Protease activation in glycerol-based deep eutectic solvents

Article abstract of DOI:10.1016/j.molcatb.2011.05.015

Deep eutectic solvents (DESs) consisting of mixtures of a choline salt (chloride or acetate form) and glycerol are prepared as easily accessible, biodegradable, and inexpensive alternatives to conventional aprotic cation-anion paired ionic liquids. These DES systems display excellent fluidity coupled with thermal stability to nearly 200 °C. In this work, the transesterification activities of cross-linked proteases (subtilisin and α-chymotrypsin), immobilized on chitosan, were individually examined in these novel DESs. In the 1:2 molar ratio mixture of choline chloride/glycerol containing 3% (v/v) water, cross-linked subtilisin exhibited an excellent activity (2.9 μmol min ^-1 g^-1) in conjunction with a selectivity of 98% in the transesterification reaction of N-acetyl-l-phenylalanine ethyl ester with 1-propanol. These highly encouraging results advocate more extensive exploration of DESs in protease-mediated biotransformations of additional polar substrates and use of DESs in biocatalysis more generally.

Full text of DOI:10.1016/j.molcatb.2011.05.015

Journal of Molecular Catalysis B: Enzymatic 72 (2011) 163–167
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Protease activation in glycerol-based deep eutectic solvents
Hua Zhaoa,∗, Gary A. Bakerb, Shaletha Holmesa
a Chemistry Program, Savannah State University, Savannah, GA 31404, USA
b Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 March 2011
Received in revised form 14 May 2011
Accepted 31 May 2011
Available online 12 June 2011
Deep eutectic solvents (DESs) consisting of mixtures of a choline salt (chloride or acetate form) and glyc-
erol are prepared as easily accessible, biodegradable, and inexpensive alternatives to conventional aprotic
cation–anion paired ionic liquids. These DES systems display excellent fluidity coupled with thermal sta-
bility to nearly 200 ◦C. In this work, the transesterification activities of cross-linked proteases (subtilisin
and ␣-chymotrypsin), immobilized on chitosan, were individually examined in these novel DESs. In the
1:2 molar ratio mixture of choline chloride/glycerol containing 3% (v/v) water, cross-linked subtilisin
exhibited an excellent activity (2.9 ␮mol min−1 g−1) in conjunction with a selectivity of 98% in the trans-
esterification reaction of N-acetyl-l-phenylalanine ethyl ester with 1-propanol. These highly encouraging
results advocate more extensive exploration of DESs in protease-mediated biotransformations of addi-
tional polar substrates and use of DESs in biocatalysis more generally.
Keywords:
Ionic liquid
Eutectic solvent
Protease
Subtilisin
© 2011 Elsevier B.V. All rights reserved.
Transesterification
1. Introduction
acids and alcohols can be combined to afford inexpensive DESs
from widely available starting materials. (ii) Biodegradable ingre-
As potential environmentally sustainable solvents, ionic liq-
uids (ILs) have emerged as fascinating and customizable reaction
media for various bio-applications, most notably enzyme catalysis
[1–3]. One hindrance to broader development of these promising
solvents in biocatalytic systems stems from the fact that archety-
pal cations employed in IL research overwhelmingly derive from
prohibitively costly heterocycles, particularly substituted imida-
zoliums, and to a lesser extent, pyridiniums, pyrrolidiniums, and
piperidiniums as well. Recently, Abbott et al. [4–6] pioneered the
development of the so-called deep eutectic solvents (DESs) which
are low-melting liquids derived from the mixture of a solid organic
salt and a suitable organic complexant, typically a hydrogen-bond
donating species such as a polyol. While the research community
is ambivalent on whether DESs should be formally classified as
ILs for the fact that they contain a significant molecular compo-
nent, they certainly share a number of attractive solvent features
which account for the popularity of their IL cousins. The exemplary
DES is illustrated by the 1:2 molar ratio mixture of choline chlo-
ride (Tm = 302 ◦C, Scheme 1a) with urea (Tm = 133 ◦C), which yields
a free-flowing fluid with a freezing point below ambient (Tf ≈ 12 ◦C)
[4]. The foremost advantages of DESs are summarized by the fol-
lowing: (i) Low-cost quaternary ammonium salts and similarly
inexpensive complexing agents such as amides, amines, carboxylic
dients can be employed, toward the development of truly green,
sustainable DESs. For instance, a popular precursor salt in DES
preparation, choline chloride (known colloquially as vitamin B4),
is used in large quantities as an additive in chicken feed [7], as well
as being implicated as an essential micronutrient for promoting
proper human health [8]. (iii) DESs can dissolve a variety of typically
water-soluble species like metal salts, amino and organic acids, and
various polyols such as glucose which remain problematic solutes
for conventional aprotic ILs [4,5,9–11]. As a result, there is serious
practical interest toward developing cholinium-based and alter-
native low-toxicity DESs as designer biosolvents for performing
catalysis.
Recently, Gorke et al. [12] reported that a number of hydro-
lases, including Candida antarctica lipase B (CALB), C. antarctica
lipase A (CALA), and epoxide hydrolase, maintained high activi-
ties in DESs based on choline chloride or ethylammonium chloride
paired with hydrogen-bond donor species such as acetamide, ethy-
lene glycol, glycerol, urea, and malonic acid. As co-solvents, DESs
were effectively shown to enhance the reaction rates and/or con-
version efficiencies of these hydrolases. Another recent study [13]
suggested that the 1:2 choline chloride/glycerol eutectic preserved
the integrity and viability of encapsulated bacteria, signifying the
possibility of performing whole-cell biocatalysis within DES media.
More recently, our groups have developed biocompatible DESs such
as 1:2 choline chloride/glycerol and 1:1.5 choline acetate/glycerol
that permitted Novozym® 435, a commercially immobilized form
of CALB, to retain high activity and stability, demonstrating high
conversions of up to 97% in the enzymatic transesterification of
∗
Corresponding author at: Chemistry Program, Box 20600, Savannah State Uni-
versity, Savannah, GA 31404, USA. Tel.: +1 912 358 4448; fax: +1 912 691 6839.
E-mail addresses: huazhao98@gmail.com, zhaoh@savannahstate.edu (H. Zhao).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.05.015

164
H. Zhao et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 163–167
providing a unified activity scale for subtilisin and ␣-chymotrypsin,
in contrast with divergent and non-standardized assay methods
advocated by individual enzyme suppliers.
HO
1a X = Cl
1b X = OAc
+
N
X
2.3. Preparation of eutectic ILs
Scheme 1. Structures of choline chloride (a) and choline acetate (b).
Choline acetate was prepared in our laboratory based on a col-
umn anion-exchange method [19], with minor modifications as
detailed in our recent report [14]. The synthesis of DESs followed
simple thermal-mixing procedures [4,6], which were also reported
elsewhere [14]. The free-flowing DES products were dried over
P2O5 at 45 ◦C for at least 2 weeks. The water content of the DESs
was determined by Karl Fischer (KF) titration (Mettler Toledo C20X
compact Coulometric; detection limit: 1 ppm water) at 20 ◦C, and
was typically found to vary between 0.5 and 1.0% (w/w). Hydranal®
Coulomat AG was used as anolyte for the KF titration.
Miglyol® oil 812 with methanol in a model biodiesel synthetic
pathway [14]. Additionally, DESs derived from cholinium salts and
glycerol tend to have substantially lower viscosities (∼80 mPa s at
50 ◦C) relative to 1:2 choline chloride/urea, which has a viscosity
near 120 mPa s at 50 ◦C [14], despite the fact that neat glycerol itself
has a viscosity at room temperature in excess of 900 mPa s.
Although proteases represent another significant category of
hydrolases with valuable roles in many biocatalytic conversions
[15], their catalytic performance in DESs has yet to be demon-
strated. As part of our continuing research in the area, we
have prepared inexpensive and biodegradable DESs derived from
choline salts (chloride or acetate, Scheme 1b) mixed with glycerol,
and examined their potential as solvent systems for conducting
protease-catalyzed transformations using cross-linked subtilisin
and ␣-chymotrypsin as biocatalysts.
2.4. Transesterification reaction
Fifty microliters of a 1-propanol solution containing N-acetyl-l-
phenylalanine ethyl ester (N-Ac-l-Phe-OEt) were mixed with DES
or organic solvent to make a 1.0 mL solution with final concen-
trations in N-Ac-l-Phe-OEt and 1-propanol of 20 mM and 0.67 M,
respectively. The reaction was initiated by addition of 20 mg of
immobilized protease or 2.0 mg of free protease. The reaction vial
was capped and held at 50 ◦C under constant stirring. At regular
intervals, 50 ␮L was taken and diluted with 100 ␮L of methanol.
After centrifugation, the clear supernatant was injected into a LC-
20AT Shimadzu HPLC using a Phenomenex® Kinetex C18 column
(100 mm × 4.6 mm, particle size: 2.6 ␮m) and eluted with 1:1 (v/v)
2. Experimental
2.1. Materials
Calbiochem® protease from Bacillus licheniformis, commonly
known as subtilisin Carlsberg, was purchased from EMD Bio-
sciences (product number 572909, ≥400 units/mg; 1.0 unit (U)
corresponds to the quantity of enzyme that hydrolyzes 1.0 ␮mol
of N-acetyl-l-tyrosine ethyl ester (ATEE) per min at pH 8.2 and
22 ◦C). The following materials were supplied by Sigma–Aldrich:
choline chloride, anhydrous glycerol, ␣-chymotrypsin from bovine
pancreas (Type II, lyophilized powder, ≥40 units/mg protein; 1.0 U
hydrolyzes 1.0 ␮mol of N-benzoyl-l-tyrosine ethyl ester per min at
pH 7.8 and 25 ◦C), and medium molecular weight chitosan (viscos-
ity of 453 mPa s for a 1% (w/w) chitosan solution in 1% (v/v) acetic
acid) (75–85% deacetylation; product # 448877, LOT # 07918TE).
methanol/aqueous acetate buffer (0.05 M, pH 4.5) at 1 mL min−1
.
The HPLC was equipped with an SPD-20A UV–visible dual wave-
length detector, with eluate monitored at 258 nm. All experiments
were run in duplicate. Standard curves of N-Ac-l-Phe and N-Ac-
l-Phe-OEt at 258 nm were nearly identical, allowing us to use
their average to establish a standard curve for N-Ac-l-Phe-OPr, a
compound not available commercially. Laszlo and Compton [20]
similarly did not differentiate the UV-responses of these three
compounds at 258 nm. Initial reaction rates were derived from
plots of product concentration versus reaction time, expressed
in the unit of ␮mol min−1 g−1 protein, based on the 10% (w/w)
protein loading on solid carrier determined earlier. The reaction
selectivity was obtained by dividing the yield of N-Ac-l-Phe-OPr
by the conversion of N-Ac-l-Phe-OEt (each determined by HPLC
analysis); N-Ac-l-Phe-OPr formation proceeds in competition with
substrate hydrolysis which yields the major by-product N-Ac-l-
Phe, as shown in Scheme 2. The selectivity was determined for the
same reaction period during which the initial reaction rate was
determined, typically within the first hour of reaction when the
substrate conversion was less than 5%.
2.2. Enzyme immobilization
A typical procedure for enzyme cross-linking followed methods
found in the literature [16]. Briefly, after washing 1.0 g of chi-
tosan with 2-propanol and water, respectively, the porous support
was added to 20 mL of phosphate buffer (0.1 M, pH 7.4) contain-
ing 0.1 g of the desired protease. After stirring the suspension at
room temperature for 2 h, 60 mL of 2-propanol was added. Follow-
ing 5 min of gentle agitation, 1.5 mL of a 25% (w/w) glutaraldehyde
solution was added into the suspension to give a 50 mM final con-
centration, and the mixture was stirred for an additional hour at
room temperature. The supernatant was finally decanted, the cross-
linked/immobilized enzyme was washed with water and diethyl
ether, respectively, and then dried. The protein loading on the solid
support was 10% (w/w) as determined by Bradford assay.
For cross-linked chitosan-immobilized subtilisin, the total yield
(defined as the total mass of immobilized enzyme, including
the solid support) was 1.29 g with an activity of 0.58 U/g pro-
tein. For immobilized ␣-chymotrypsin, the corresponding yield
was 1.31 g with 0.60 U/g activity. The pure subtilisin showed
an activity of 1.24 U/g. 1.0 U corresponding to the hydrolysis
of 1.0 mM N-glutaryl-l-phenylalanine p-nitroanilide (GPANA) to
produce 1.0 ␮mol of p-nitroaniline (molar extinction coefficient:
8900 M−1 cm−1 at 410 nm [17]) per min at pH 7.4 and 35 ◦C [18].
This assay was chosen because it represents a convenient and well-
accepted method for quantifying a protease’s hydrolytic activity,
2.5. Equilibration of water activity
The solvent in question (e.g., t-butanol) was equilibrated over
a saturated salt solution in a closed vessel for 7 days at 25 ◦C. The
hygrostats used to achieve various thermodynamic water activi-
ties were based on these salts: LiCl (aw = 0.11), MgCl2 (aw = 0.33),
NaCl (aw = 0.75), and KNO3 (aw = 0.94) [21–23]. The analytical water
contents present in the solvent were then measured using KF coulo-
metric titration conducted at 25 ◦C, with details as specified above.
3. Results and discussion
3.1. Physical properties of eutectic ILs
DESs selected for investigation include 1:2 choline chlo-
ride/glycerol (herein, all DES ratios denote molar ratios), and 1:1.5

H. Zhao et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 163–167
165
O
HN
HN
O
O
+ 1-PrOH
O
HN
+
Pr
O
O
O
O
- EtOH
+ EtOH
HN
O
Enz
+ H2O
O
Enz-OH
OH
O
Scheme 2. Protease-catalyzed transesterification of N-acetyl-l-phenylalanine ethyl ester with 1-propanol in competition with the hydrolysis reaction.
or 1:2 choline acetate/glycerol. Recent studies [11,24] based on
the visual inspection of freezing point (Tf) suggest the eutec-
tic composition of choline chloride/glycerol to be 1:2, coincident
with a Tf near −40 ◦C. Recent differential scanning calorimetry
(DSC) melting point determinations indicate a eutectic composi-
tion close to 1:1.5 for the choline acetate/glycerol DES, however
[14]. Beyond the advantages of low cost and low toxicity, these
glycerol-based DESs also reveal lower viscosities than urea-based
eutectics such as 1:2 choline chloride/urea [14]. In addition,
glycerol-based DESs are now known to be compatible with hydro-
lases such as CALB, CALA, and epoxide hydrolase [12,14], making
them a logical choice for initiating studies of protease activity
within DESs.
The thermal stabilities of these DESs were determined by ther-
mogravimetric analysis (TGA), using the quantitative measure of
decomposition temperature (Tdcp), which signifies the tempera-
ture for which 90% of the initial mass remains for a given ramp
rate during the TGA scanning experiment, in this case a con-
stant heating rate of 10 ◦C min−1. As shown in Fig. 1, these DESs
are generally stable up to nearly 200 ◦C, sufficient for nearly
any biocatalytic investigation conceivable. The Tdcp values for 1:2
choline chloride/urea, 1:2 choline chloride/glycerol, 1:1.5 choline
acetate/glycerol, and 1:2 choline acetate/glycerol all fall in the
range from 205 to 216 ◦C.
3.2. Transesterification activity and selectivity in eutectic ILs
Proteases typically remain active in hydrophobic ILs, such as
those based on the bis(trifluoromethylsulfonyl)imide [Tf2N−] or
hexafluorophosphate [PF6−] anions, in the presence of suitable
amounts of water, but are reportedly inactive in most hydrophilic
ILs, unless in dilute aqueous solution in which case the hydrolytic
activity naturally predominates [1–3]. For this reason, it remains a
considerable challenge to perform enzymatic transformations on
polar substrates such as sugars, which typically only have nominal
solubility in conventional ILs [25,26]. Although recent studies have
reported high activities for lipase in DESs [12,14], it is known that
lipases are generally far more robust than proteases within the non-
aqueous milieu. Indeed, lipases have even been shown to maintain
their catalytic properties in some hydrophilic ILs [1,3]. On the other
hand, the catalytic behavior of proteases within DESs remains com-
pletely unexplored. In order to address this deficiency, this study
examines the synthetic activities of two of the most well-studied
proteases, subtilisin and ␣-chymotrypsin, in glycerol-based DESs
using the standard transesterification reaction of N-Ac-l-Phe-OEt
with 1-propanol. Scheme 2 outlines this model reaction, showing
the fast formation of an acyl-enzyme intermediate, followed by the
competition between esterification and hydrolysis reactions.
The transesterification activities of free and cross-linked
(immobilized) subtilisin were examined in t-butanol alongside
several DESs, as summarized in Table 1 and Fig. 2. The reaction tem-
perature of 50 ◦C was elected on the basis of two considerations: (a)
proteases have been previously shown to retain their activity at this
temperature [27,28]; and (b) DESs have relatively low viscosities at
this temperature [14]. Our experimental data suggest the following
trends for subtilisin activity in DESs: (1) immobilized subtilisin
activity and selectivity remain poor in t-butanol containing up to
10% (v/v) water (Fig. 2a). As Fig. 3 makes clear, the thermodynamic
water activity (aw) in t-butanol is correlated with the water
content, with 7.6% (v/v) water corresponding to an aw above 0.9
at 25 ◦C. (2) Conversely, as can be seen in Fig. 2b, activities close
to 2.0 ␮mol min−1 g−1 are achieved in 1:2 choline acetate/glycerol,
but only at the expense of selectivity which falls to 25% at 3% (v/v)
water. (3) Low activities of 0.42–0.90 ␮mol min−1 g−1 paired with
selectivities near 99% were found for the cross-linked subtilisin in
1:1.5 choline acetate/glycerol (Table 1). (4) As shown in Fig. 2c, a
high activity of 2.9 ␮mol min−1 g−1 with simultaneously excellent
selectivity (98%) is attained in 1:2 choline chloride/glycerol con-
taining 3% (v/v) water. (5) Free subtilisin exhibits superb synthetic
activity in 1:2 choline chloride/glycerol at high water contents
(Fig. 2d), however, the selectivity falls precipitously as water
content increases and is only 14% at 3% (v/v) water. The covalent
cross-linking of free proteases onto a solid carrier clearly results
in some losses in transesterification activity, a result typical of
100
ChOAc/glycerol (1:1.5)
ChOAc/glycerol (1:2)
ChOCl/urea (1:2)
80
ChOCl/glycerol (1:2)
60
40
20
0
0
100
200
300
400
500
Temperature (°C)
Fig. 1. Thermogravimetric analysis of DESs conducted on a TA Instruments 2950.
DES samples (35–50 mg) were micropipetted into open platinum pans and then
heated from ambient temperature up to 500 ◦C at a heating rate of 10 ◦C min−1 under
a protective atmosphere of nitrogen gas (50 mL min−1 flow rate) (ChOAc for choline
acetate; ChOCl for choline chloride).

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H. Zhao et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 163–167
Table 1
Activities of subtilisin and ␣-chymotrypsin in DESs.
Solvent
Protease
Water content (v/v) (%)
Activity (␮mol min−1 g−1
)
Selectivity (%)
t-Butanol
Subtilisin on chitosan
Subtilisin on chitosan
Subtilisin on chitosan
Subtilisin on chitosan
Free ␣-chymotrypsin
␣-Chymotrypsin on chitosan
␣-Chymotrypsin on chitosan
Subtilisin on chitosan
2
2
3
4
2
2
3
3
0.50
0.42
0.40
0.90
0.028
0.031
0.75
2.9
29
99
99
99
99
99
99
98
Choline acetate/glycerol (1:1.5)
Choline acetate/glycerol (1:1.5)
Choline acetate/glycerol (1:1.5)
Choline chloride/glycerol (1:2)
Choline chloride/glycerol (1:2)
Choline chloride/glycerol (1:2)
Choline chloride/glycerol (1:2)
Notes: The transesterification between N-Ac-l-Phe-OEt (20 mM) and 1-propanol (0.67 M) was catalyzed by 20 mg of immobilized protease or 2.0 mg of the free enzyme in
1.0 mL of solvent at 50 ◦C. The reaction mixture was periodically analyzed by HPLC. The initial reaction rate (i.e., activity) was calculated from the rate of product formation
and the selectivity calculated from the ratio of production yield over substrate conversion.
enzyme immobilization [29], however, our data clearly point to
seriously improved selectivity for this model transesterification
reaction in DESs upon subtilisin immobilization, making the
cross-linked enzyme a far superior choice for application in DESs.
Compared with subtilisin, ␣-chymotrypsin was much less active
in 1:2 choline chloride/glycerol in both free and immobilized
forms. As shown in Table 1, at 2% (v/v) water, the activities
of free and cross-linked ␣-chymotrypsin were only 0.028 and
0.031 ␮mol min−1 g−1, respectively; even at a 3% (v/v) water
level, the immobilized ␣-chymotrypsin remained about four-fold
less active than the similarly immobilized subtilisin (0.75 versus
2.9 ␮mol min−1 g−1).
anions like chloride and acetate, their molar concentrations are
notably reduced by the addition of 1.5–2 molar equivalents of
glycerol which tends to form hydrogen bonds with these anions.
Therefore, the tendency of these anions to negatively impact the
protease is greatly diminished. As a result, the denaturing tendency
of these DESs is much lower than for common hydrophilic ILs car-
rying denaturing anions. (2) We also suggest that the hydrophilic
nature of the DESs allows them to structurally “tie up” water
molecules, limiting their availability as nucleophiles in the com-
peting hydrolysis. Consequently, the actual thermodynamic water
activity (aw) of DESs is anticipated to be lower than that of con-
ventional hydrophobic organic solvents and ILs. This postulate has
been experimentally validated by a number of enzymatic studies
carried out in hydrophilic ILs [28,30,31]. It is also well established
The high subtilisin activity in DESs can be rationalized by a num-
ber of factors. (1) Although these DESs contain enzyme-denaturing
14.0
12.0
10.0
8.0
100
80
60
40
20
0
a
14.0
12.0
10.0
8.0
100
80
60
40
20
0
b
Activity
Activity
Selectivity
Selectivity
6.0
6.0
4.0
4.0
2.0
2.0
0.0
0.0
0
2.0
5.0
7.0
10.0
0.6
2.0
3.0
4.0
Water content (%, v/v)
Water content (%, v/v)
c
14.0
100
80
60
40
20
0
14.0
100
80
60
40
20
0
d
Activity
12.0
10.0
8.0
12.0
10.0
8.0
Selectivity
Activity
Selectivity
6.0
6.0
4.0
4.0
2.0
2.0
0.0
0.0
1.0
2.0
3.0
4.0
5.0
1.0
2.0
3.0
4.0
Water content (%, v/v)
Water content (%, v/v)
Fig. 2. Protease activity and selectivity in different solvents at various water contents (%, v/v): (a) cross-linked (immobilized) subtilisin in t-butanol; (b) immobilized subtilisin
in 1:2 choline acetate/glycerol; (c) immobilized subtilisin in 1:2 choline chloride/glycerol; (d) free subtilisin in 1:2 choline chloride/glycerol. The transesterification between
N-Ac-l-Phe-OEt (20 mM) and 1-propanol (0.67 M) was catalyzed by 20 mg of immobilized protease (or 2.0 mg of the corresponding free protease) in 1.0 mL of solvent at 50 ◦C.

H. Zhao et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 163–167
167
10.0
8.0
6.0
4.0
2.0
0.0
Acknowledgements
This research was supported by the National Institutes of Health
under a RIMI grant (5P20MD003941) subproject to HZ.
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Fig. 3. The relationship between water content and thermodynamic water activity
(aw) of t-butanol at 25 ◦C.
that the enzyme’s activity is far more dependent upon aw than on
the analytical water content per se [32]. (3) In the present case,
the enzyme-host (chitosan) additionally contains hydroxyl groups,
which allow the support to absorb water molecules via hydrogen-
bond interactions, further reducing aw in the reaction system.
Therefore, a higher selectivity might reasonably be expected for the
chitosan-immobilized proteases than for freely dissolved enzyme.
4. Conclusions
Glycerol-based DESs derived from choline chloride (or acetate)
are shown to possess relatively low viscosities and high ther-
mal stability. Furthermore, these eutectic melts can be tailored
as effective solvents for carrying out protease-mediated synthe-
sis. In particular, cross-linked subtilisin exhibited a high activity
and selectivity in 1:2 choline chloride/glycerol in the presence
of 3% (v/v) water whereas free subtilisin gave poor selectivity
in the same DES. Subtilisin was also found to be more active in
1:2 choline chloride/glycerol than in choline acetate/glycerol mix-
tures, and subtilisin was also more compatible with DESs than
was ␣-chymotrypsin. The excellent suitability of DESs for subtilisin
biocatalysis is thought to be tied to the low molar concentration
of the denaturing anion (relative to their IL peers), reducing the
propensity for hydrogen bond formation between these detrimen-
tal anions and the protein itself, and the hydrophilic features of the
chitosan host which aid in controlling the local water activity in the
vicinity of the enzyme.
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