Low ionic liquid concentration in water: A green and simple approach to improve activity and selectivity of lipases

Article abstract of DOI:10.1039/c4ra07625g

The activity of several commercial and genetically engineered lipases, already immobilized on octyl-Sepharose, in aqueous solutions containing ionic liquids (ILs) in a molar concentration of 0.01 mol L^-1 was investigated. Strong variations in t

Full text of DOI:10.1039/c4ra07625g

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Low ionic liquid concentration in water: a green
and simple approach to improve activity and
selectivity of lipases
Cite this: RSC Adv., 2014, 4, 49115
Marco Filice,a Oscar Romero,†a Olga Abian,bcd Blanca de las Rivase
a
*
and Jose M. Palomo
The activity of several commercial and genetically engineered lipases, already immobilized on octyl-Sepharose,
in aqueous solutions containing ionic liquids (ILs) in a molar concentration of 0.01 mol Lꢀ1 was investigated.
Strong variations in the hydrolytic activity values were found depending on the lipase, the ionic liquid and the
substrate used. The most interesting results were found in the engineered variants of Geobacillus
thermocatenolatus lipase (GTL). The activity of GTLs-L230C variant in the monodeacetylation of per-
acetylated glucal increased seven fold when using 1-ethyl-3-methylimidazoliumhexafluorophosphate [emim
[PF6] and fivefold when using 1-ethyl-3-methylimidazolium methyl sulphate [emim][MeSO3]. In general, ILs
ꢀ
containing BF4 had a very negative effect on the enzyme activity. Moreover, improvements in the enantio
and regioselectivity of the immobilized lipases were obtained by adding ILs to the reaction medium. The
regioselectivity of Candida rugosa lipase in the hydrolysis of peracetylated thymidine improved (from 72% to
81% yield of C-5 monodeprotected product) in the presence of [emim][PF6]. More interestingly, the addition
of 1-butyl-2,3-dimethylimidazolium hexafluorophosphate [bdmim][PF6] improved the regioselectivity of
immobilized GTLs-L230C variant in the hydrolysis of peracetylated glucal, from 78% to 96% yield of the C-3
monodeprotected product. Circular dichroism and fluorescence experiments revealed conformational
changes in the tertiary structure of the enzyme induced by the ionic liquid which could explain these
phenomena.
Received 25th July 2014
Accepted 24th September 2014
DOI: 10.1039/c4ra07625g
www.rsc.org/advances
process is more complex and normally exists an equilibrium
between these two lipase conformations which depends on the
Introduction
medium conditions to be shied mainly to the closed or the
open form.7
Lipases, acylglycerol hydrolases (EC:3.1.1.3), are one of the most
used enzymes in biotransformations.1–5 The catalytic mecha-
nism of a lipase is based on the movement of an oligopeptide
chain “lid” in its structure. In homogeneous aqueous media the
lipase exists mainly in a closed conformation, where the lid
maintains the enzyme active site secluded from the medium.6
However in the presence of hydrophobic interfaces, this peptide
has a tendency to shi generating an open conformation of the
lipase, which exposes a large number of hydrophobic residues
to the medium for the adsorption on this interface.6 This
Based on that, this equilibrium has successfully modied by
different strategies. Immobilization protocols to rigidify or x a
particular conformation has been successfully used to improve
the activity and specially the enantio and regioselectivity of
these enzymes.8–10 Other methodologies such as chemical
modication of the enzyme surface11,12 or changes in experi-
mental parameters (pH, T or the addition of co-solvents) have
been quite useful to alter the lipase catalysis.8,13
In this sense, ionic liquids (ILs) have emerged as a very
attractive alternative to environmentally harmful organic
solvents.14
aDepartamento de Biocatalisis, Instituto de Catalisis (CSIC), Marie Curie 2.
Cantoblanco. Campus UAM, 28049 Madrid, Spain. E-mail: josempalomo@icp.csic.es;
´
´
In particular, the large number of applications of ILs with
lipases has been published,15–18 however the use of full or even
higher concentration of ILs may decrease, or also completely
inactivates the enzymes, since they have the capacity to interact
with the water molecules around the enzyme surface19 and in
some cases, with the enzyme itself20 making difficult a real
application of them. In this context, there is a strong and urgent
need to increase the catalytic properties of lipases in ILs.
Fax: +34 915854760
b
´
´
IIS Aragon, Instituto Aragones de Ciencias de la Salud (IACS), Zaragoza, Spain
cInstitute of Biocomputation and Physics of Complex Systems (BIFI), Joint Unit IQFR-
CSIC-BIFI, Universidad de Zaragoza, Zaragoza, Spain
d
´
´
´
´
Centro de Investigacion Biomedica en Red en el Area Tematica de Enfermedades
´
Hepaticas y Digestivas (CIBERehd), Spain
e
´
´
Laboratorio de Biotecnologıa Microbiana, Instituto de Ciencia y Tecnologıa de
´
alimentos y Nutricion, (ICTAN-CSIC), Madrid, Spain
† Present address: Institute of Biomaterial Science, Helmholtz Zentrum
Geesthacht, Teltow, Germany.
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Table 1 Specific activity of commercial lipases immobilized on octyl-
Sepharose in the hydrolysis of 1 in the presence of different ILsa
ILs
PFL
CAL-B
CRL
—
31.30
55.78
95.61
85.71
91.21
71.72
74.01
64.96
592
258
748
555
726
449
882
936
8.44
—
13.76
14.85
12.33
—
[emim][BF4]
[emim][PF6]
[emim][NO3]
[emim][MeOSO3]
[bmim][BF4]
[bdmim][BF4]
[bdmim][PF6]
—
15.21
a
ꢀ1
The initial rate in mmol mgprot minꢀ1. It was calculated at 10–20%
yield. IL without enzyme was tested and no conversion was detected.
The experiments were performed by triplicate and error was less than
5%.
Scheme 1 Green biocatalytic preparation of monodeacetylated drug
intermediates.
genetically engineered mutants from GTL [GTL-C65S/C296S
(GTLs), BTL-C65S/C296S/A193C (GTLs-A193C), GTL-C65S/
C296S/L230C (GTLs-L230C) and BTL-C65S/C296S-S196C (GTLs-
S196C)] was evaluated in the presence of seven different ionic
liquids: 1-ethyl-3-methylimidazolium tetrauoroborate [emim]
[BF4], 1-ethyl-3-methylimidazoliumhexauorophosphate [emim]
[PF6], 1-ethyl-3-methylimidazolium nitrate [emim][NO3], 1-ethyl-
3-methylimidazolium methyl sulphate [emim][MeOSO3], 1-
butyl-3-methylimidazolium tetrauoroborate [bmim][BF4], 1-
butyl-2,3-dimethylimidazolium tetrauoroborate [bdmim][BF4]
and 1-butyl-2,3-dimethylimidazolium hexauorophosphate
[bdmim][PF6] as ionic liquid molar concentration of 0.01 mol Lꢀ1
in aqueous media at pH 5.
Recently, the use of microemulsion of ILs in water enhanced
the lipase activity based on the interfacial activation mecha-
nism.21 In a preliminary work in the development of an efficient
synthesis of a carbohydrate derivative we observed that the
addition of small amounts of ILs in the reaction media
considerably improved the enzyme activity of R. miehei lipase,
though any improvement of regioselectivity was not achieved in
this case.22
Herein we have demonstrated that the application of
micromolar concentration of ILs in aqueous solution can alter
the activity and for the rst time the enantio and regioselectivity
of lipases. This method improves lipase catalysis without
requiring any pretreatment of the enzyme or manipulation or
modication of the ILs.
In order to establish a general application, this method was
evaluated using three of the most used commercial lipases,
from Pseudomonas uorescens (PFL), from Candida antarctica B
(CAL-B) and from Candida rugosa (CRL). Furthermore four
engineered mutants of Geobacillus thermocatenulatus lipase
(GTL), a more complex enzyme constituted by two different lids
and very hydrophobic pocket, were used. In all cases the lipases
were immobilized on the hydrophobic commercial matrix octyl-
Sepharose, xing their open conformation. For the evaluation of
The addition of ILs was positive for the PFL activity in the
hydrolysis of 1. A 3 fold increase of the activity was found in the
presence of [emim][PF6] and [emim][MeOSO3] (Table 1).
However, with 2 and 3 positive resultsꢀwere only found with ILs
ꢀ
containing PF6ꢀ, NO3 or MeOSO3 ions, whereas a high
decrease in the enzyme activity was observed using BF4-based
ILs (Tables 2 and 3).
The prole was quite similar for CAL-B. Moreover the use of
[bdmim][PF6], a more hydrophobic IL, showed a good increase
of the enzyme activity in the hydrolysis of 1 and 3 (Tables 1 and
3). In the case of CRL, the effect of BF4ꢀ was even more negative
lipase properties, a deacetylation process, a quite useful Table 2 Specific activity of commercial lipases immobilized on octyl-
Sepharose in the hydrolysis of 2 in the presence of different ILsa
biotransformation in organic synthesis, was selected. Three
different acetylated compounds, (ꢁ)-O-acetyl-1-phenylethanol
ILs
PFL
CAL-B
CRL
(1), 3,5-di-O-acetyl-thymidine (2) and 3,4,6-tri-O-acetyl-glucal
(3), were used as model substrates to evaluate the enantio and
regioselectivity of the lipases on these new conditions, creating
interesting selective monodeacetylated products, useful
building blocks in the synthesis of more complex pharmaceu-
tical molecules (Scheme 1).23,24
—
19.40
17.70
19.40
17.43
18.93
14.68
18.43
18.75
28.1
9.3
29.6
28.1
29.6
9.3
10.5
—
11
8.5
17.5
—
[emim][BF4]
[emim][PF6]
[emim][NO3]
[emim][MeOSO3]
[bmim][BF4]
[bdmim][BF4]
[bdmim][PF6]
10.4
15.6
—
8.46
Results and discussion
a
ꢀ1
The initial rate in mmol mgprot hꢀ1 ꢂ 10ꢀ1. It was calculated at 10–
20% yield. IL without enzyme was tested and no conversion was
detected. The experiments were performed by triplicate and error was
less than 5%.
The catalytic activity of a set of seven different pure and immo-
bilized lipases, three commercial (PFL, CRL, CAL-B) and four
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Table 3 Specific activity of commercial lipases immobilized on octyl-
Sepharose in the hydrolysis of 3 in the presence of different ILsa
ILs
PFL
10.5
1.44
12
11.4
9.6
0.9
1.20
10.95
CAL-B
CRL
—
4.88
0.30
7.95
8.45
8.10
0.30
0.33
7.95
17.10
1.20
12.30
10.80
15.10
0.60
[emim][BF4]
[emim][PF6]
[emim][NO3]
[emim][MeOSO3]
[bmim][BF4]
[bdmim][BF4]
[bdmim][PF6]
Scheme 2 Enzymatic kinetic resolution of 1.
Table 5 Enantioselectivity of immobilized lipases catalyzing hydrolysis
of (ꢁ)-1 catalyzed in the presence of different ILs
0.60
8.85
E valuea (ee, %)
a
ꢀ1
The initial rate in mmol mgprot hꢀ1. It was calculated at 10–20%
yield. IL without enzyme was tested and no conversion was detected.
The experiments were performed by triplicate and error was less than
5%.
ILs
PFL
CALB
CRL
—
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
>100(>99)
14(87)
—
22(91)
19(90)
19(90)
—
[emim][BF4]
[emim][PF6]
[emim][NO3]
[emim][MeOSO3]
[bmim][BF4]
[bdmim][BF4]
[bdmim][PF6]
Table 4 Specific activities of immobilized GTL variants on the regio-
selective hydrolysis of 3 by in the presence of different ILsa
ILs
GTLs GTLs-A193C GTLs-S196C GTLs-L230C
—
18(89)
—
5.1
6.9
6.6
8.6
15.7
34.9
32.1
17.4
28.4
25.9
20.1
10.0
8.0
21.6
21.8
12.2
15.6
10.2
12.4
5.4
10.8
50.1
70.6
27.3
53.8
21.3
9.2
a
ee of product calculated at 15% conversion.
[emim][BF4]
[emim][PF6]
[emim][NO3]
[emim][MeOSO3] 6.1
[bmim][BF4]
[bdmim][BF4]
[bdmim][PF6]
6.4
8.5
3.6
Additionally, the lipase selectivity was improved because of
the presence of ILs. For CAL-B and PFL, the enantioselectivity in
the kinetic resolution of 1 (Scheme 2) was excellent (ee > 99%,
E value > 100) and it was conserved in the presence of all ILs. In
the case of CRL, the ILs also slightly enhanced the enantiose-
lectivity of the enzyme, from E value of 14 (87% ee) to 22 (91%
ee) with [emim][PF6] (Table 5).
23.8
a
ꢀ1
The initial rate is expressed in mmol mgprot minꢀ1 ꢂ 10ꢀ3. It was
calculated at 10–20% yield. IL without enzyme was tested and no
conversion was detected. The experiments were performed by
triplicate and error was less than 5%.
In the deprotection of acetylated thymidine 2 (Scheme 3), the
regioselectivity of CAL-B or CRL was improved with all ILs tested
(Table 6). The yield in monodeprotected C-5 product 5 synthe-
sized by CAL-B was improved from 61 to 79% with the addition
of [bdmim][PF6]. The yield in monodeprotected C-3 product 6
synthesized by CRL was improved from 72 to 80% with the
to the enzyme and no activity in the presence of ILs containing
this anion was found for this enzyme in the hydrolysis of 1 and 2
(Tables 1 and 2). In general, the addition of ILs such as [emim]
[MeOSO3] or [emim][PF6] was the most positive for these
enzyme activity.
Other ILs concentrations such as 0.02, 0.03 or 0.04 mol Lꢀ1
were tested with no differences in results (data not shown).
More interesting variations in the enzyme activity with the
addition of ILs were obtained for four different GTL variants in
the hydrolysis of 3 (Table 4). Interestingly, the effect of ILs was
signicantly higher in the GTLs variants with a unique cysteine
in the lipase lid site in different positions (GTLs-A193C, GTLs-
S196C and GTLs-L230C). The enzymatic activity of GTLs-L230C
increased in the presence of almost all ILs tested, in particular 7
fold in the presence of [emim][PF6] and 5 fold with [emim]
[MeOSO3]. These activity improvements are the highest one
described in the literature for this enzyme in this reaction.25
[emim][PF6] improved the activity of the other variants, up to 3
fold for GTLs-S196C and 2 fold for GTLs-A193C.
The presence of ILs containing BF4ꢀ in the GTL catalysis did
not cause the negative effect observed for the other lipases and,
for example, [emim][BF4] enhanced the activity for all variants
from 3 to 5 fold (Table 4).
Scheme 3 Enzymatic hydrolysis of peracetylated thymidine.2.
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Table 6 Regioselective hydrolysis of 2 catalyzed by immobilized Table 7 Regioselective hydrolysis of 3 catalyzed by immobilized
lipases in the presence of different ILs commercial lipases in the presence of different ILs
PFLa,b CALBa,c
CRLa,d
PFL
CALB
CRL
7a (%)
7a (%)
8a (%)
ILs
5a (%) 6a (%) 5a (%) 6a (%) 5a (%) 6a (%)
ILs
—
28
29
29
26
51
53
53
54
54
19e
19e
52
61
3
—
3
4
10
—
9
9
72
—
98
>99b
>99
>99
>99
>99b
>99b
>99
>99
10c
>99
>99
98
>99
24d
>99
>99
>99
19d
22d
91
f
f
f
f
[emim][BF4]
[emim][PF6]
[emim][NO3]
—
75
69
73
—
81
78
76
[emim][BF4]
[emim][PF6]
[emim][NO3]
[emim][MeOSO3]
[bmim][BF4]
[bdmim][BF4]
[bdmim][PF6]
[emim][MeOSO3] 26
1
9
f
f
f
f
f
15e
14e
28
—
—
—
3
—
—
10c
10c
99
[bmim][BF4]
[bdmim][BF4]
[bdmim][PF6]
f
f
f
—
—
—
79
8
80
a
a
Yield of the monodeprotected product at 100% conversion. The rest of
Yield of the monodeprotected product at 100% conversion aer 2 h.
yield corresponds to the bihydrolyzed product (thymidine). b Complete
b
The rest of yield corresponds to the bihydrolyzed product. 4 h.
c
d
c
15% conversion. d 27% conversion.
reaction: 50 h. Complete reaction: 96 h. Complete reaction: 24 h.
e
35% conversion. f No reaction observed.
Table 8 Regioselective hydrolysis of 3 catalyzed by different immo-
bilized GTL variants in the presence of different ILs
7a (%)
ILs
GTLs GTLs-A193C GTLs-S196C GTLs-L230C
—
79
77
87
72
74
80
89
76
75
87
73
79
84
80
85
74
80
74
85
80
78
93
88
93
92
71
75
96
[emim][BF4]
[emim][PF6]
[emim][NO3]
[emim][MeOSO3] 77
[bmim][BF4]
[bdmim][BF4]
[bdmim][PF6]
73
89
74
Scheme 4 Enzymatic deacetylation of 3.
a
Yield of the monodeprotected 3 at 100% conversion. The rest of yield
corresponds to the bihydrolyzed product.
addition of [emim][PF6] or [bdmim][PF6]. The PFL regiose-
lectivity in this process was not modied by any ILs.
In the monodeprotection of the fully acetylated glucal 3
(Scheme 4), the excellent regioselectivity of PFL, CAL-B or CRL
to produce 7 or 8 respectively was conserved in the presence of
the ILs (Table 7).
Under this catalytic aspect, the highest improvements on
enzyme regioselectivity were observed by adding the ILs on the
different GTL variants catalyzing the hydrolysis of 3 (Table 8).
The enzyme variants with the mutated unique cysteine
exhibited quite interesting increase in the regioselectivity in
comparison with the non mutated one, where slight changes
were observed. Hence, the presence of ILs enhanced the regio-
selectivity of GTLs-A193C and GTLs-L230C variants, whereas
non positive results were found for GTLs-S196C.
With the purpose of giving a possible explanation of the
results under a structural point of view, near circular dichroism
(CD) and uorescence assays were performed in some of the
most interesting examples to analyze the effect of the presence
of ILs on the protein tertiary structure (Fig. 1 and 2).
First, in the case of commercial lipases, we observed how the
near CD of CAL-B and PFL were altered by the presence of the
ionic liquid (Fig. 1). [emim][BF4] caused a strong shi of the
maximum peak in uorescence for PFL, from 333 to 369 nm,
and could be related to changes in the neighboring protonated
acidic groups (Asp or Glu) (Fig. 1D). Also, the CD spectrum was
quite altered and this could mean changes in the aromatic
amino acid residues of the protein: mobility, environment
(H-bonding, polar groups and polarisability) and their spatial
disposition in the protein (Fig. 1C).
The yield of 7 produced by GTLs-A193C was increased from
74% to 87 or 89% with the addition of [bmim][BF4] or [emim]
[PF6] respectively (Table 8).
However, the best regioselectivity improvement for GTLs-
L230C was achieved in the presence of [bdmim][PF6], from 78 to
96% yield of 7. High yields of 7 using this variant were obtained
using [emim][PF6] or [emim][NO3] (92–93%) (Table 8). These
results represent the rst example of the regioselectivity
improvement of this GTL variant in the hydrolysis of 3, which
have not been achieved even with more sophisticated method-
ologies such as site-specic peptide modications.25
Therefore these spectroscopic results and the observed
decrease in the enzyme activity (nine fold) (Table 3) could
suggest that the protein adopts a more closed conformation in
the presence of [emim][BF4].
The CD spectrum of CAL-B was altered in the presence of
[emim][MeOSO3] (Fig. 1A), although the uorescence signal did
not change (Fig. 1B). In this case, the activity almost duplicated
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Fig. 1 CD and fluorescence analysis of lipases in the presence of ILs in water. (A) CD spectra (near UV spectrum) of CALB (black line), CALB +
[emim][MeOSO3](red). (B) Fluorescence spectra of CALB and CALB + [emim][MeOSO3]. (C) CD spectra (near UV spectrum) of PFL (black line), PFL
+ [emim][BF4](red). (D) Fluorescence spectra of PFL, PFL + [emim][BF4].
Fig. 2 CD and fluorescence analysis of GTLs-L230C variant in the presence of ILs in water. (A) CD spectra (near UV spectrum). (B) Fluorescence
spectra. GTLs-L230C (black), GTLs-L230C + 0.1% Triton X-100 (red), GTLs-L230C + [emim][BF4] (light blue), GTLs-L230C + [bdmim][BF4]
(green) and GTLs-L230C + [bdmim][PF6] (dark blue).
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its value in the presence of ionic liquid (Table 3). This suggests
that the ionic liquid interacts with the protein but this inter-
action does not alter the open/closed protein conformation.
In the case of GTLs-L230C variant, the structural changes
were studied in the presence of three ionic liquids, [emim][BF4],
[bdmim][BF4] and [bdmim][PF6], evaluating the effect of the IL
cation and the anion (Fig. 2).
GTLs-L230C variant CD spectra undergoes an important
decrease of its signal in the presence of these three ILs, being
the most remarkable the case of [emim][BF4], followed by
[bdmim][PF6] and then [bdmim][BF4] (Fig. 2A). This order is the
same found in the activity data (50.1, 23.8 and 9.2 respectively)
(Table 4). In addition, the best regioselectivity (93 and 96%)
were found with [emim][BF4] and [bdmim][PF6] respectively, ILs
with the largest changes occurring in CD spectra. In uores-
cence spectra, no signicant variation was found using ILs, only
the signal pick was wider for [emim][BF4] (Fig. 2B).
These results revealed the key role of the IL cation on the
structure, indicating that [emim]+ is the best cation for activity
improvement and [bdmim]+ for the selectivity, in both cases
combined with [PF6]ꢀ. Thus, spectroscopic data conrm that
the presence of ILs promotes conformational changes on this
GTLs-L230C variant structure that are different from those
occurring in the presence of detergent (where the lid is
completely opened) (Fig. 2).
In lipases, the movement of the lid is a critical point in
catalysis but also involves a reorganization of the whole protein.
Recently we have demonstrated how modications on this oli-
gopeptide create new lipase structures with new different
properties.25 Therefore, the genetic incorporation of a single
cysteine in different positions of enzyme lid resulted crucial for
the improvements achieved in the presence of the IL. The
position of the mutation on the lid was also important. The
introduction of a cysteine in residue 230 was the best option
instead of 193 or 196, probably because it is surrounded by
several hydrophobic residues participating together with the
thiol in the interaction with the cation of the IL and in conse-
quence shiing the movement of the lid (Fig. 3).
Conclusion
Therefore, herein we present a simple and green method to
improve the activity and enantio and regioselectivity of lipases
in a different way. The enzyme activity of different lipases was
improved from good to excellent values by the IL addition in
some cases preserving the excellent enzyme selectivity and in
other cases a further enhancement of the lower enantio or
regioselectivity of the lipase was observed.
The addition of this very small amount of ILs in aqueous
solution did not represent problems in the separation of the
product, toxicity, etc., being an interesting and useful applica-
tion technology for greener biocatalytic processes useful in
organic and pharmaceutical chemistry.
Circular dichroism and uorescence experiments revealed
conformational changes in tertiary structure of the enzyme
induced by the ionic liquid, indicating a feasible alteration of
the lipase lid movement.
Experimental
Materials
The lipase from Candida antarctica (fraction B) was kindly
donated by Novozymes (Denmark). Lipase from Pseudomonas
uorescens was from Amano S.A. Octyl-Sepharose (4BCL) was
purchased from GE-Healthcare (Uppsala, Sweden). Lipase from
Candida rugosa, (ꢁ)-O-acetyl-1-phenylethanol (1), p-nitro-
phenylbutyrate (pNPB), thymidine and 3,4,6-tri-O-acetyl-1,5-
anhydro-2-deoxy-D-arabino-hex-1-enitol (3) were from Sigma-
Aldrich. 1-Ethyl-3-methylimidazolium methyl sulphate [emim]
[MeOSO3], 1-ethyl-3-methylimidazolium nitrate [emim][NO3], 1-
ethyl-3-methylimidazolium hexauorophosphate [emim][PF6],
1-ethyl-3-methylimidazolium tetrauoroborate [emim][BF4], 1-
butyl-2,3-dimethylimidazolium tetrauoroborate [bdmim][BF4]
and 1-butyl-2,3-dimethylimidazolium hexauorophosphate
[bdmim][PF6] were from Fluka. Peracetylated thymidine 2 was
synthesized as previously described.25
Enzymatic activity assay
The activities of the soluble and immobilized lipases were
analyzed spectrophotometrically measuring the increment in
absorbance at 348 nm (isosbestic point) produced by the release
of p-nitrophenol (pNP) (3 ¼ 5150 Mꢀ1 cmꢀ1) in the hydrolysis of
0.4ꢃmM pNPB in 25 mM sodium phosphate buffer at pH 7 and
25 C. To initialize the reaction, 0.05–0.2 mL of lipase solution
or suspension was added to 2.5 mL of substrate solution.
Enzymatic activity is given as micromole of hydrolyzed pNPB
per minute per milligram of enzyme (IU) under the conditions
described above.
Purication and immobilization of commercial lipases on
hydrophobic supports
The enzyme was puried from crude extract by interfacial
adsorption as previously described.26 2.3 mL of commercial
solution of RML (7 mgprotein per mL, 25% of lipase content
analyzed by Bradford's assay27) were added to 19 mL of 25 mM
Fig. 3 Crystal structure of GTL in open conformation. The mutated
positions A193, S196 and L230 are marked in green, whereas hydro-
phobic residues on the lid are marked in red. Figure was drawn using
Pymol 0.99 program and pdb codes 2W22.
49120 | RSC Adv., 2014, 4, 49115–49122
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phosphate buffer pH 7 and one gram of octyl-Sepharose was buffer pH 7 with 0.5% Triton X-100 (v/v) per gram of support.
added. The reaction was maintained in stirring for 3 h. Aer Aer that, the detergent was removed by ultracentrifugation
that, the suspension was ltered by vacuum and the solid was and the lipase variants were immobilized on octyl-Sepharose at
washed several times with distilled water.
pH 7 in 25 mM sodium phosphate buffer for 1 h at 25 ꢃC (>95%
ꢀ1
30
For the others lipases, the purication/immobilization immobilization yields) with a nal loading of 5 mglip gcat
.
protocol was the same. In each case, it is important to calcu- The remaining pNPB activity for the different immobilized GTL
late the right amount of crude lipase extract to add with the nal variants was more than 95% of the initial activity.
objective to obtain 1 g of octyl-Sepharose derivative loaded with
4 mg of pure protein. The lipase content (analyzed by Bradford's
assay27) for each commercial crude extract was: CAL-B: 3.5
mgprotein per mL (100% of lipase content); CRL: 9.5 mgprotein per
mL (100% of lipase content); PFL: 8.8 mgprotein per mL (100% of
lipase content).
In each case, more than 98% of the enzyme was immobi-
lized, obtaining biocatalysts with an enzyme loading of
4 mglipase per g support. SDS-PAGE analysis of the protein
adsorbed to the octyl-Sepharose only showed a single band with
a molecular weight corresponding to that of the different native
lipases.26
Enzymatic biotransformations
2 mM of 1–3 in 25 mM NaH2PO4 98%/CH3CN 2% and 5 eq. of
ꢃ
ionic liquid at pH 5 and 25 C were prepared. 0.4 g of immo-
bilized lipase were added to 2 mL of the previous solution to
initialize the reaction. The hydrolytic reaction was carried out
under mechanical stirring and the pH value was controlled by
automatic titration. Hydrolysis reactions were followed by HPLC
using a HPLC spectrum P100 (Thermo Separation products). At
least triplicates of each assay were made.
The HPLC analysis was performed using a Kromasil-C18 (250
ꢂ 4.6 mm and 5 mm ø) column. Analyses were run at 25 ꢃC using
an L-7300 column oven couple to a UV detector L-7400. For
substrate 1 and 3, the hydrolytic conversion was determined at
215 nm and the mobile phase was an isocratic mixture of 25%
acetonitrile/75% 10 mM ammonium phosphate buffer at pH
3.8; ow rate 1.0 mL minꢀ1. Under these conditions, the
The octyl-lipase immobilized preparations were used as
catalysts.
Site-directed mutagenesis of Geobacillus thermocatenulatus
lipase (GTL)
All site-directed mutagenesis experiments were carried out by PCR retention times were: 3 ¼ 30 min, 7 ¼ 6.8 min and 8 ¼ 7.3 min.
using mutagenic primers. Briey, to introduce the amino acid The enantiomeric excess of product S-4 was determined by
change, the corresponding pair of primers was used as homolo- chiral reverse-phase HPLC, using pure commercial enantiomers
gous primer pair in a PCR reaction using a specic plasmid as as standards. The column was chiracel OD-R and the mobile
template and Prime Start HS Takara DNA polymerase. The product phase was an isocratic mixture of 35% acetonitrile and 65% 10
of the PCR was digested with endonuclease DpnI that exclusively mM sodium phosphate buffer at pH 7. The analyses were per-
restricts methylated DNA.28 E. coli DH10B cells were transformed formed at xed ow of 0.45 mL minꢀ1 by recording the absor-
directly with the digested product. The plasmid with mutated gtl bance at 225 nm.
were identied by sequencing and then transformed into E. coli
For substrate 2, the hydrolytic conversion was determined at
BL21 (DE3) cells to express the corresponding proteins. Firstly, 260 nm and the mobile phase was a gradient mixture of A: 10
C65S was created, and the resulting plasmid was used as template mM NH4H2PO4 90%/CH3CN 10% pH 4.2; B: CH3CN 90%/H2O
to create the double mutant C65S/C296S-GTL.29 This plasmid 10%; method: 0–6 min 100% A; 6–14 min 65% A and 35% B;
(pT1BGTLmutCys) was used as template to construct additional 14–22 min 100% A; ow rate 1.0 mL minꢀ1. Under these
mutations (A193C, S196C, L230C) using different mutagenic conditions, the retention times were: 2 ¼ 13.3 min, 5 ¼ 8.4 min,
primers: A193C (Ala/cys 193-5,50-GAAAGCGtgcGCTGTCGCCAG; 6 ¼ 7.6 min and thymidine ¼ 2.1 min.24
Ala/cys 193-3 50-CTGGCGACAGCg caCGCTTTC), S196C (Ser/
Cys196-5 50-GTTGAAAGCGGCG GCTGTCGCCtgcAA TGTGCCGTA
Product characterization
CACGAGTCAAG'Ser/Cys196-3,50-CTTGACTCGTGTACGGCA CATT
50-O-Acetylthymidine (5). This compound was characterized
gcaGGCGACGCCGCCGCTTTCAAC) and L230C (Leu/Cys 230-5 50-
how previously reported in literature.24
CATTATTTTGAACGGtgcAAACG, Leu/Cys 230-3 50-CGTTTgcaCCG
30-O-Acetylthymidine (6). This compound was characterized
TTCAAAA TAATG).
how previously reported in literature.24
4,6-Di-O-acetyl-D-glucal (7). 1H-NMR (500 MHz, CDCl3) d:
Cloning, expression, purication and immobilization of GTL
6.35 (dd, J ¼ 6.2 Hz, 1H, H-1), 4.95 (dd, J ¼ 8.4, 6.2 Hz, 1H, H-4),
variants
5.41–5.50 (m, 1H, H-3), 4.84 (dd, J ¼ 6.2, 5.2, 3 Hz, 1H, H-2), 4.27
The gene corresponding to the mature lipase from G. thermo- (dd, J ¼ 12.9, 6.2 Hz, 1H, H-3), 4.11 (ddd, J ¼ 3.1 Hz, 1H, H-5),
catenulatus (GTL) was cloned into pT1 expression vector as 4.20–4.37 (m, 2H, H-6A, H-6B), 2.55 (bs, 1H, OH), 2.16 (s, 3H,
previously described.28 Cells carrying the recombinant plasmid CH3), 2.11 (s, 3H, CH3).
pT1GTL were grown at 30 ꢃC and over expression were induced
3,4-Di-O-acetyl-D-glucal (8). 1H-NMR (500 MHz, CDCl3) d:
ꢃ
by raising the temperature to 42 C for 20 h. The enzyme was 6.49 (dd, J ¼ 6.1 Hz, 1H, H-1), 5.41–5.50 (m, 1H, H-3), 5.22 (dd,
puried from E. coli crude extract by interfacial adsorption on J ¼ 9.0, 6.5 Hz, 1H, H-4), 4.81 (dd, J ¼ 5.9, 2.8 Hz, 1H, H-2), 3.98–
butyl-Sepharose as previously described.30 The lipase was des- 4.09 (m, 1H, H-5), 3.66–3.86 (m, 2H, H-6A, H-6B), 2.07–2.13 (2s,
orbed from the support adding 20 mL of 25 mM phosphate 6H, 2CH3).
This journal is © The Royal Society of Chemistry 2014
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Paper
10 M. Marciello, M. Filice and J. M. Palomo, Catal. Sci. Technol.,
2012, 2, 1531.
11 C. A. Godoy, B. de las Rivas, M. Filice, G. Fernandez-Lorente,
J. M. Guisan and J. M. Palomo, Process Biochem., 2010, 45,
534.
12 M. L. E. Gutarra, O. Romero, O. Abian, F. A. G. Torres,
D. M. G. Freire, A. M. Castro, J. M. Guisan and
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13 Z. Yang and Z.-L. Huang, Catal. Sci. Technol., 2012, 2, 1767.
14 P. Dominguez de Maria, Angew. Chem., Int. Ed., 2008, 47,
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15 M. Sandoval, A. Cortes, C. Civera, J. Trevino, E. Ferreras,
M. Vaultier, J. Berenguer, P. Lozano and M. J. Hernaiz, RSC
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Circular dichroism
Circular dichroism (CD) spectra of the different lipases with or
without ILs were recorded in a Chirascan spectropolarimeter
(Applied Photophysics) at 25(ꢁ1) ꢃC. Near-UV spectra were
recorded at wavelengths between 250 and 310 nm in a 1 cm
path-length cuvette, with 10 mM protein solutions in phosphate
buffered saline, pH 7.2 (PBS; bioMerieux).
Fluorescence spectroscopy
Fluorescence measurements were performed in a Varian Cary
Eclipse Fluorescence Spectrophotometer (Agilent Technologies)
monitoring the intrinsic tryptophan uorescence in the
different GTL variants, using an excitation wavelength of 280
nm, with excitation and emission bandwidths of 5 nm, and
recording uorescence emission spectra between 300 and 400
nm. All spectroscopic measurements were made in water.
16 A. Shariati, R. A. Sheldon, G.-J. Witkamp and C. J. Peters,
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18 R. A. Sheldon, Chem. Commun., 2008, 3352.
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Acknowledgements
¨
20 M. Klahn, G. S. Lim and P. Wu, Phys. Chem. Chem. Phys.,
This work has been sponsored by CSIC and the Spanish
Ministry of Economy and Competitiveness (Project EUIN2013-
51161). The authors also thank to Dr Ramiro Martinez from
Novozymes for the generous gi of lipases.
2011, 13, 18647.
21 S. P. M. Ventura, L. D. F. Santos, J. A. Saraiva and
J. A. P. Coutinho, Green Chem., 2012, 14, 1620.
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49122 | RSC Adv., 2014, 4, 49115–49122
This journal is © The Royal Society of Chemistry 2014