphate buffer (10 mM, pH 6.9) or sodium acetate
buffer (10 mM, pH 3.8) at 500 rpm for 10 min and centrifuged
at 8000 rpm (4600g) for two min to allow phase separation
to occur. The lower fluorous phase was then collected and
the concentration of surfactant analyzed by measuring the
absorbance at 240 nm (e240 = 59.4 M-1 cm),10 as described above.
This measurement was repeated in triplicate.
Dynamic light scattering (DLS)
This paper reports new data on the solubilisation of CMT
using HIP and describes efficient recycling of the HIP solubilised
protease for transesterification reactions catalysed in both a FBS
and scCO2.
DLS measurements were carried out at 25 ◦C on a Zetasizer
Nano S at Malvern Instruments. The particle size was taken as a
mean value of three measurements. CMT dissolved in aqueous
buffer; AOT and CMT-AOT dissolved in hexane; and CMT-
KDP and KDP dissolved in PFMC were measured at 0.02 mM,
2.3 mM, 0.02 mM, 0.7 mM and 0.02 mM, respectively. The data
was analyzed with the following fixed parameters:23 refractive
index 1.333, 1.372, 1.277, 1.39, 1.4 and 1.45 (water, hexane,
PFMC, AOT, KDP and protein respectively) and viscosity (cP)
1.0019, 0.294 and 1.561 (water, hexane and PFMC respectively).
Materials and methods
a-Chymotrypsin (CMT, type II from bovine pancreas,
51 U mg-1) was obtained from Aldrich. Chemicals: N-acetyl-L-
phenyl alanine ethyl ester (APEE), n-butanol and rac-2-butanol
were purchased from Aldrich. The organic surfactant sodium
bis(2-ethyl-2-hexyl)succinate (AOT) was purchased from Fluka
and the fluorinated surfactant KDP 4606 was kindly donated by
Estimation of water content using coulometric Karl Fisher
titration
R
DuPontꢀ. Perfluoromethylcyclohexane (PFMC) was purchased
from Fluorochem. All organic solvents were purchased at the
highest purity available from Aldrich. All commercially available
reagents and solvents were used without further purification.
High purity CO2 was purchased from CryoService. Gases
hydrogen, helium and air were purchased from BOC Gases.
The water content of hexane, PFMC, CMT-AOT in hexane,
and CMT-KDP in PFMC based on coulometric Karl Fisher
titration was estimated using a Mitsubishi moisture meter CA-
100 following the manufacture’s method. Samples (2 to 10 mg)
were added into the titration solution using a syringe and the
mass of sample introduced was indicated to the titrator. Titration
with iodine was performed under stirring at 700 rpm. The
percentage of water in the samples was measured in triplicate.
The sensitivity of the Coulometric Karl Fisher titrator is 1 ppm
water content.24
Safety note
Experiments with scCO2 involve high pressures and should only
be carried out in equipment with the appropriate pressure rating
and safety operating procedures.
Transesterification of N-acetyl-L-phenylalanine ethyl ester
Solubilisation of CMT-KDP in PFMC
Transesterification of N-acetyl-L-phenylalanine ethyl ester
(APEE) with n-butanol or rac-2-butanol was catalysed by the
protease a-chymotrypsin (CMT, MW = 51 000). The protein
was used either in solubilised form as its HIP complex (CMT-
AOT in hexane or CMT-KDP in PFMC) or as the suspended na-
tive form in hexane, hexane-PFMC (v/v 1 : 1) or scCO2 (100 bar)
at 40 ◦C for two hours. The reaction studied is shown in
Scheme 1.
The preparation of protein–surfactant complexes has been
described previously.13 CMT (51 U, 0.02 mM) dissolved in
sodium phosphate buffer (1 mL, 10 mM, pH 6.9), 5 mM CaCl2
(1 mL) was stirred with AOT (2.3 mM) in hexane (1 mL) or KDP
(0.7 mM) in PFMC (1 mL) at room temperature at 500 rpm
for 10 min and centrifuged at 8000 rpm (4600g) for 2 min
to allow phase separation to occur. The lower fluorous phase
was then collected and diluted ten-fold in PFMC (1 mL) and
the UV absorbance of the protein was measured at 280 nm
(value20,21 of e240 = 50 585 M-1 cm-1) using an Agilent 8453 UV
spectrophotometer. Comparable experiments were set up using
AOT (1 mg mL-1, 2.3 mM) in hexane (1 mL) and the amount of
CMT-AOT in hexane was determined by UV spectroscopy. All
experiments were performed in triplicate.
Determination of the critical micellar concentration (CMC) of
KDP 4606 in PFMC
Scheme 1 Transesterification of N-acetyl-L-phenylalanine ethyl ester
(APEE) (4.2 mM) with n-butanol or rac-2-butanol (0.45 M) was
catalysed by a-chymotrypsin (CMT, 102 U, 0.04 mM) in hexane or
hexane–PFMC v/v 1 : 1 (2 mL) or scCO2 (100 bar, 8.5 mL) at 40 ◦C for
two hours.
The method used to determine the critical micellar concentration
(CMC) of the fluorinated surfactant by UV-visible spectroscopy
has been described previously.22 Solutions of KDP in the range
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Table 1 Solubilisation of CMT-AOT in hexane and CMT-KDP in
CMT (51 U, 1 mg) solubilised in PFMC (1 mL) was added
to the substrates N-acetyl-L-phenylalanine ethyl ester (APEE)
(8.5 mmol, 2 mg) and n-butanol (0.9 mmol, 80 mL) or rac-2-
butanol (0.9 mmol, 80 mL) in hexane or hexane/PFMC v/v
1 : 1 (2 mL). A comparable reaction was set up in scCO2
(100 bar, 8.5 mL) and the system was left stirring at 40 ◦C
for two hours. When the reaction was finished, the system was
cooled down to 0 ◦C in an ice bath to allow phase separation to
occur. Aliquots of the upper organic phase were collected and
analyzed as described above. A fresh batch of substrates was then
added to the lower fluorous phase and the reaction was repeated
as described above. The yields of products were determined
using a Shimadzu GLC-2010 GLC equipped with an AOC
20i autosampler and fitted with a RTX5-FAST column (fused
silica, crossbond 5% diphenyl–95% dimethyl polysiloxane, 10
m ¥ 0.1 mm ¥ 0.1 mm) from Restek. A temperature programme
was used for each run; the oven temperature was increased from
100 ◦C to 250 ◦C at 25 ◦C per minute and held at 250 ◦C for two
minutes. Samples of 1.0 mL were injected onto the column with
a split ratio of 75 : 1. The injector temperature was 250 ◦C and
the FID temperature was 350 ◦C.
PFMC
Complexation efficiencya (%)
Concentration of CaCl2 in
buffer (mM)
CMT-AOT in
hexane
CMT-KDP in
PFMC
0
5
68
96
3
3
29
51
4
4
a Complexation efficiency (%) = [mass of protein solubilised in organic
(or fluorous) phase]/[initial mass of protein] ¥ 100
and sample ionisation was carried out using electron impact (EI)
at 70 eV.
Results and discussion
Solubilisation of CMT-KDP in PFMC
There are suggestions in the literature that the presence of a salt,
such as CaCl2 in the aqueous buffer from which an enzyme is
extracted strongly influences both the solubility and activity of
the enzyme in the receiving organic solvent.12,16,26 It has been
postulated that at high salt concentrations, that the salt matrix
may act as a rigid structure that would protect the enzyme from
the organic solvent.26 Also, the salt matrix, being highly polar,
might help to maintain the native structure of the enzyme in
organic media.26
CMT was therefore dissolved in a sodium phosphate buffer
(10 mM, pH 6.9) with or without addition of 5 mM CaCl2.
The protein was then extracted into hexane using AOT or into
PFMC using KDP and the complexation efficiency of CMT-
AOT in hexane and CMT-KDP in PFMC were estimated by
measuring the UV absorbance of the protein at 280 nm. The
results obtained are summarised in Table 1.
Adding CaCl2 to the aqueous buffer was observed to facilitate
the extraction of the protein into the organic (or fluorous) phase
since the complexation efficiency was observed to be increased
by about 40–50% in both cases. Under comparable conditions,
the amount of enzyme solubilised in hexane using AOT (96% of
the starting material) was higher than that obtained using KDP
in PFMC (51%).
The differences in complexation efficiencies observed between
the two surfactants tested may be due to the structure of the
surfactants (see Fig. 1). Parameters such as the size, polarity
and hydrophobicity12 of the fluorinated tail on the surfactant are
likely to influence the contact between surfactant and protein.
Batch reactions
Enzymatic reactions in organic solvents and PFMC were carried
out in 2 mL septum-sealed Supelco glass micro reaction vessels
equipped with magnetic stirrer bars and stirred at 500 rpm in a
silicon oil bath thermostated to the indicated temperature using
a Yellowline MST basic hot plate equipped with a Yellowline
TC1 thermostat.
Reactions in scCO2 were performed in a stainless-steel high
pressure batch reactor specially designed at the University of
Nottingham.25 The batch reactor (autoclave) equipped with a
magnetic stirrer bar has an internal volume of 8.5 mL and was
placed on an IKA Labotecknick RCT basic stirring plate. Solid
substrates and reactants and stirrer bar were added into the
reactor prior to sealing. Liquid substrates and reactants were
added using a Gilson Pipetman micropipette into the autoclave
then sealed using a safety valve. A band heater, thermocouple,
input and output pressure pipes were then connected to the
reactor to enable temperature and pressure control of the system.
The system was then heated to the desired temperature and
liquid CO2 was pumped into the reactor using a high pressure
NWA PM-101 Pickel pump until the desired pressure was
achieved. The reaction mixture was stirred for the period of
time specified and the system was then depressurised by placing
the autoclave in a dry ice–acetone bath. The residual mixture in
the autoclave was dissolved in acetone (3 mL) and centrifuged
for 2 min. at 8000 rpm (4600g).
Aliquots (50 mL) were diluted in acetone (1 mL) and substrates
and products were analyzed by gas-liquid chromatography
(GLC) using a Shimadzu GC 2010 chromatograph equipped
with a Shimadzu AOC-20Si autosampler using helium as the
carrier gas. The structure of products was further identified
using GLC and GLC-MS by comparing the retention times
and fragmentation patterns with those of authentic samples.
GLC-MS was performed using a ThermoFinnegan Polaris-Q
trap GC-MS equipped with a DB-5 (30 ¥ 0.25 ¥ 0.25 mm film
thickness) fused silica column. Helium was used as a carrier gas
Determination of the critical micelle concentration (CMC) of
KDP 4606 in PFMC
By plotting absorbance against concentration of KDP10 it was
possible to determine the CMC of KDP in PFMC as 48 mM at
room temperature (maximum on the graph shown in Fig. 2).
The concentration of KDP (0.7 mM) used to solubilise CMT
in PFMC in our experiments is far below the measured CMC
(48 mM) which suggests that the solubilisation process used does
not lead to the formation of micelles.
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Table 2 Water content of hexane, AOT and CMT-AOT in hexane,
PFMC, KDP and CMT-KDP in PFMC estimated using coulometric
Karl Fisher titration
Sample
Water content (ppm)
Hexane
34
45
4
5
8
AOT in hexane
CMT-AOT in hexane
PFMC
KDP in PFMC
CMT-KDP in PFMC
41
<1a
<1a
<1a
a Sensitivity of the coulometric Karl Fisher Titrator 1 ppm.24
The experiments were performed in triplicate and the results
obtained were reproducible as shown in Table 2.
Fig. 2 UV absorbance versus concentration for KDP 4606 at 240 nm for
the determination of the critical micelle concentration (CMC), indicated
by dashed line.
Less than 50 ppm water was detected in a sample of CMT-
AOT in hexane which is mainly due to the water content of
hexane. The amount of water in the PFMC samples was below
the detection limit of the instrument (1 ppm).24 To ensure that
the fluorinated solvent PFMC was not interfering with the
detection of Karl Fisher titration, known quantities of water
were purposely added to PFMC and the water content was
measured using Karl Fisher titration. In these cases, the amount
of water added was accurately detected by the machine.
The very low amount of water detected for CMT-KDP
in PFMC may explain the formation of protein aggregates
observed by DLS (Fig. 3). In a recent paper, Akbar et al.
describe the solubilisation of subtilsin Carlsberg and CALB in
isooctane with or without extraction from an aqueous buffer
solution.27 They observed that the proteins directly solubilised in
a solution of AOT in isooctane containing 0.2% of water would
form clusters/aggregates in the organic solvent. However, the
enzymes dissolved by extraction from an aqueous buffer solution
would appear as single proteins surrounded by the surfactant in
isooctane.
Dynamic light scattering (DLS)
The particle size of CMT solubilised in phosphate buffer
(10 mM, pH 6.9), CMT-AOT complexes in hexane and CMT-
KDP complexes in PFMC were determined using DLS. The
results are shown in Fig. 3.
Fig. 3 DLS traces showing different particle sizes of CMT in different
media (native CMT in buffer, CMT-AOT in hexane and CMT-KDP in
PFMC), indicating that CMT-KDP is clustered under these conditions.
These results are in agreement with our own observation
that proteins tend to form aggregates when the amount of
water is limited in their environment, such as the in very
hydrophobic fluorous solvent PFMC. Akbar et al. suggested
that this aggregation might provide physical protection of the
enzymes, thus maintaining them in an active form and able to
catalyse the transesterification of a natural product with vinyl
butyrate in different organic solvents. In their case, the enzyme
aggregates seemed to be more active for the reaction than the
single protein extracted from the buffer.
It seems clear that both protein aggregation and water content
are key parameters in determining the enzyme activity. In order
to assess if the CMT-KDP HIP complexes formed in PFMC are
catalytically active, a CMT catalysed transesterification reaction
was studied.
The particle diameter of CMT dissolved in aqueous buffer
was determined to be 2.5 nm; consistent with the size of a
single molecule of CMT. The diameter of CMT-AOT particles
in hexane was found to be around 6.0 nm which is similar to the
size of a single molecule of CMT (2.5 nm) plus two molecules
of AOT (size of AOT 1.2 nm).15 This suggests the presence of
individual CMT particles surrounded by the surfactant AOT in
hexane.
Particles of CMT-KDP in PFMC were found to have a
diameter of 25 nm which would correspond to the formation
of an aggregate of about 100 protein molecules surrounded by
the surfactant KDP in PFMC. KDP is estimated to have a length
of ~1.4 nm. The aggregation observed for the solubilised CMT
in PFMC might be related to the very low water content of the
hydrophobic fluorinated solvent.27
Transesterification of N-acetyl-L-phenylalanine ethyl ester
Estimation of water content using coulometric Karl Fisher
titration
The activity of a-chymotrypsin (CMT) solubilised in either
hexane (CMT-AOT) or hexane-PFMC (CMT-KDP) for
the transesterification of N-acetyl-L-phenylalanine ethyl ester
(APEE) with n-butanol or rac-2-butanol was investigated.
The activity of the solubilised protein was compared to that
The water content of hexane; AOT in hexane and CMT-AOT
in hexane; PFMC; KDP in PFMC; and CMT-KDP in PFMC
were determined using the coulometric Karl Fisher titration.
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of the suspended protease (native CMT) and control reac-
tions with only the surfactants: AOT in hexane; or KDP
in PFMC were also set up. The results are summarized in
Fig. 4.
the enzyme complexes formed with AOT and with the larger
surfactant KDP restricting access more.
As explained above (see Fig. 3), particles of CMT-KDP
in PFMC were found to form aggregates of a diameter of
25 nm, whereas the estimated size for CMT-AOT complexes
(6.0 nm) suggested the presence of individual CMT molecules
surrounded by the surfactant AOT in hexane. This difference in
the particle size might explain the higher activity observed for
CMT solubilised in hexane when compared to that in PFMC,
as the number of accessible active sites in the latter will be much
reduced as it is aggregated.
In the paper published by Akbar et al., however, subtilisin
Carlsberg or CALB enzyme aggregates formed were found to
be more active than individual molecules extracted from the
buffer for the transesterification of bergenin with vinyl butyrate
in different organic solvents.27 Their study and our results both
demonstrate that solubilised enzymes aggregates can be more
active than the suspended protein. Possibly, aggregation may
provide a physical protection of the enzymes, thus maintaining
them in an active form.27
The water content of enzyme–surfactant preparations is also
a key factor in the activity of enzymes in non-aqueous media.
This was determined using coulometric Karl Fisher titrations
(see Table 2) and less than 1 ppm of water was detected for CMT-
KDP in PFMC compared to about 50 ppm for CMT-AOT in
hexane. The higher water content of CMT-AOT particles might
also lead to the higher catalytic activity observed compared to
CMT-KDP. Interestingly, our results suggest that an amount
of water less than 1 ppm is sufficient to permit the for-
mation of catalytically active protein-surfactant (CMT-KDP)
particles.
The reusability of CMT-KDP dissolved in PFMC was
investigated for the transesterification reaction in the fluorous
biphasic system (hexane–PFMC) and in scCO2 (100 bar) for
2 hours at 40 ◦C. The results achieved are presented in
Fig. 5. The solubilised protease CMT-KDP in PFMC was
successfully reused for the transesterification of APEE with
both alcohols tested, and no loss of activity was observed
over four cycles in the hexane–PFMC biphasic system or in
scCO2. CMT-KDP in PFMC exhibited good stability in both
media investigated; in particular, its activity in scCO2 did not
appear to be affected by the pressurisation and depressurisation
procedures.13
Fig. 4 Transesterification of APEE (4.2 mM) with either n-butanol or
rac-2-butanol (0.42 mM) catalysed by surfactant AOT only ( ) or KDP
only ( ) or native CMT ( ) or solubilised CMT-AOT ( ) or CMT-
KDP ( ) (0.01 mM) at 40 ◦C with stirring for two hours in the FBS
hexane-PFMC v/v 1 : 1 in a 2 mL septum sealed glass vessel or in scCO2
(100 bar) in a 8.5 mL high pressure stainless steel batch reactor. Yields
were estimated by GLC equipped with a RTX-5 Fast column. Note that
although CMT-KDP is not as active as CMT-AOT, it has higher activity
than the native enzyme. However, unlike CMT-AOT, CMT-KDP can be
recycled.
The HIP solubilised CMT exhibited higher activity than the
suspended enzyme for the transesterification of APEE with the
two alcohols tested in both a FBS and scCO2. Under homo-
geneous conditions (hexane–PFMC and scCO2), the dispersion
of the enzyme–surfactant complexes into the reaction medium
as well as the contact between the enzyme and the substrates
are believed to be increased, thus enhancing the yield of the
reaction.13 With both alkyl alcohols tested, the yields of the ester
products are slightly higher in scCO2 compared to the hexane–
PFMC which might be due to better mass transfer of substrates
to the enzyme active site in scCO2.
The CMT-AOT complexes in hexane appear to be around
three times more active than the CMT-KDP complexes in
PFMC and this trend was observed in both scCO2 and
hexane–PFMC. It is believed that the protein adopts an active
configuration14,16,17 in both media tested and that the difference
in activity observed between CMT-AOT and CMT-KDP could
be due to a difference in the accessibility of the active sites in
Conclusions
The technique of hydrophobic ion pairing (HIP) has been
successfully used to solubilise a-chymotrypsin (CMT) using
fluorinated surfactant KDP in PFMC. The transesterification
of APEE catalysed by the HIP solubilised protease has been
investigated in FBS and scCO2. Under comparable conditions,
we have shown that CMT solubilised in either hexane or
PFMC via HIP gives higher product yields than the suspended
enzyme. The CMT-KDP surfactant enzyme complexes which
form catalytically active CMT-KDP aggregates in PFMC retain
their activity over at least four reaction cycles, suggesting long-
term recyclability.
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Acknowledgements
We wish to thank Dr Helen R. Hobbs for the prelimi-
nary work performed on this project. We are also grate-
ful to all our colleagues in the Clean Technology Group
at the University of Nottingham, the School of Chemistry
Workshop, Mark Guyler and Paul Hamley for their help
and technical support. Finally, we thank Marie Curie Pro-
gramme SubClean Probiomat (MEST-CT-2004-007767) for
funding.
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