Improved synthesis of disaccharides with Escherichia coli β-galactosidase using bio-solvents derived from glycerol

Article abstract of DOI:10.1016/j.tet.2011.08.009

A noticeably increase in activity, keeping total regioselectivity was found in the synthetic behaviour of Escherichia coli β-galactosidase in glycerol-based solvents using a 1:7 molar ratio of donor (pNP-β-Gal): acceptor (GlcNAc). Yields of up to 97% of β(1→6) with different solvents were found. These reactions take place without noticeable hydrolytic activity and with total regioselectivity, representing a considerable improvement over the use of aqueous buffer or conventional organic solvents. There is a clear dependence of the catalytic results on the solvent structure, which is analysed in terms of polarity and hydrophobicity.

Full text of DOI:10.1016/j.tet.2011.08.009

Tetrahedron 67 (2011) 7708e7712
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Improved synthesis of disaccharides with Escherichia coli
b-galactosidase using
bio-solvents derived from glycerol
a
ꢀ
b
c
a
María P eꢀ rez-S aꢀ nchez , Alvaro Cort eꢀ s Cabrera , H eꢀ ctor García-Martín , J. Vicent Sinisterra ,
c
a,
*
Jos eꢀ I. García , María J. Hern aꢀ iz
Department of Pharmaceutical and Organic Chemistry, Faculty of Pharmacy, Complutense University of Madrid, Campus de Moncloa, 30100 Madrid, Spain
Unidad de Bioinform aꢀ tica, Centro de Biología Molecular ‘Severo Ochoa’ (CBMSO), CSIC, Universidad Aut oꢀ noma de Madrid (UAM), C/Nicol aꢀ s Cabrera 1, 28049 Madrid, Spain
Instituto de Ciencia de Materiales de Arag oꢀ n, CSIC-Universidad de Zaragoza, C/Pedro Cerbuna 12, E-50009 Zaragoza, Spain
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 June 2011
Received in revised form 28 July 2011
Accepted 3 August 2011
Available online 7 August 2011
A noticeably increase in activity, keeping total regioselectivity was found in the synthetic behaviour of
Escherichia coli
b
-galactosidase in glycerol-based solvents using a 1:7 molar ratio of donor (pNP-
b-Gal):
acceptor (GlcNAc). Yields of up to 97% of
b
(1/6) with different solvents were found. These reactions take
place without noticeable hydrolytic activity and with total regioselectivity, representing a considerable
improvement over the use of aqueous buffer or conventional organic solvents. There is a clear de-
pendence of the catalytic results on the solvent structure, which is analysed in terms of polarity and
hydrophobicity.
Keywords:
Biocatalysis
Solvent effects
Ó 2011 Published by Elsevier Ltd.
Glycerol-derived solvents
Molecular dynamics
Transglycosylation
1. Introduction
some interesting features, such as their ready availability from
biodiesel production, easy derivatisation and tuneable properties.
11
Biocatalysis and solvents play an important role in green pro-
cesses, since they provide a valuable alternative to classic organic
chemistry.1 First, enzymes offer suitable tools for industrial re-
actions, which can be carried out under milder conditions, without
using of heavy metals and with a great control over chemo-, regio-
and stereoselectivity using appropriate enzymes. Second, there are
numerous potential advantages in employing enzymes in organic
solvents, such as increased solubility of nonpolar substrates, shift-
ing of thermodynamic equilibria to favour synthesis over hydroly-
sis, decrease of water-dependent side reactions, enhanced thermal
stability of enzymes and elimination of microbial contamination,
Oligosaccharides are involved in a wide range of biological
processes including bacterial and viral infection, cancer metastasis,
the blood-clotting cascade and many other crucial intercellular
e4
12e15
recognition events.
As the understanding of these biological
functions increases, the need for practical synthetic procedures of
oligosaccharides in large quantities has become a major subject.
Organic chemical methods for obtaining them have been devel-
16e18
oped,
but they involve several elaborate protection and
deprotection procedures. Glycosyl hydrolases (glycosidases) can be
used to synthesize oligosaccharides in a kinetically controlled re-
action, where a glycosyl donor is used to transfer its glycosyl resi-
due to a sugar acceptor present in the reaction medium.
5
among others. On the other hand, the presence of organic solvents
in enzymatic reactions can alter the activity and specificity of the
enzyme. There are numerous examples in the literature wherein
solvents physical properties, such as dielectric constant, dipole
moment and hydrophobicity are related to various effects on en-
In this work, we present a study of the influence of the use of
green solvents derived from glycerol on the activity and selectivity
of
b-galactosidase of Escherichia coli in the enzymatic synthesis of
disaccharides.
6
e9
zyme activity, specificity and enantioselectivity.
Among the
different families of the so-called bio-solvents, organic solvents
derived from renewable sources are receiving an increasing at-
tention. In particular, glycerol and glycerol derivatives present
2
. Results and discussions
10
Novel carbohydrate compounds can effectively be synthesized
using enzymatic methods. Glycosidases can be used to synthesize
oligosaccharides in a kinetically controlled reaction, where a gly-
cosyl donor is used to transfer its glycosyl residue to a sugar
*
Corresponding author. E-mail address: mjhernai@farm.ucm.es (M.J. Hern aꢀ iz).
0
040-4020/$ e see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.tet.2011.08.009

M. P ꢀe rez-S aꢀ nchez et al. / Tetrahedron 67 (2011) 7708e7712
OH
7709
acceptor present in the reaction medium.19 In this sense,
b-galac-
OH
OH
tosidase from E. coli has been proven to be a valuable biocatalyst for
galactosyl transfer from suitable donors onto a N-acetylglucos-
amine as acceptor.20 The regioselectivity of enzymatic trans-
galactosidation depends on the source of the glycosidase used. The
O
O
HO
HO
+
HO
O
OH
OH
NHAc
NO2
synthesis of Gal-
employing -galactosidase from E. coli, A. oryzae and Bacillus cir-
culas have been previously reported.
b-(1/6)-GlcNac or/and Gal-b-(1/4)-GlcNac
b
pNF- -Gal
GlcNAc
2
0e22
-
galactosidase
In a recent work we have shown that the use of co-solvents
derived from glycerol in the reaction medium is able to direct the
regioselectivity of the transglycosydation reaction, allowing to
from E .coli
OH
HO
OH
obtain only the b(1/6) regioisomer, in the case of the B. circulans
O
3
b
-galactosidase.2
In the present work, we have extended the use of this kind of
glycerol-based solvents to the study of oligosaccharides synthesis
catalysed by -galactosidases from E. coli. Two commercial solvents
glycerol and 2,2,2-trifluoroethanol, TFE) have also been included
for comparative purposes (Fig. 1).
O
OH
HO
HO
O
b
OH
(
NHAc
-
(1-6 )
Scheme 1. General scheme of transglycosylation reaction catalysed by
from E. coli.
b-galactosidase
HO
OH
F3C
OH
OH
Glycerol
Table 1
Commercial TFE
Transglycosylation yields obtained with
b-galactosidase from E. coli using Tris/HCl
pH 7.3 and 2 M of glycerol-based solvent in the same buffer
O
O
O
O
a
N
T
b
Solvent
log P
0.00
3
E
pNP-Gal
Gal
Galb(1/6)
GlcNAc
OH
OH
1
2
Only buffer
Glycerol
TFE
1
2
3
4
5
6
78.50
42.50
27.10
13.00
7.00
1.00
0.81
0.90
0.61
0.48
0.44
0.59
0.70
0.55
0.46
0.45
0.15
0.70
0.07
8
63
65
100
96
73
100
7
15
100
100
100
81
24
37
35
d
d
d
d
d
d
d
d
d
d
58
68
d
d
d
4
27
d
93
85
d
d
d
19
15
À1.30
0.71
À0.60
0.14
0.27
1.14
1.42
1.71
1.93
2.07
2.48
2.56
3.77
O
O
O
O
O
O
O
CF3
CF3
OH
3
OH
4
6.60
F3C
CF3
F3C
O
O
12.00
14.60
13.70
5.20
5.60
4.70
OH
5
O
6
7
8
9
O
O
O
O
O
O
OH
8
OH
7
10
10.40
8.60
11
27
F3C
CF3
a
Dielectric constant of the pure solvent.
C
O
C
b
N
F
F
2
E
T
of the pure solvent.
O
2
OH
10
9
However, solvents 5 and 6 lead to the best yields for the synthesis of
Gal- -(1/6)-GlcNac (93% and 85%, respectively), with no traces of
hydrolysis product. This means an important increase of activity
according to natural behaviour of this enzyme in buffer 10 mM Tris/
HCl buffer at pH 7.3, but also an improvement in the selectivity of
the reaction (no hydrolysis product), keeping total regioselectivity
F
F
b
2
2
C
C
CF3
F3C
C
O
O
C
F
F
2
2
OH
11
Fig. 1. Structure of different bio-solvents from glycerol employed in transglycosylation
reaction with -galactosidase from E. coli.
to the
b-(1/6) isomer. Solvents 5 and 6 have two trifluoromethyl
b
(
CF ) groups in their structure, which seems to have a beneficial
3
effect to the transglycosylation reaction. The presence of only one of
these groups (solvent 4) has no effect at all. On the other hand, an
increase in the length of fluorinated chains (solvents 10 and 11) was
not beneficial for the activity in transglycosylation, either. This re-
sult is difficult to rationalize in terms of solvent polarity only, given
the similar values displayed by 5, 6, 10 and 11. A factor that could
have influence would be solubility in water. Solvents with long
fluorinated chains tend to be immiscible with water, so the pres-
ence of two separated liquid phases (buffer and fluorinated solvent)
cannot be discarded in the case of 10 and 11, which would explain
the different behaviour obtained.
Transglycosylation reaction catalysed by a commercial prepa-
ration of the -galactosidase from E. coli was carried out following
the general procedure described in the Experimental section and
monitored by HPLC (Scheme 1). The concentration of bio-solvent
was fixed to 2 M in the mixture with the Tris/HCl buffer. The re-
sults obtained for the transglycosylation reaction with the b-ga-
lactosidase from E. coli in the presence of different solvents derived
from glycerol are summarized in Table 1.
The use of only buffer as the reaction medium leads to a good
conversion of the substrate (92%), but there is a considerable
amount of hydrolytic product (galactose), so that the total yield in
disaccharide is a moderate 68%. The use of either glycerol or TFE
results in the total loss of transglycosylation activity, and only ca.
b
In order to check the influence of the co-solvent proportion on
the enzymatic activity, different concentrations of 5 (0.5, 1 and 2 M)
and 6 (0.5,1, 2 and 3 M) were also tested, and the results obtained in
these experiments are shown in Table 2.
3
5% hydrolysis product is observed. The total activity shows
therefore a marked decrease.
Most of glycerol derivatives (1, 2, 4, 7, 8 and 9) give rise to a total
loss of enzymatic activity when used as co-solvents in this reaction.
As can be seen, in the case of solvent 5 the best yield in di-
saccharide was obtained with a concentration 1 M in glycerol-
based solvent (97% yield with total substrate conversion).

7710
M. P ꢀe rez-S aꢀ nchez et al. / Tetrahedron 67 (2011) 7708e7712
Table 2
glutamic residue and O6 of NAG (N-acetylglucosamine) was mea-
Transglycosylation yields obtained with b-galactosidase from E. coli using Tris/HCl
sured during the simulation time, and it was found to be shorter
and have fewer fluctuations in the case of the mixture, mainly at the
beginning of the simulation (Fig. 3). This difference could explain
the product enhancement obtained through a better disposition in
the active site of NAG that may be caused by a favourable NAG-
solvent interaction.
10 mM pH 7.3 and glycerol-based solvents 5 and 6 in the same buffer at different
concentrations
Solvent (concn)
pNP-Gal
Gal
Galb(1/6)GlcNAc
5
5
5
6
6
6
6
(0.5 M)
(1.0 M)
(2.0 M)
(0.5 M)
(1.0 M)
(2.0 M)
(3.0 M)
76
d
d
3
d
d
3
24
97
93
25
57
84
d
7
75
40
16
100
Table 3
d
Distance from C1 of modified residue Glu-537 to O6 of NAG
d
Solvent
concn)
(2.0 M)
Water
Average
distance (A)
Standard
deviation (A)
Maximum
distance (A)
ꢁ
ꢁ
ꢁ
(
5
4.33
4.50
0.56
1.01
6.9
8.7
However, with lower concentrations (0.5 M), the transglycosylation
yield downs to ca. 25%. In the case of solvent 6, the maximum yield
in the transglycosylation reaction is obtained with a concentration
of green solvent of 2 M (84%), and this yield systematically de-
creases with solvent concentration. On the other hand, no activity
was observed when the concentrations of this green solvent were
increased up to 3 M, indicating the necessity of working at the
adequate co-solvent concentration to obtain optimal results.
To get some insights on the origin of the activity boost observed
in some of the glycerol-derived solvents tested, a molecular dy-
namics study was carried out, comparing pure water with the
5
ewater mixture, as solvating media of the b-galactosidase of E.
coli. Stability of the enzymeesubstrate complex was analysed in
both media measuring the root mean square deviation (RMSD) of
a
carbons through the simulation time. Both solvated systems have
similar values of RMSD and radius of gyration, indicating that there
is no difference in the protein flexibility (see Supplementary data,
Fig. S1).
To investigate any possible displacement or motion that could
explain the enhancement of the reaction, the RMS fluctuations
(
RMSF) for C-a atoms were computed (Supplementary data,
Fig. S2). The results show that some parts of the protein may
have more flexibility in the water system (Fig. 2). However, the
surrounding environment of the active site remains with similar
flexibility in both systems. The superimposition of the average
structures of water-only and wateresolvent 5 complexes on the
original structure shows little displacement on active site residues
confirming the absence of difference between systems.
Fig. 3. Distance between C1 carbon of modified residue Glu-537 and O6 oxygen of
NAG.
3. Conclusions
To sum up, an enzymatic activity boost, keeping total regiose-
lectivity, was found in the synthetic behaviour of E. coli
sidase in glycerol-based solvents. Yields of up to 97% of Gal-
1/6)-GlcNac were obtained with total substrate conversion.
b-galacto-
b
-
(
These reactions take place without noticeable hydrolytic activity
and with total regioselectivity, representing a considerable im-
provement over the use of aqueous buffer or conventional organic
solvents. These results reinforce the concept of driving enzymatic
syntheses to the desired product simply by adjusting the reaction
medium with small amounts of green solvents. Molecular model-
ling studies point to a better disposition of the substrate in the
active centre, with origin in solvation effects, as a possible expla-
nation for the behaviour observed.
4
4
. Experimental section
.1. General
Fig. 2. LacZ monomer of E. coli. In yellow, residues 263e273, which present a high
RMSF in water. In red, residues 714e722, which present a moderate RMSF, in water too.
Commercially available b-galactosidase from E. coli and other
chemicals were obtained from Sigma.
Since structural differences were not found between simula-
tions, a detailed analysis of ligand disposition was done (Table 3).
Distance from catalytic glutamic residues to the acceptor is usually
found to be correlated with regioselectivity and activity changes.
In the present work the distance between C1 of the modified
UVevis spectra were recorded on a UV-2401 PC Shimadzu. HPLC
Agilent 1100 with UVevis detector using Mediterraneasea 18
15 cmÂ0.46 5 mm column (Teknokroma) with water/acetonitrile
(75:25) as a mobile phase at a flow of 0.7 mL/min. HPLC Jasco with
2
4
light scattering detector using Lichrosorb NH
2
5 mm 25Â0.47

M. P ꢀe rez-S aꢀ nchez et al. / Tetrahedron 67 (2011) 7708e7712
7711
column (Teknokroma) with acetonitrile/water (80:20) as a mobile
phase at a flow of 1.0 mL/min. NMR spectra were recorded on
Bruker 500 MHz spectrometers. The structure of the enzymatically
synthesized disaccharides was assigned by protoneproton shift
correlations, carboneproton shift correlation.
phenol-b-galactopiranose was initially docked into the active cen-
tre of the enzyme to determine the position of the molecule to
simulate the glycosyleenzyme intermediate. We selected the best
pose according to geometrical (close enough to the nucleophilic
2
6
residue Glu-537) and energetic criteria (favourable energy) and
set up a covalent link between the nucleophilic glutamic residue
and the galactose. In the second docking, the glycosyleenzyme was
used to perform a docking with the NAG. The best solution was
selected taking only into account energetic criteria, and it was used
as the starting structure for the subsequent molecular dynamics
studies. Docking procedures were carried out with Autodock 4
4
.2. Solvents
Glycerol-based solvents were synthesized using the same pro-
11
cedures previously described. The alcoholysis of either epichlo-
rohydrin (symmetrical derivatives) or the appropriate glycidol
ether (unsymmetrical derivatives) gave the corresponding 1,3-
dialkoxy-2-propanols. O-methylation of some selected targets
leads to the corresponding 1,2,3-trialkoxypropanes. All the solvents
were purified by distillation and used in high pure form. The
complete listing of the solvents used in this work is shown in Fig. 1.
2
7
software, using a 60Â60Â60 grid points box around nucleophilic
glutamic residue (Glu-537), to embrace all residues implicated in
ꢁ
the mechanism, and with a grid spacing of 0.375 A. A Lamarckian
genetic algorithm with the standard parameters was selected.
4.5.3. Solvent parameterisation. The glycerol derivative (5) used for
4
.3. Enzyme activity assay
the simulation was parameterised for the GROMOS 96 43a1 force
field as it was described in our recent work. The mixture of glycerol
derivate and water was previously equilibrated 300 ps at 298 K and
1 atm before solvating the system.
Protein concentration was determined by the Bradford25
method using bovine serum albumin as the standard. Hydrolytic
activity was determined spectrophotometrically by quantification
of pNP (p-nitrophenol) liberated from correspondent p-NP-
p-nitrophenyl- -galactopyranoside) in Tris/HCl buffer 10 mM pH
.3. A sample of enzyme solution (20 L) was added to 80 L of Tris/
-Gal. The reaction mixture was
incubated for 3 min at 37 C. Absorbance was measured at 410 nm.
One enzymatic unit was defined as the amount of protein that
b
-Gal
4.5.4. Molecular dynamics. Molecular dynamics simulations were
set up with the best solution obtained from docking and the gly-
cosyleenzyme intermediate. Parameters adopted in GROMOS 96
43a1 were used for the protein, while the parameters required for
the galactoseeglutamic residue were derived in a consistent
(
7
b-D
m
m
HCl buffer to containing 5 mM pNP-
b
ꢀ
2
8
manner with the force field. NAG parameters were generated by
2
9
hydrolyses 1
m
mol of pNP-
b
-Gal per minute under the conditions
the Dundee PRODRG 2.5 Server. For all systems, an initial mini-
misation was performed with 500 steps of SD followed by 1500
steps of PolakeRibiere Conjugate Gradients (CG). The minimized
complexes were solvated in different cubic boxes, fulfilling the re-
quirement of a minimum distance of 1.2 nm between any atom of
the complex and the faces of the box. In one case, the monomer was
solvated with SPC water molecules, to reproduce the buffer con-
ditions, and in the other, a mixture of glycerol derivate and SPC
water molecules was used, with the aim to reproduce the condi-
tions for the regioselectivity change. Following, each box was
minimized with 500 steps of SD and 3000 steps of CG and equili-
brated 100 ps at 298 K and 1 atm (NPT conditions). Finally, a 5 ns
production simulation for the water system was carried out while
a 20 ns production simulation for the mixture was performed. All
MD simulations were set up with GROMACS software suite
described before.
4
.4. General procedure for transglycosylation reactions using
b-galactosidase from E. coli
A solution of 100 mg (0.17 M) p-NP-b-D-Gal (donor) and 550 mg
(
1.25 M) of N-acetylglucosamine (acceptor) in 1 mL of glycerol-
ꢀ
based solvent (2 M)-buffer mixture was pre-equilibrated to 30 C.
Afterwards, 155 mmol/min (U) of b-galactosidase from E. coli were
added to the reaction mixture. Reaction was monitored by HPLC
UVevis and final products obtained by HPLC with a light scattering
detector (ELSD) were analysed. The reaction was stopped by heat-
ꢀ
ing to 100 C for 5 min. Final reaction mixture obtained was loaded
on activated carbon (50% m/m) and Celite (50% m/m) column
3
0
(
50 cmÂ2 cm, 25 cm column height) eluted with 3 volumes of
milliQ water, 3 volumes of 5% ethanol (in water) and 3 volumes of
5% ethanol (in water). Disaccharide enriched fractions were de-
(v4.0.7).
1
Acknowledgements
termined by TLC on silica gel 60 (Merck, Darmstadt, Germany), with
isopropanol/nitromethane/water (10:9:2) as eluent with detection
This work was supported by two research projects of the
by spraying the plates with 10% aq H
2
SO
4
in methanol and heating.
Spanish MICINN (Ministerio de Ciencia e Innovaci oꢀ n de Espa n~ a)
Ten, these fractions were collected in 15% ethanol; they were
pooled and analysed by HPLC-ELSD as described above. H NMR
CTQ2009-11801 and CTQ2008-05138, and one European project
(FP-62003-NMP-SMF-3, proposal 011774-2).
1
spectrum (D
2
O) was recorded to purified disaccharide on a Bruker
5
4
4
00 MHz spectrometer.
Supplementary data
.5. Molecular Modelling
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tet.2011.08.009.
.5.1. Protein model. The X-ray structure of the b-galactosidase of E.
coli was obtained from the Brookhaven Protein Data Bank (PDB ID:
PX3). The enzyme is composed by four identical units assembled
in a tetramer. To simplify the study the monomer A was selected.
The structure was relaxed with 2000 steps of Conjugative Gradient
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