Optimised N-acetyl-D-lactosamine synthesis using Thermus thermophilus β-galactosidase in bio-solvents

Article abstract of DOI:10.1016/j.tet.2012.11.053

Synthesis of N-acetyl-D-lactosamine (Gal-β[1→4]GlcNAc, LacNAc) catalyzed by β-galactosidase from Thermus thermophilus (TTP0042) is affected by side reactions that give as result very low yields (about 20%) of LAcNAc when the reaction is performed in buffer. The process is improved (up to 91% of disaccharide yield) when the reaction takes place in the presence of solvents from biomass (bio-solvents) at 2.0 M concentration. Most of the solvents tested increased the LacNAc synthesis and reduced the undesired side reactions. In order to understand the possible effects of these solvents over the enzyme regioselectivity, we developed a conformational study of the enzyme structure in the presence of a selected bio-solvent by circular dichroism and fluorescence. According to this study, we were able to conclude that the presence of bio-solvents in the reaction media modifies the enzyme secondary and tertiary structure and this may be the cause of the regioselectivity changes observed in the transglycosylation reaction.

Full text of DOI:10.1016/j.tet.2012.11.053

Tetrahedron 69 (2013) 1148e1152
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Optimised N-acetyl-D-lactosamine synthesis using Thermus
thermophilus
b-galactosidase in bio-solvents
a
b
c
b
a,
*
ꢀ
ꢀ
Manuel Sandoval , Concepcion Civera , Jose Berenguer , Francisco García-Blanco , María J. Hernaiz
a Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, Complutense University of Madrid, Campus de Moncloa, 28040 Madrid, Spain
b Department of Physical Chemistry II, Faculty of Pharmacy, Complutense University of Madrid, Campus de Moncloa, 28040 Madrid, Spain
c
ꢀ
ꢀ
Centro de Biología Molecular “Severo Ochoa” (CBMSO), CSIC, Universidad Autonoma de Madrid (UAM), C/Nicolas Cabrera 1, 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of N-acetyl-D-lactosamine (Gal-b[1/4]GlcNAc, LacNAc) catalyzed by b-galactosidase from
Received 25 September 2012
Received in revised form 9 November 2012
Accepted 15 November 2012
Available online 21 November 2012
Thermus thermophilus (TTP0042) is affected by side reactions that give as result very low yields (about
20%) of LAcNAc when the reaction is performed in buffer. The process is improved (up to 91% of di-
saccharide yield) when the reaction takes place in the presence of solvents from biomass (bio-solvents)
at 2.0 M concentration. Most of the solvents tested increased the LacNAc synthesis and reduced the
undesired side reactions. In order to understand the possible effects of these solvents over the enzyme
regioselectivity, we developed a conformational study of the enzyme structure in the presence of a se-
lected bio-solvent by circular dichroism and fluorescence. According to this study, we were able to
conclude that the presence of bio-solvents in the reaction media modifies the enzyme secondary and
tertiary structure and this may be the cause of the regioselectivity changes observed in the trans-
glycosylation reaction.
Keywords:
Bio-solvents
b-Galactosidase
N-Acetyl- -lactosamine
D
Thermus thermophilus
Transglycosylation
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
galactopyranoside (pNP-b-Gal) as donor and N-acetyl-D-glucos-
amine (GlcNAc) as acceptor (Scheme 1),17 but a side reaction is also
involved during this synthesis, producing some amounts of self-
Carbohydrates are main molecules related in cell to cell recog-
nition processes, they appear as glycoproteins, glycolipids or pro-
teoglycans anchored in the cell membrane.1 Due to its strategical
situation, oligosaccharides are involved in many biological pro-
cesses, such as: embryogenesis, neuronal proliferation and
apoptosis.2e4 As the understanding of these biological functions
increases, the need for practical synthetic procedures of oligosac-
charides in large quantities has become a major issue.
condensation of the donor (Gal
b[1/3]Gal-b-pNP and Galb[1/6]
Gal-b
-pNP).18
Synthesis of disaccharides and glycoconjugates can be per-
formed by organic chemical methods,5e7 but these procedures are
characterized by several protection and deprotection steps. For that
reason the synthesis of these carbohydrates is often achieved by
enzymatic methods, using: glycosyltransferases, glycosidases and
glycosynthases as biocatalysts.8e11
b-Galactosidase (b-D-galactoside galactohydrolase, EC 3.2.1.23)
catalyses the hydrolysis of the
b-galactoside linkages of di-
saccharides into their monomers but also the transgalactosylation
reaction to produce galactooligosaccharides.12e14 Thermus thermo-
Scheme 1. Synthesis of N-acetyl-D-lactosamine (LacNAc) catalyzed by
TTP0042 from Thermus thermophilus.
b-galactosidase
philus
which can synthesize N-acetyl-
GlcNAc) via transglycosylation
b
-galactosidase (TTP0042),15,16 is a thermophilic enzyme,
D-lactosamine (LacNAc, Gal
b[1/4]
using p-nitrophenyl-
b-D-
There has been reported, that many enzymes can maintain their
activity in a variety of organic solvents.19 Recently some
-galac-
b
tosidases have been tested in presence of environmental friendly
* Corresponding author. Tel.: þ34 91139 41821; fax: þ34 9139 41822; e-mail
solvents made from glycerol,20e22 solvents from biomass (bio-
address: mjhernai@farm.ucm.es (M.J. Hernaiz).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.11.053

M. Sandoval et al. / Tetrahedron 69 (2013) 1148e1152
1149
solvents)21e24 and ionic liquids (ILs).21,22,25 As a result of these
studies, it was found that the use of these solvents decreases
unfavourable side reactions and shifts the equilibrium towards the
product side, therefore minimising hydrolysis.
In the present work, we have evaluated the effect of different
bio-solvents (solvents made from glycerol and from biomass) in the
reaction media for transglycosylation using TTP0042 enzyme as
a catalyst. To understand the effect of these liquids over the syn-
thetic behaviour of the enzyme, we performed a conformational
study by circular dichroism and fluorescence techniques between
TTP0042 and selected bio-solvents.
As can be seen in Table 1, most of the solvents shifted the re-
action equilibrium towards synthesis of LacNAc with good yields
(>65%) respect the reaction performed in buffer (17%). This result
shows an important effect of the solvent in the reaction media and
also confirms the sensibility of this enzyme to different solvents.
Unfortunately, not all the solvents allow the reactions, and the
enzyme seems to be inactive from the beginning of the reaction,
these solvents: S3, S7 and S11 behave like inhibitors or denaturants
of the enzyme. Solvents: S3, S5 and S13 did not complete the re-
action and conversion yields were about 55e50% (data not shown).
All other solvents present high conversion yields (about 95e100%,
data not shown). There is not a clear relationship between solvents
structures and the regioselectivity change found in the reactions
performed. The three groups of solvents tested display good results
with most of their solvents. So, there is a sensibility of the enzyme
for the chemical environment, but also, the enzyme seems to be
active at very different values of LogP and also polarities. As im-
portant results, the highest values of LacNAc yields were obtained
under biphasic conditions and moderate-low polarities of solvents
(S9, S10 and S11, Table 1).
2. Results and discussion
2.1. Effect of bio-solvents over LacNAc synthesis
The effect of three different groups of solvents (Scheme 2) was
screened in order to evaluate their effect over TTP0042 b-galacto-
sidase during transglycosylation reaction; 1-glycerol based solvents
(cyclic structures, S1, S2 and S3); 2-glycerol based solvents (open
chain, [S4eS12]); 3-N,N-dymethyl amide based solvents (S13eS15).
Main features of these solvents (density, Log P and solubility 2 M)
and reaction yields are summarized in Table 1.
2.2. Effect of concentration of solvents in the synthesis of
LAcNAc
In order to check the influence of the co-solvent proportion on
the enzymatic activity, different concentrations of S2, S4, S9, S12,
S13 and S15 (0.25, 0.5, 1.0, 1.5 and 2 M) were also tested, and the
results obtained in these experiments are shown in Fig. 1. Taking
into account the solvent effects in the disaccharide synthesis, we
selected the following solvents: S2, S4, S9, S12, S13 and S15, in
order to evaluate their effect in the transglycosylation reactions at
different concentrations of each solvent. These solvents are repre-
sentative of the three kinds of solvents screened and also display
different reactions systems, biphasic and monophasic.
Most of the solvents tested, presented their higher effect about
2 M of solvent, there was no difference related to the nature of the
system (biphasic and homogeneous systems). The addition of bio-
solvent to the mixture of reaction causes a change in the enzyme
traditional regioselectivity. Some solvents (S4, S9, S12 and S13)
presented good disaccharide yields when 1.50 M concentrations
were used. This effect was previously found for this enzyme by
using ionic liquids as co-solvents and was explained as a confor-
mational change in the secondary and tertiary structure of the
enzyme due to the presence of co-solvents.25 In order, to confirm
this hypothesis for bio-solvents, we decided to analyse conforma-
Scheme 2. Molecular structure of solvents used in this study.
tional changes in the a-helix from the secondary structure of the
Table 1
Transglycosylation yields (%) catalyzed by TTP0042 in presence of 2 M of solvents and 50 mM of sodium phosphate buffer pH 6.0
Medium
Solvent
density
(g mLꢀ1
Log P
System
composition
% Self
condensated
% Self
condensated
b(1/6) Galb(1/6)GalpNP
% Hydrolysis
(galactose)
% LacNAc
% Galb(1/6)GlcNAc
)
Galb(1/3)GalpNP
Buffer
S1
S2
S3
S4
S5
S6
S7
S8
1.00
1.24
1.41
1.06
1.07
0.94
0.91
1.12
1.36
1.27
0.90
0.89
0.89
1.06
1.15
0.91
0.00
ꢀ0.057
ꢀ0.024
0.030
ꢀ0.60
0.14
0.27
1.14
1.42
1.71
Monophasic
Monophasic
Monophasic
Monophasic
Monophasic
Monophasic
Monophasic
Biphasic
Biphasic
Biphasic
Biphasic
Biphasic
73
33
0
0
5
22
0
0
0
1
0
0
1
7
35
0
10
5
6
0
1
0
0
0
0
3
0
0
2
2
0
0
0
7
0
0
4
0
0
0
3
1
4
0
0
3
0
4
17
55
77
0
75
78
0
0
0
17
0
15
0
0
0
6
4
22
0
0
91
91
74
0
94
80
65
68
S9
S10
S11
S12
S13
S14
S15
1.93
2.07
2.48
Biphasic
3
8
0
28
ꢀ0.69
Monophasic
Monophasic
Biphasic
1.41
1.42

1150
M. Sandoval et al. / Tetrahedron 69 (2013) 1148e1152
100
90
80
70
60
50
40
30
20
10
0
of the enzyme in the presence of solvent S4 at 2 M shows a signif-
icant change in ellipticity (ꢀ3600 deg cm2 dmolꢀ1) at 209 nm.
This result shows an effect of the solvent over the enzyme
structure. For this experiment, the enzyme displayed two maxi-
mum values of negative absorption at: 217 nm and 209 nm. These
CD bands were used as indicators of changes in the secondary
structure of the protein. As shown in Fig. 3, the protein decreased
ellipticity when the concentration of S4 is increased. These mea-
surements confirm the solvent effect in the enzyme secondary
structure.
S2
S4
S9
S12
S13
This result suggests that the enzyme modifies its native struc-
ture during the transglycosylation reaction due to the addition of
bio-solvents. This structural modification of the secondary struc-
ture of the enzyme could be responsible of the regioselectivity
change found in the disaccharide synthesis reactions previously
performed.
0,00
0,50
1,00
1,50
2,00
Concentration of bio-solvent (M)
Fig. 1. Synthesis of LacNAc at different solvent concentrations.
The interaction between solvent and enzyme could be similar to
the found previously when TTP0042 have proven to be sensitive to
the addition of ionic liquids, these solvents increase the enzyme
regioselectivity and improve the disaccharide yields when they
have been employed. This phenomenon also has been explained as
modification of the enzyme secondary and tertiary structure by the
addition of those solvents that make the enzyme more flexible
during synthesis conditions.32
protein by circular dichroism26e28 and in the tertiary structure by
fluorescence emission due to tryptophan excitation.29,30
2.3. Conformational effects of solvents over enzyme second-
ary structure
In order to measure the effect of water soluble solvents in the
secondary structure of the enzyme, solvents: S2, S4, S13 and S14
were used. These solvents were selected because of their important
effect in the enzymatic synthesis of LacNAc and also for their sol-
ubility in water at the desired concentration (2.0 M). For this, we
analysed their CD spectra at 2.0 M concentrations and their effect in
the recording (noise) for the spectra.
According to this result, only S4 was found able to use it in the
CD study for solvent effect on the enzyme secondary structure,
because it presented a null observed ellipticity at 2 M concentra-
tion, while S2, S13 and S14 exhibit important changes in the CD
spectra and also incremented the noise of the recorded (data not
shown). According to this result, we measured the effect of differ-
ent concentrations of S4 over the enzyme secondary structure at
60 ꢁC. The final concentrations were: 0.00, 0.25, 0.50, 1.00, 1.50 and
2.00 M. These conditions were selected in order to use the same
concentrations and temperature previously developed during the
transglycosylation study. As a result of this experiment, we find
a modification of the enzyme secondary structure due to the ad-
ditions of S4 (Fig. 2).
2.4. Conformational effects of solvents over enzyme tertiary
structure
The effect of the S4 solvent over the enzyme fluorescence
spectrum was investigated. For this experiment, we recorded the
emission spectra for the enzyme in buffer solution at 60 ꢁC and
then, several additions of S4 were carried out and the respective
fluorescence spectra of them were done.
As a result of this experiment, we noticed that the enzyme was
shifting the emission spectra to the blue, from 334 nm as maximum
emission wavelength in buffer solution up to 329 nm in 2 M solu-
tion of S4 (Fig. 4). This result reveals that the tertiary structure of
the enzyme was modified affecting the tryptophan exposition to-
wards more hydrophobic environment and producing important
changes over its native emission spectra in fluorescence. The dis-
placement of the maximum emission wavelength in the fluores-
cence of this enzyme by using S4 as co-solvent was confirmed over
1.00 M concentrations (Fig. 5), at lower concentrations no differ-
ence was seen. It supposes an important modification of the ter-
tiary structure of the enzyme under our reaction conditions (2 M of
S4), which could be related to the change in the enzyme
At the beginning of the procedure (buffer solution), the enzyme
displays a typical a
-helix spectrum.31 The CD spectra for the en-
zyme in buffer solution exhibit a profile with a minimum at 209 nm
with a ꢀ6700 deg cm2 dmolꢀ1 ellipticity. In contrast, the CD spectra
8000
-2000
Buffer
0.25 M
1.50 M
0.50 M
2.00 M
6000
4000
2000
0
209 nm
1.00 M
-2500
217 nm
-3000
-3500
-4000
-4500
-5000
-5500
-6000
-2000
-4000
-6000
-8000
0,00
0,50
1,00
1,50
2,00
200
210
220
230
240
250
Concentration of S4 (M)
Wavelength (nm)
Fig. 2. CD spectra for TTP0042 at different concentrations of S4.
Fig. 3. Effect of S4 concentration over negative ellipticity bands at 209 nm and 217 nm.

M. Sandoval et al. / Tetrahedron 69 (2013) 1148e1152
1151
affects the a-helix of the secondary structure of the protein and also
333 nm
(1.00 M)
329 nm
(2.00 M)
affects the tryptophan exposition of the tertiary structure of the
enzyme. These conformational changes over secondary and tertiary
structure of the enzyme may be the responsible for the modifica-
tion of the enzyme regioselectivity found in the transglycosylation
reactions.
The isolation of the target molecule LacNAc from the reaction
mixture proved to be feasible using a simple purification procedure.
These results have opened up promising possibilities to follow in
trying to make the enzymatic synthesis of regioselective LacNAc
industrially feasible. However, further research is clearly required
to explore the process implications of using bio-solvents.
334 nm
(0.00 M)
1.00
0.90
0.80
1
0.8
0.6
0.4
0.2
0
0.70
320
330
340
350
4. Experimental section
4.1. General
0.00 M
1.00 M
2.00 M
p-Nitrophenol (pNP), pNP-b-Gal and GlcNAc were purchased
from Sigma Aldrich. Solvents from glycerol were kindly donated by
310
360
410
Wavelength (nm)
460
ꢀ
Prof. Jose I. García, solvents from Bio-mass were a gift from Cognis
IP Management GmbH, now part of BASF. All other chemicals were
from analytical grade. UVevisible spectra were recorded on a UV-
2401 PC Shimadzu. HPLC Jasco using NH2P50-4E amino column
(Asahipak, Japan) with acetonitrile/water (80:20) as a mobile phase
at a flow of 0.8 mL/min. Chromatograms were recorded using an
UVevis, circular dichroism (CD) and evaporative light scattering
(LS) detectors. NMR spectra were performed on Bruker Avance
700 MHz spectrometer.
Fig. 4. Fluorescence emission spectra at 65 ꢁC for TTP0042 at different concentrations
of S4.
335
334
333
332
331
330
329
328
4.2. Enzyme activity and protein purification
Recombinant TTP0042 enzyme was obtained as describe be-
fore.17,32 Protein concentration was determined by Bradford
method,33 using BSA as standard. Hydrolytic activity was de-
termined by quantification of pNP liberated from 5 mM solution of
pNP-
using continuous method.17,34 One enzyme unit was defined as the
amount of protein that releases 1 mol of pNP- -Gal per minute
b
-Gal in phosphate buffer 50 mM pH 7.0 at 80 ꢁC and 410 nm
0
0,5
1
1,5
2
m
b
Concentration of S4 (M)
under the conditions previously described.
Fig. 5. Maximum emission fluorescence wavelength of TTP0042 at different concen-
trations of S4.
4.3. Disaccharide synthesis
Transglycosylation reactions were carried out in 1.0 mL of so-
lution (2 M solvent)-sodium phosphate buffer (50 mM, pH 6.0),
regioselectivity. According to the experimental results, we consider
that the enzyme modifies its secondary and tertiary structure
during the transglycosylation reactions, due to the conditions in-
volved in these processes. This, might cause a different synthesis of
products respect the same reaction when the enzyme reacts in
water medium.
with 51.2 mg (0.17 M) pNP-
GlcNAc (acceptor) mixture was preequilibrated to 60 ꢁC. After-
wards, 36 -galactosidase were
mol minꢀ1 (U) of T. thermophilus
b-Gal (donor) and 188 g (0.85 M) of
m
b
added to the reaction mixture and reaction was incubated during
3 h, then, was stopped (and homogenized in the case of biphasic
systems) by addition of methanol. Reaction yields were determined
by HPLC-ELSD. The crude mixture was then directly loaded onto
a carboneCelite column eluted with a linear gradient of 0e15% (v/
v) of ethanol in water. Solvents were eliminated and disaccharide
(LacNAc) was dissolved in D2O to be characterized by 1H and 13C
NMR spectroscopy. NMR spectra for LacNAc were consistent to
previous reports.17,20,35
3. Conclusions
In this study we have proven that the presence of green solvents
from biomass modify the traditional regioselectivity of TTP0042
b
-galactosidase towards the synthesis of N-acetyl-D-lactosamine.
There were several solvents with important yields in the
disaccharide synthesis and there was no relation in the nature of
the system and the modification of the enzyme regioselectivity,
because both: soluble and insoluble solvents were able to modify
the enzyme synthetic activity towards the synthesis of LacNAc. The
concentration of the solvent is an important factor in order to im-
prove the synthesis of the disaccharide, because at lower concen-
trations of bio-solvents the synthesis of LacNAc was reduced, the
optimal concentration of bio-solvent for these reactions seems to
be 2.0 M. The addition of S4 to the enzyme solution (in buffer)
4.4. Effect of ILs concentration on enzyme activity
According to results obtained in transglycosylation, some sol-
vents were selected in order to study their effect in the synthesis of
disaccharide, these solvents are: S2, S4, S9, S12, S13 and S15 (see
Scheme 2). Thus, transglycosylations reactions were done as de-
scribed above, using different concentrations of selected solvents.
[0.00, 0.25, 0.50, 1.00, 1.50 and 2.00 M].

1152
M. Sandoval et al. / Tetrahedron 69 (2013) 1148e1152
4.5. Solvents effects in secondary structure by circular di-
chroism measurements
fluorescence spectrum was done for TTP0042 (1.0 mg mLꢀ1) in
sodium phosphate buffer 10 mM at pH 6.00. The emission spectrum
of S4 was negligible with respect to the spectrum of the enzyme in
buffer solution. Then, several additions of S4 were performed in the
cuvette in order to evaluate the effect of this solvent in the tryp-
tophan exposition of the enzyme.
In order to understand the effect of solvents in the secondary
structure of the protein, we developed a circular dichroism (CD)
study, by imitating of the reaction parameters, under these condi-
tions, we try to find a rational explanation for the previous results
in transglycosylation.
Acknowledgements
4.6. General procedures
This work has been supported by three research projects, two of
ꢀ
the MICINN (‘Ministerio de Ciencia e Innovacion’), CTQ2009-11801,
A Jasco-710 dichrograph, previously calibrated with D-10-
camphorsulphonic acid was used to record the far-UV CD spec-
tra of TTP0042. Measurements were performed in 0.1 cm cells at
60 ꢁC (temperature was selected in order to simulate reaction
conditions). Five accumulations were acquired for each spectrum.
The ellipticity was measured with a 1 nm bandwidth and a 2 s
response. For this study, recombinant TTP0042 was used, this is
a protein with a molecular weight of 50664.2 Da (higher than
native enzyme due to polypeptidic histidine tag attached to the
enzyme) and 450 residues. The mean residue ellipticity (MRE) was
expressed in deg cm2 dmolꢀ1 and calculated using the following
equation:
BIO2010-18875 and MAT 2008-02542; and one from UCM/
Santander CCG07-UCM/MAT-2681 (Gp950247). M.S. thanks a PhD
fellowship granted by Universidad Nacional de Costa Rica.
References and notes
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MRW$qobs
MRE ¼
10$l$c
ꢀ
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MRW¼molecular weight per residue (112,587)
l¼cuvette pathlength
qobs¼observed ellipticity in degrees
l¼pathlength in cm
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4.7. CD spectra for selected solvents
The following solvents: S2, S4, S13 and S14 were chosen to
evaluate their CD spectra and their effect in the recording (noise)
for the spectra. These solvents were selected because of their im-
portant effect (at 2.0 M) in the enzymatic synthesis of LacNAc.
ꢀ
ꢀ
4.8. Solvents effects over enzyme CD spectra
19. Jongejan, J. A. Organic Synthesis with Enzymes in Non-aqueous Media; Wiley-
VCH GmbH KGaA: 2008, pp 25e46.
ꢀ
ꢀ
ꢀ
20. Perez-Sanchez, M.; Sandoval, M.; Cortes-Cabrera, A.; García-Marín, H.;
From the previous procedure, S4 was chosen as the best solvent
to perform a CD study. First a CD spectra for the enzyme solution
(1.0 g mLꢀ1), in sodium phosphate buffer 10 mM at pH 6.00, was
recorded. Then, several aliquots of S4 were added to the solution
and the mixture was homogenized. Then the CD spectra were
recorded. The final concentrations of S4 used in this study were:
0.00, 0.25, 0.50, 1.00, 1.50 and 2.00 M.
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study
Fluorescence emission spectra were obtained on a PTI modular
spectrometer. 295 nm was set as excitation wavelength because it is
highly selective for tryptophan. Emission was acquired from 310 to
500 nm. The excitation and emission bandwidth were adjusted at
2 nm, measurements were done in 0.20 cm pathlength cell at 25 ꢁC
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