Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 116861

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Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Spectroscopic FTIR and NMR study of the interactions of sugars

with proteins
Mark Rozenberg a,⁎, Shifra Lansky a, Yuval Shoham b, Gil Shoham a
a
Institute of Chemistry, The Hebrew University, Jerusalem 91904, Israel
b
Department of Biotechnology and Food Engineering, Technion, Haifa 32000, Israel

a r t i c l e i n f o a b s t r a c t

Article history: FTIR and NMR spectra were measured in parallel for specific two-components mixtures of various proteins with
Received 14 November 2018 different sugar molecules, such as arabinose, glucose, and sucrose. In the FTIR spectra of arabinose with some of
Received in revised form 20 January 2019 these proteins, the bands assigned to the vibrational modes of the C\\H and C\\OH groups disappeared, and new
Accepted 17 February 2019
ones, related to an arabinose–protein C\\N mode, appeared. Similar changes were observed in the FTIR spectra of
Available online 19 February 2019
lyophilized mixtures of arabinose with different amino acids. In additional FTIR spectra, measured for other pro-
Keywords:
tein-sugar mixtures, the bands correlated to the ring modes of arabinose, in the range 1150–1000 cm−1, disap-
Protein-sugar interactions peared, and two new very strong narrow bands became dominant, indicating ring opening or some kind of
Arabinose arabinose decomposition. Contrary to the prevailing opinion that complexes between sugars and proteins are
Galactose formed mainly by hydrogen bonds, the IR and NMR spectra of the sugar-protein mixtures studied here suggest
Xylose that significant chemical reactions also take place between the interacting sugar and the protein. Two types of
FTIR sugar-protein chemical reactions can be distinguished on the basis of these IR spectra, leading to the formation
NMR of a new C\\N bond and to the decomposition of sugar skeletal bonds. The new IR bands suggest that the latter
reaction results in the formation of new bonds, which are related to new polyether moieties. These results high-
light the often ignored non-specific chemical reactions that take place between sugars and proteins, and demon-
strate that the simultaneous application of FTIR and NMR spectroscopic analyses can detect and further
characterize these types of sugar-protein interactions.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction
Application of FTIR for the study of protein interactions with sugars
has a long history, as it is of prime interest in biochemistry, food and me-
dicinal chemistry [1,2]. The main interest of these studies has usually
been focused on the stabilizing effect of carbohydrates on the secondary
structure of the protein, and accordingly the main region of interest was
that of the Amide-I band in the infrared spectra of the target proteins
[3–5].
Less commonly studied, however, were the specific changes in the
carbohydrate bands in these sugar-protein mixtures [6,7]. When this
was done, the spectral range studied was that of the most intense carbo-
hydrate bands (1400–1000 cm−1), including the so-called “fingerprint”
region (down to 900 cm−1), where the bands are usually not well re-
solved [8]. The previous spectroscopic assignments in the IR spectra of
carbohydrates have been summarized in a seminal report about 33
years ago [9], and up to now, there have been no significant changes
in this general field [10]. The analytical application of IR spectroscopy
of carbohydrates is quite widespread in the food industry [11], mainly
⁎ Corresponding author.
E-mail address: mark.rozenberg1@mail.huji.ac.il (M. Rozenberg).
for the detection and determination of the structures of the various
fructans produced in the framework of biocatalytic processes [12].
In this paper, sugar–protein interactions are studied by FTIR spec-
troscopy, based on the observation of changes in the skeletal C\\H
(C\\OH) sugar bands below 1200 cm−1 in the sugar-protein mixtures.
The reliable assignment of the bands in this range to the CH– and
COH-related modes of several sugars was partially done by Koenig et
al, using deuterium exchange of skeletal C\\H(D) groups [13]. The in-
formative benefits of the bands in this range as representatives of a fu-
ranose ring [14] has been previously shown for the levansucrase
protein [15,16], in a study of the relevant interactions of this protein
with its sucrose substrate [12].
In the past, the studies of protein-sugar interactions have been fo-
cused mainly on the stabilization of such complexes via specific hydro-
gen bonds, as based on the corresponding crystal structures of the
protein-sugar complexes (e.g. [17–20]). The results presented in the
current study demonstrate, however, that sugar–protein interactions
could include not only these specific hydrogen bonds, but also some sig-
nificant chemical changes that take place in the structure of the sugar
and, sometimes, changes involving specific moieties of the protein.
These results are further supported by NMR spectroscopy, which
was done here in combination with the FTIR study. Such combination

https://doi.org/10.1016/j.saa.2019.02.085
1386-1425/© 2019 Elsevier B.V. All rights reserved.
2 M. Rozenberg et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 116861
proved to be very useful since the NMR spectra of sugars are usually
very complex, especially in combination with proteins in solution
[21,22], and the correlation of these spectra with the corresponding
FTIR spectra allowed for an easier and more reliable assignment of the
relevant peaks.
It should be noted that initially we intended to characterize the spe-
cific interactions that take place between various sugar-degrading en-
zymes, or sugar-binding proteins, and their specific sugar substrates or
sugar products. This was expected to give us valuable information
concerning the sugar-protein interactions in the specific binding site
of these proteins. As such, we selected the arabinanase AbnB [18,23],
an arabinan-degrading enzyme, and its non-active catalytic mutants
AbnB-D17A and AbnB-E201A [18], as a representative enzyme to
study its interactions with arabinose, the product of its catalytic degra-
dation reaction. Following the same rational, we selected the xylanase
XT6 [21,24–28], a xylan-degrading enzyme, to study its active site inter-
actions with xylose, one of the products of the catalytic reaction. Simi-
larly, we selected the levansucrase LevanSR, a levan-producing
enzyme [15,16], to study its active site interactions with sucrose, the
substrate of its catalytic reaction, as well as with glucose and fructose,
the products of its parallel sucrose-degrading catalytic reaction. Sucra-
lose was also tested with LevanSR, as it is a closely related substrate an-
alog of this enzyme. In addition to these three enzymes, we selected two
sugar-binding proteins, AraP [23] and GanP [20,29,30], for the study of
their specific interactions with their binding substrates, arabinose and
galactose, respectively. In retrospect, it turned out that the interactions
that we could follow with both FTIR and NMR are probably more rele-
vant for non-specific protein-sugar interactions, and we hence mixed
those representative proteins with all of the target sugars studied
here, in order to determine whether these interactions are specific or
general. These wider studies of protein-sugar interactions, indicated
that unexpected interactions (and probably surface chemical reactions)
take place between the target proteins and the wider array of sugars
studied here, as further explained and discussed below.
2. Experimental
All the sugars were purchased from Sigma and used here as such,
without further processing or purification. The proteins selected, were
kindly provided by our collaborators from the Technion – The Israel In-
stitute of Technology, Haifa. These representative proteins were usually
prepared with a Tris buffer, although in some cases we used an inor-
ganic buffer, which did not contain any organic additives. For the FTIR
measurements of the dried powders, we prepared the samples by ly-
ophilization of 0.5–1 ml of the corresponding aqueous solutions, con-
taining 1–2 mg of sugar, 1–2 mg of the target protein and 200 mg of
KBr (with different relative concentrations of protein and sugar).
These samples were then dried in a desiccator with molecular sieves
and pressed to form KBr pellets at pressures not higher 10 t/cm2.
These general preparation procedures are further detailed in our previ-
ous FTIR studies of the interactions between amino acids and peptides
[31–33], as well as for the interactions between DNA bases, nucleotides
and DNA oligomers [34–37].
The same procedure was used for the pure protein samples and the
pure sugar samples, which were studied separately. Spectra were mea-
sured on a Bruker Equinox 55 FTIR spectrometer in transmission mode
at a resolution of 2 cm−1 and processed with the OPUS program. Deute-
rium exchange (D-exchange) of arabinose, xylose and fructose (de-
noted below as D-crystals) was performed by re-crystallization from
heavy water (Sigma) with subsequent crystallization from ethanol-OD
(Sigma). The aqueous-solution spectra of several sugar-protein mix-
tures were measured in cells of 0.03–0.01 mm thickness with CaF2 or
KRS-5 windows, in the range of 1200–900 cm−1. Spectra of the pro-
tein-sugar mixtures in aqueous solution, (e.g. AbnB-arabinose or
LevanSR-sucrose; see below) were similar to those of the solid mixtures
lyophilized with KBr in the corresponding range. As a reference, the
bands at about 1000 cm−1 and at 1650–1500 cm−1 were used for the
sugars and the proteins, respectively.
Following the IR measurements, the KBr pellets containing the sam-
ples of sugar, protein or sugar-protein mixture were dissolved in D2O,
and NMR H1 spectra were measured with the Bruker 500 MHz spec-
trometer at room temperature, with the HDO or TSP-d4 signals as stan-
dards. After dilution of the KBr-sugar-protein mixture, separation into
two phases in the NMR tubes was observed – a colorless or light yellow
viscous phase, which contained the coagulated proteins in the resulting
salt solution and a transparent liquid with the sugar. For the latter, the
NMR spectra were measured. Thus, the proteins do not seem to inter-
fere with the NMR sugar spectra. The most intense signal, at 4.78 ppm
in all the figures below, is related to H2O contamination in D2O. It is
noted that the signal position of the HDO proton is displaced from
0.05 to 0.15 ppm in solutions of different proteins, and it is therefore
more accurate to take the absolute chemical shift from the measure-
ment with an inert standard (e.g. TSP-d4). From a comparison of the
spectra with the literature, it follows that the presence of KBr does not
affect the NMR spectra, at least for pure sugars (the chemical shift varies
by not more than 10−2–10−3 ppm). When it was possible, the IR spectra
of the viscous phase were also obtained. In some cases, we were able to
record the NMR spectra of several protein-sugar mixtures without their
prior lyophilization (and without KBr), as measured in aqueous (H2O/
D2O) solutions (with water suppression). These kinds of studies have
been done for AraP with arabinose, for GanP with glucose and galactose,
and for AbnB and the inactive mutant AbnB-E201A with arabinose, in
order to compare their spectroscopic results with the corresponding
solid mixtures in KBr. Nevertheless, these samples turned out to be
quite complex in the range of the chemical shifts of the sugar CH pro-
tons of 3–6 ppm, and unfortunately their analysis was too complicated
to be unequivocally effective.
The FTIR and NMR spectra of the pure sugar samples of arabinose,
xylose, glucose, galactose, fructose, sucrose and sucralose, observed
here after lyophilization with KBr, agree well with the corresponding
reference data [38]. All samples were exposed to temperatures not
higher than 30°C.
Additional FTIR and H1-NMR spectra of several other sugar-protein
mixtures, studied in the framework of the current project, are not in-
cluded here for clarity, yet they are available in the Supplementary In-
formation (SI).
3. Results
3.1. Refinement of the FTIR spectra of arabinose and xylose
The assignment of the vibrational mode bands in the arabinose and
xylose spectra can be done only on the basis of published spectral stud-
ies of related sugar monomers [10,13,39,40]. The group of bands above
1000 cm−1 is assigned to mixed C\\O\\C, C\\CH and C\\OH stretching
and deformational modes of the arabinose ring. The group of bands
below 1000 cm−1 is assigned to C\\H deformations, including the
anomeric CH protons, and also to out-of-plane deformational modes
of OH groups, differently bound with hydrogen bonds of different
strengths [41–43]. In order to distinguish between the bands in skeletal
modes and hydroxyl OH modes, experiments with deuterium exchange
were also performed and the results are shown in Fig. 1, where the arab-
inose spectra are shown at 300 K before (trace 1) and after D-exchange
(trace 2).
The H-arabinose crystal bands at 1066–1051, 844, 784, 713 and 680
cm−1, together with a broad background with a center at ca 650 cm−1,
disappear in the D-crystal and shift to 768, 619, 578 and 499 cm−1, re-
spectively, with a frequency isotopic shift of about 1.36–1.38, displaying
their connection with hydroxyl-OH proton modes in the arabinose crys-
tal. The isotopic analog of the band at 844 cm−1 possibly overlaps with
the bands at 608-578 cm−1 and noticeably intensifies the latter. The
very narrow isolated bands at 893 and 784 cm−1 maintain their
 M. Rozenberg et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 116861 3

1081.
1096.3

1008.6
1149.4

768.5
1057.8

897.7

578.5

499.5
609.4
1364.4

1263.1
1380.8

821.5

419.4
absorbance

1475.3

1308.5

689.4
842.7
1449.2
2

1132

993.2
1091.5

783.9
1066.4
1051

677.9
842.7
1315.2

891.9

602.6
1256.4

579.5
1372.1
1354.7

501.4
1474.3

713.5
1240
1424.2

429.1
1401

943
1

1500 1400 1300 1200 1100 1000 900 800 700 600 500 400
-1
wavenumber, cm

Fig. 1. Spectra of crystalline arabinose at 300 K, before (1) and after deuterium exchange (2). The range of interest is marked by a frame.

position in the D-crystal (there is no band in the H-crystal, which could 3.3. FTIR spectra of the protein-sugar mixtures
be isotopically correlated with them) and can be safely assigned to the
C\\H (C\\OH) deformation skeletal modes. The assignment of the In Fig. 2A, we compare the FTIR spectra of crystalline arabinose
893 cm−1 band to the anomeric CH can be suggested on the basis of a (trace 1), pure buffer (trace 2), pure protein (trace 3) and a mixture of
classical experiment [13] in which the band closest to our band at 911 arabinose with the arabinanase AbnB [18] (trace 4). The corresponding
cm−1 in the glucose spectra was assigned to an anomeric CH group NMR spectra are shown in Fig. 2 (A1).
with deuterium exchange at the carbon (C1) atom. The analogous ex- Clearly, the IR spectra of the buffer and pure protein do not contain
periment with glucose - CH2(CD2) confirmed the assignment of the any interfering bands. The bands of arabinose at 892 cm−1 and 784
band in the glucose spectrum at 1011 cm−1 to the CH2 group.1 In the cm−1 (assigned above to skeletal CH modes) have reduced intensity.
spectrum of a pure OH xylose crystal (Fig. S1) the narrow bands at Other bands – the narrow one at 842 cm−1 and the broad one at the
977, 934 and 898 cm−1, which noticeably reduce the intensity at deute- center at 678 cm−1 – disappear because of the destruction of the crys-
rium exchange, can be assigned to the out-of-plane deformational pro- talline H-bond structure on dissolution. The band at about 1080 cm−1
ton modes of CO-H groups bound with hydrogen bonds of different increases slightly at the expense of the vanished structure of a crystal-
energy. The xylose band at 759 cm−1 does not change in this case and line spectrum, but the strong bands at 1138 and 1000 cm−1 maintain
can be assigned to the CH mode, which is possibly analogous to the their intensity. Simultaneously, two new weak bands at 1340 and
893 cm−1 band of arabinose. The bands at 893 and 784 cm−1, assigned 1257 cm−1 appear in the spectrum. No significant changes are observed
above to C\\H (CH2) skeletal modes, were chosen as an indicator of in the range of 1700–1500 cm−1, as related to the Amide-I and Amide-II
arabinose interaction with the protein in this study. modes. Similar spectral changes in the arabinose spectrum are observed
in the mixture of the xylanase XT6 [24,27] protein with arabinose.
The NMR spectra show noticeable changes in the positions and in-
3.2. Spectra of proteins tensities of the signals which are assigned to 3 and 5 protons in the
range of 3.65–3.70 ppm [38].
In general, the main features observed in the FTIR spectra of proteins As compared to arabinose, the mixture of isomeric xylose with the
present less assignment difficulties, as they are described and discussed arabinanase AbnB [18], as well as with the xylanase XT6 [27] (the latter
in numerous relevant publications (see e.g. [44]). Obviously, the Amide- is shown in Fig. S1), show no changes in the NMR spectrum, in full
I, Amide-II and Amide III bands (as observed near 1650, 1550 and 1300– agreement with the corresponding IR spectrum. The latter represents
1200 cm−1, respectively), are the most intense. The other bands are of a simple sum of the two components of the mixture.
relatively low intensity and the proteins are practically transparent In Fig. 3, the FTIR (A) and NMR (A1) corresponding spectra are pre-
below 1400 cm−1. For the NMR studies of the target proteins with sented for arabinose in a mixture with the non-active catalytic mutants
sugars, there are no significant NMR signals in the protein spectra of the arabinanase AbnB (AbnB-D17A and AbnB-E201A [18]). All bands
when measured in D2O solutions (after the dissolution of their corre- previously assigned to the skeletal modes of arabinose in the range
sponding KBr pellets), evidently since most of the protein precipitated 1200–700 cm−1 disappear, and two strong narrow bands at 1037 and
in the NMR tube (probably due to the KBr ionic strength), so practically 1056 cm−1 become dominant in the spectra. In parallel, substantial
only the sugar spectra could be recorded in these measurements. changes in the NMR spectra take place (in Fig. 3A) near the position of
the chemical shift at 3.7 ppm. The signals of the anomeric protons (ca
4.6 and 5.3 ppm), together with several high-field signals, disappear
completely. Very similar transformations are observed in the FTIR spec-
1
It should be noted that in a very recent review, the band at 844 cm−1 was also
assigned to the skeletal mode [10], despite the fact that deuterium exchange shifts its po- tra of the mixtures of both arabinose and galactose with the arabinose
sition to approximately 600 cm−1, with the usual isotopic shift of 1.38. binding-protein AraP [23] (SI, Fig. S3, C and D).
4 M. Rozenberg et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 116861

3.6
A A1

1652.7

3.725
3.722
3.897

3.69
1075.1

3.893

3.716

3.526
1531.2

3.97

3.709
3.923

3.51
3.506
3.919
3.966
4.021

3.838
3.886

3.831
1645

3.668
3.879

3.674
4.044

3.648
3.642
3.818
4.047

3.811
4.03

3.946
1138.8

3.906
1002.8
1403.9
1448.3

4.064
1252.5

512
1341.2
1530.2

785.9
3
4

3.667
944

3.674
absorbance

3.89 3.894

3.708
1449.2
1402

3.71
1239

3.684
3.694
3.687
1618

3.681

3.528
4.021

3.512
3.956
3

3.92

3.508
3.493
476.3

3.916

3.835
3.952
623.9

3.827
4.044

3.873

3.644
3.88
4.047

3.815
3.808
2

3.639
3.9
1094.4

3.789
1262.2

802.2
2
1091.5
1132

993.2

783.9
1051

677.9
842.7
1315.2

891.9

602.6
1256.4
1372.1
1474.3

501.4
1401

1
1

1800 1600 1400 1200 1000 800 600 400 4.1 4.0 3.9 3.8 3.7 3.6 3.5
-1 chemical shift, ppm
wavenumber, cm

Fig. 2. A - FTIR and A1 - NMR spectra of arabinose with the AbnB protein. In panel A – pure arabinose (1), buffer (2), pure protein AbnB (3), a mixture arabinose - protein (4). The range of
interest is marked with a frame. In panel A1 - pure AbnB protein (1), pure arabinose (2), mixture of arabinose –protein (3).

In the mixture of the glucose/galactose binding-protein GanP [29] and in good correlation with the results reported in similar FTIR exper-
with glucose, it is apparent that the strong band at 996 cm−1 disappears iments [8]. These results are also confirmed here by the corresponding
and a new strong band at 1019 cm−1 appears, as illustrated in Fig. S3-A. NMR spectrum. In the IR spectra of the mixture of sucrose with LevanSR,
The spectra of the arabinose sugar were measured also in its mix- which are shown in Fig. 5 A, new bands with clear dependence on con-
tures with the small amino acids L-glutamine, L-histidine and L-tyro- centration appear near 1017 cm−1 and in the range of 918-703 cm−1;
sine, in order to compare them with the spectra obtained from the the latter are characteristic of a pure furanose sugar [8,12], one of the
corresponding arabinose-protein mixtures. The spectrum of the arabi- possible side products of the LevanSR enzymatic reaction. The changes
nose–glutamine mixture is shown in Fig. 4. The arabinose bands at in the IR spectrum correlate well with the corresponding NMR spec-
893 and 784 cm−1 disappear and a new, quite intense, narrow band ap- trum, where strong changes at 3.80–4.20 ppm can be observed,
pears at 1261 cm−1 (Fig. 4A, trace 3). The changes in the arabinose spec- confirming the formation of levan, the polymeric fructan produced nat-
trum below 1000 cm−1, as reflected in its mixtures with both proteins urally by this enzyme [15,16].
and amino acids, seem therefore to be identical. In the NMR spectrum The IR spectrum of sucralose also clearly shows some interaction
of the arabinose–glutamine mixture, which is shown in Fig. 4 (A1), no with the LevanSR protein, as the intense band at 1055 cm−1 (which
changes were observed. is very close to the main fructose band at 1054 cm−1) appears, and
The relative sensitivity of the IR and NMR spectra to the possible re- the bands of C\\H at 890–860 cm−1 and C\\Cl at 775–749 cm−1 be-
action of sugars with proteins is demonstrated in Fig. 5, as reflected in come essentially weaker. Nevertheless, these spectral changes
the spectra of the levansucrase LevanSR [15,16] in its mixtures with are not as clear and as pronounced as in the case of sucrose, so
two sugars, the natural sugar dimer sucrose and its synthetic analog su- that we can not tell if some degree of a sucralose polymerization
cralose. In the case of the sucrose mixture, the spectroscopic results is taking place. In this case, however, the NMR spectrum happens
demonstrate clearly the formation of a levan polymer (the expected to be noticeably less sensitive, so no significant spectral changes
product of the enzymatic reaction), as shown here in the FTIR spectrum are observed.
3.93

A1
4.118

3.971

A
3.976
3.966
1056.81054.9

4.106
1632.4 1631.5

4.267

3.744
1037.5
1552.4

3.93
595.9
628.7
1294
1551.5

4.118
1403.9

594.9
absorbance

466.7

3.971
1459.8

627.7

3
1133

3.873
3.857
802.2

4.267

3.744
3.803
4.206
1294

421.4
1451.2
1403

1132 1133

2
803.2
1091.5

4.629
993.2

4.61
783.9
1066.4
1050.1

894.8

3.759
925.7

3.75
677.9

3.783
3.797
3.794
4.097

3.774
842.7

3.765
1315.2

3.973

3.762
891.9

602.6
1256.4
1372.1
1354.7

579.5

3.592
3.967

3.612
44.005

3.918
1474.3

501.4
1236.1

3.909
5.326
713.5

3.586
4.04
5.335

3.568
1424.2

429.1

3.953

3.723
943

1800 1600 1400 1200 1000 800 600 400 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5
-1 chemical shift (ppm)
wavenumber, cm

Fig. 3. FTIR (A) and NMR (A1) spectra of arabinose, alone (1), and with the proteins AbnB-D17A (2) and AbnB-E201A (3). The signal at 4.78 ppm belongs to HOD contamination in D2O,
which is used here as a standard.
 absorbance

1800
1733.7
absorbance
absorbance 1686.4 1685.5

1800
1639.2
1627.6 1

1750

signal as a standard.
1800
1585.2 1586.2

1600
1544.7
1659.4 1649.8

1700
1628.6 1634.4 1486.8 1485.9
1474.3
1640.2 1451.2 1450.2

1600
1424.2 1410.7
1552.4 1409.7

1650
1548.6 1640 1401

1600
1372.1

1400
1514.8 1354.7 1359.6 1358.6
1479.1
1536 1333.5 1334.5
1459.8 1460.8 1459.8 1460.8 1315.2 1315.2

1600
1428 1426.1 1280.5
1261.2

wavenumber, cm
1449.2 1256.4 1259.3
1403.9 1403.9

-1
1240
1231.3

1400
1373.1 1203.4 1203.4

1550
1355.7 1540
1384.6

1200
1162.9

1400
1303.6
1360.5
1295.9 1294 1132 1131 1135.9
1325.8 1103.1 1104

1500
1261.2 1091.5 1085.7 1085.7
1243.9 1066.4 1072.2
1255.4 1051 1052.9 1052
1208.2 1028.8
1003.8

1450

1200
1164.8 993.2999.9

1
2
3
4
5
6
1000
1133 1138.8 1132 1136.8

wavenumber, cm

1200
943 944

-1
1093.4 1145.5 925.7
891.9 896.7
1061.6 1050.1 1054.9 1099.2
1044.3 1034.6
1026.9 842.7 848.5 843.7
1060.7
1002.8 1002.8 810 809
982.6

1000
wavenumber, cm
969.1 1017.3 783.9

800
777.2

-1
1000
918.9

wavenumber, cm
4
5
6
906.4 713.5

-1
891
863
918.9 677.9 677.9 678.8
857.2 653.8
856.2 621 621.9
602.6 596.9 596.9

800
600
775.2 817.7 579.5
749.2 738.6 538 538

800
780.1
713.5 501.4 505.3
479.2 478.3
663.4 703.9 453.2 455.1 456.1
626.8 627.7 429.1
418.5 423.3
621.9
596.9 595.9 593
400

600
1
2
3

594.9

600
528.4
498.5
553.5
A

471.5 524.5

425.2

400
417.5

1
2
3
4
400
A

5.6
5.6
5.505
5.497 5.504
5.496
4.044
5.541
5.531 5.513
5.503
4.021 4.018

5.4
5.4
4.0

3.956

5.2
3.953

5.2
3.92
3.9133.917

5.0
3.894

5.0
4.953 3.887 3.891
4.933 3.88 3.877
3.87

4.8

4.8
4.754 4.766 3.835 3.832
4.726 3.825
3.815
3.804
*

HDO 4.674 4.607 HDO 4.686 3.792

4.6
3.789

4.6
* *

4.575 4.571
4.567 3.78

chemical shift, ppm

4.45 4.438
4.462 4.448
4.438
4.421 4.421

4.4
chemical shift (ppm)

4.4
chemical shift, ppm

4.343
4.321
4.292
4.28
4.273 3.71
4.263 3.708
3.705
4.243
4.235 4.239
4.232 4.195 3.692

4.2
4.222
4.215 4.218
4.211 4.173 4.174 4.165 3.684 3.685
3.681
4.152 4.159
4.149
4.14
M. Rozenberg et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 116861

4.2
4.175 4.173 4.134
4.128 3.672
4.141 4.158 4.156 4.11
4.104
4.095 3.667 3.665
4.139 4.079
4.12
4.103 4.119 4.108
4.103
4.097
4.092 4.109
4.081 4.096
4.091 4.039
4.031
4.079 4.022
4.013 4.031 4.024 4.049
4.017 3.644 3.641
4.001
3.992 3.997
4.0
3.982
3.973
3.964 3.966 3.984
3.979
3.956
3.949 3.942 3.94 3.95
3.936

4.0
3.98
3.973 3.98
3.972 3.904 3.917 3.928
3.912
3.96
3.952 3.959
3.952 3.887
3.931
3.919 3.93 3.918 3.865
3.905 3.836 3.825
3.801 3.8113.804
3.795
3.858
3.8

3.856 3.781
3.833
3.827 3.831
3.825 3.763
3.748 3.758
3.802 3.806 3.743
3.794 3.7943.8

3.8
3.771 3.77 3.782 3.706
3.697 3.696
3.766 3.782 3.756 3.766 3.681
3.672 3.679
3.673
3.743 3.743
3.617

1
2
3.594
3.6

3.569 3.528 3.525

3.539
3.519
3.516 3.512 3.509
3.496 3.505
3.5

3.493

B1
3.489
3.4
1
2
A1
1
2
A1

Fig. 4. FTIR (A) and NMR (A1) spectra of arabinose (1), pure glutamine (2) and mixture of arabinose –glutamine (3); Stars (*) in the A1 panel relate to the glutamine signals.

arabinose, traces 3–6 – LevanSR + sucrose at increasing concentration; all spectra normalized relative to the band at 1640 cm−1 (Amide-I). The arrow on inset shows the decreasing
Fig. 5. IR (left side - X) and NMR (right side - X1) spectra of LevanSR with sucrose (A and A1) and sucralose (B and B1). In IR spectrum A – trace 1- pure LevansSR; trace 2- LevanSR +

all the NMR spectra – trace 1- pure sugar, trace 2- LevanSR + sugar. Chemical shifts were measured in panel A1 relative to the HDO signal, and in panel B1, relative to the TSP-d4
5

of the Amide-II bands in parallel with increasing of the levan quantity. In IR spectra B - trace 1 – pure buffer, trace 2- pure sugar, trace 3 – pure LevanSR, trace 4 - LevanSR +sugar. In
6 M. Rozenberg et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 116861

As a reference study, we examined the interactions of LevanSR with

arabinose and fructose, two sugar monomers which are not related to

1208.2
1430
its natural substrate, sucrose. In this case, the spectra of arabinose in

1328.7
its mixture with the LevanSR protein (Fig. S2, A and A1) show some no-

1570.7
ticeable changes using both methods. Yet, in the case of fructose (Fig. S2,
B and B1), the changes in the NMR spectrum of the mixture are more

1430.9

658.6
noticeable than in the IR, where the spectrum for a pure fructose and

742.5
701
1208.2
the spectrum for fructose in its mixture with the protein are practically

1259.3
identical.
The band at 1017 cm−1 in the FTIR spectrum of the sucrose-LevanSR
5

1094.4
mixture can be safely assigned to the C\\O\\C glycoside bond that is

absorbance
formed between the fructosyl rings of the levan polymer chain. The

662.4
band at 1019 cm−1, which is observed in the spectra of glucose with

1260.3

801.3
1211.1
1425.1

742.5
the glucose/galactose binding-protein GanP (SI, Fig. S3, A and A1), is 4

1096.3
very close to the 1017 cm−1 band in the sucrose-LevanSR mixture
(Fig. 5A) and can possibly indicate the formation of an analogous

798.4
C\\O\\C glycosidic bond in this case as well. The nearby band at 990–

1207.2
1445.4 1441.5

624.8
1000 cm−1 was observed in aqueous solutions and was assigned to
3

872.6

667.2
the glycosidic bond of disaccharides [45], but in general, the visibility

1105

699.1
of this narrow band depends on the quality of the levan crystal [46]. Re-

1207.2
garding the protein part of the mixture, a clear change is observed in the
2

804.2
1261.2
bands related to the polypeptide main-chain of the LevanSR protein
(most likely resulting from global conformational changes), as reflected

1029.8
1100.2
1331.6
in a decrease in the band intensity of the Amide-II NH band at 1500

1577.5
cm−1 (Fig. 5A; inset). These changes become progressively more pro-

802.2
1

nounced, in parallel with the increase in the levan band at 1017 cm−1.
These parallel spectral changes indicate that whatever is happening to
the main-chain of the protein, is likely to be linked to the progress of
the enzymatic reaction and the accumulation of the levan polymer. To
the best of our knowledge, this interesting phenomenon was never de-
tected and followed by FTIR spectroscopy before, although supported by
the observation of a pH-linked protein fibrilization, as detected by cryo-
microscopy for a related levansucrase [16]. The FTIR measurement of
the corresponding LevnSR-sucrose system in the aqueous solution,
shown in Fig. S4 (inset), confirms the parallel spectral changes
discussed above. The high similarity of both spectra also demonstrates
that this type of FTIR measurements are completely independent of
the sample preparation methodology, at least for the protein and the
sugar components involved here.
The FTIR spectra of the colored viscous phase separated in D2O, men-
tioned above in the Experimental section, are shown in Fig. 6. They all
demonstrate, in varying degrees, the new band that appears at 1260
cm−1, which is characteristic of a newly formed C\\N bond, and
which is similarly seen in the spectra of the glutamine-arabinose mix-
ture discussed above. Both observations suggest that a chemical reac-
tion is taking place between the sugar and the protein, similar to the
reaction that takes place between the sugar and the amino-acid. The de-
velopment of a yellowish color in both cases supports the occurrence of
a chemical reaction.
4. Discussion
On the basis of the FTIR and NMR spectral results presented above, it
is possible now to conclude that on mixing the target proteins with sim-
ple monomeric sugars, two types of reactions take place, which could be
clearly observed in the corresponding spectra. The first one is the forma-
tion of a new C\\N bond, probably between the sugar and the protein,
without destruction of the basic sugar structure, as observed in the mix-
tures of arabinose with glutamine and its mixture with the proteins
AbnB, LevanSR and XT6. The second type of reaction seem to affect the
ring structure of the sugar, as observed in the mixtures of arabinose
with the proteins AbnB-D17A, AbnB-E201A and AraP, in the mixture
of galactose with AraP, and in the mixture of glucose with GanP. It is
not improbable that both reactions occur simultaneously or consecu-
tively to a variable extent. These two types of reactions and their
742.5
1600 1400 1200 1000 800 600
-1
wavenumber, cm
Fig. 6. IR spectra of the viscous phase of the protein-sugar samples after dissolution of the
KBr pellets in D2O for NMR measurements. 1- AbnB-E201A + arabinose, 2- XT6 + xylose,
3- XT6 + arabinose, 4- AbnB-D17A + arabinose, 5- AbnB + arabinose; The arrow shows
the new (C\ \N) band position at 1260 cm−1.
validation by the current spectroscopic experiments are further
discussed below.
The arabinose bands at 892 cm−1 and 842 cm−1, which can be
assigned to skeletal C\\H and C\\OH deformational modes, disappear
in the spectra of all arabinose mixtures with the AbnB, XT6, AraP and
LevanSR proteins. This means that some kind of a reaction is taking
place with the participation of skeletal protons, possibly with the
anomeric proton C1\\H. At the same time, the new band at 1260–
1340 cm−1 is being formed and can tentatively be assigned to the new
Amide-III mode with significant contribution of a new C\\N stretching
mode, which is characteristic of a glycosyl-amine moiety [1,47]. In this
range, the IR band was observed and assigned to the asymmetric
mode Casn-N-Cgluc in the reaction products, when mixing the free
amino acid asparagine and glucose [48]. The same effect is seen in the
present work, following the spectra of arabinose when mixed with
amino acids, where the new C\\N band appears at 1260 cm−1. This
new C\\N band is seen, to variable degrees, also in the protein part of
some of the proteins studied here, after their separation in D2O solutions
(Fig. 6). Chemically, this process is known as the Maillard Reaction [1,2].
At a close position in the spectrum, the new band appearing at about
1230 cm−1 was observed in the cation of deoxyfructose glycine, which
was used as a model for the Amadory product and could be assigned
to the same mode and indicated a newly formed C\\N bond [49].
Although known for a relatively long time, the Maillard reaction has
been usually observed and reported in non-standard conditions, as for
example in elevated temperatures. To the best of our knowledge, this
is the first time that such a reaction is reported for mixtures of proteins
 M. Rozenberg et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 116861
and sugars at room temperature and in close to normal physiological
conditions. As such, this type of reaction may prove to be more common
and more significant than appreciated in the past, and should be espe-
cially considered for its more general physiological, nutritional and
medical consequences.
In the spectra of arabinose with the proteins AbnB-D17A, AbnB-
E201A and AraP, as well as in the spectra of galactose with the protein
AraP, new spectral changes are observed (in addition to those men-
tioned above), which indicate some kind of a decomposition of the
sugar ring frame. These changes are reflected in the bands at about
1130–1150 and 1000 cm−1, which are typical of cyclic sugars, and
which disappear in the sugar-protein mixtures listed above, while
new bands appear in the spectra. These are presented in Table 1
below, with possible specific assignments. These new bands are similar
to those observed in polyethylene glycols and in similar linear mole-
cules, such as (poly)alcohols and (poly)ethers [50]. The band at 1019
cm−1 in the spectrum of glucose in its mixture with GanP (Fig. S3) is
very close to 1017 cm−1, which is clearly seen in the spectra of the
LevanSR–sucrose mixture in the solid Fig. 5(A). As such, this band can
be assigned to the C\\O\\C band of any molecule analogous to the
levan polymer. The high similarity of these bands in different sugar-pro-
tein mixtures, as for example for the GanP-glucose and for the LevanSR-
sucrose systems (Table 1), indicate that in these cases the reaction prod-
ucts should also be considerably similar.
Another aspect of the current study is the parallel application of both
FTIR and NMR techniques, two very different methodologies that their
combination can potentially be complementary and advantageous. In
this respect, it should be noted that for the newly formed C\\N bond
discussed above, the FTIR spectra clearly shows the formation of N-
glycosylamine moiety, but only relatively weak changes could be seen
in the NMR spectrum of the sugar-glutamine mixture, probably indicat-
ing that the environment of the relevant C\\H protons changes rela-
tively little, at least in the initial stage of the interaction [1].
Nevertheless, the NMR spectra seem to support the IR data, as the
main NMR spectral changes take place for all the systems in the range
of 3.7–3.9 ppm. These new signals appear in the range that is character-
istic for protons located in alcohols and ethers (R-O-CH3), yet a more
comprehensive NMR analysis is needed in order to fully characterize
these specific changes.
Generally speaking, direct correlation between IR and NMR results is
not trivial, mainly due to the different physical basis of the two tech-
niques, influencing in a different way the molecular-vibrational spectra
(for FTIR) and the proton-nuclear-spin spectra (for NMR). The first
seems to relate better to the formation or destruction of a chemical
bond in the molecules under study. In our case, when looking at the tar-
get sugar, it can be suggested that we are dealing with the breaking of
the C\\H (anomeric) or other CH(CH2) bonds, and the formation of a
new C\\N bond. If a decomposition of the sugar takes place, the relevant
C\\C or C\\O bonds are broken and their corresponding bands disap-
pear from the spectra. In the NMR method, the detailed interpretation
is considerably more difficult, mainly because of mutually interacting
protons, especially in the case of regions of the sugar spectra that over-
lap with significant protein bands. The absence of any changes in the
NMR spectrum, despite the new bands that appear in the IR spectrum,
seem to indicate that the formation of the new C\\N bond is taking
place in the vicinity of the relevant C\\H group, yet without influencing
Table 1
The frequencies of the new bands in the sugar-protein IR spectra.
AraP-arabinose AbnB-D17A-arabinose AbnB-E201A-arabinose
1057.0 1055.0 1058.0
1037.0 1036.0 1037.0
7
the magnetic properties of all the other protons in this region. This phe-
nomena is well demonstrated in the simpler mixture of arabinose with
the glutamine amino acid (Fig. 4, A and A1), since identical changes are
observed in the IR spectra of both the protein–sugar mixture and the
glutamine–sugar mixture, as reflected in the appearance of the new
band at 1260 cm−1. While this new band clearly confirms the formation
of the new C\\N bond by the FTIR spectra, no significant spectral
changes are observed in the corresponding NMR spectra, demonstrating
the much lower sensitivity of the NMR technique for these kind of
chemical changes.
Another aspect to be considered in this respect is the fact that the
FTIR experiments were done mainly in the solid (within KBr disks),
while for the NMR experiments we dissolved these solid pellets in aque-
ous solution. This was done to keep identical ratios between the protein
and the sugar components, and to keep all the other components of the
original solution studied. A number of reference experiments have been
done to make sure that these technical differences do not influence the
chemical interpretation of the results. One of such control experiments
examined the effect of KBr on the behavior and spectra of the studied
protein-sugar mixtures, confirming that the addition of KBr does not af-
fect either the protein or the sugar. Similarly, it was confirmed that the
solubilization of the protein and the sugar in aqueous solutions does not
effect the main features of their spectra. As an example, for the LevanSR
reaction with sucrose (resulting in the formation of polymeric levan),
the FTIR spectrum of the solid lyophilized from a KBr solution was
found to be practically identical to that measured in aqueous solution
without KBr (e.g. compare Figs. 5A and S4). Moreover, the addition of
KBr to the sugar-protein solution caused often a significant precipitation
of the protein in the NMR tube, reducing its soluble concentration in the
aqueous (D2O) solution and improving the observed NMR spectra of the
(modified) sugar component of the mixture. The fact that the NMR
spectra of the soluble part of the sugar-protein solution are of better
quality after separation of the protein in a saline solution, represents a
new potential approach for the study of otherwise quite complicated
such systems. This approach could make the interpretation of the
resulting spectra much easier, especially since the solubilization and
the KBr were proved to have no significant effect.
Another aspect to be considered is the different behavior of the sugar
monomers studied here, as for example the differences observed for L-
arabinose and L-xylose. One of the reasons for these differences could
be linked to the different hydrogen-bond structures of these sugar mol-
ecules, as explained in the following for arabinose and xylose. Neutron
studies of β-L-arabinose crystals [51] indicate the presence of four H-
bonds with H⋯O bond lengths of 1.735, 1.801, 1.820 and 2.201 Å. The
X-ray crystal structure of α-xylose also contains four H-bonds with
O⋯O bond lengths of 2.743, 2.749, 2.808 and 2.687 Å [52]. If an average
O\\H bond length of 0.97 Å [53] is subtracted from these values, the
resulting H⋯O bond lengths will be 1.773, 1.779, 1.838 and 1.717 Å, re-
spectively, essentially shorter by about 0.1–0.3 Å, and correspondingly
stronger, as compared to those of the β-L-arabinose. In this respect, it
has been established [22–24,54–56], that the shortest and, accordingly,
the strongest H-bonds have a higher frequency of the bending proton
(OH⋯O) ν4 mode. In the case of the arabinose spectrum (Fig. 1), the
FTIR bands with frequencies of 892, 842, 678 and 620 cm−1, which
were assigned to this mode by deuterium exchange, correlate well
with the independently measured H-bond lengths. From their peak
AraP-galactose Mode GanP–glucose LevanSR-sucrose Mode
1059.0 ν (-C-OH) 1144.0 1145.0 δ(-CH2-)
1036.0 ν (-C-C-) 1102.0 1099.0 ν (C-O-C)
1057.0 1060.0 ν (-C-OH)
1019.0 1017.0 ν (-C-O-C-)
8 M. Rozenberg et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 116861
positions and their relative intensities, it hence follows that most of the
H-bonds in the arabinose crystal have energies in the range of 23–51
kJ·mol−1. In contrast, in the FTIR spectra of xylose (Fig. S1-A), there
are no bands with frequencies lower than 897 cm−1, which are sensitive
to deuterium exchange and could be hence assigned to the bending-
proton mode of the C-OH group. The lowest H-bond energy for xylose
is therefore approximately 54 kJ·mol−1, significantly larger than most
of the H-bond energies in arabinose. Since the H-bond energies in
water are comparable with the weak H-bonds in solid arabinose, the
arabinose H-bonds can potentially be broken once it is dissolved in
aqueous solution, explaining the disappearance of the corresponding
bands in the spectra. For xylose, the H-bonds of solid xylose are much
stronger and generally remain unaffected by water, accounting for the
observation that the corresponding crystal bands retain their intensities
in solution, even after mixing with the protein. This probably suggests
that, even in aqueous solution, the xylose molecules are mostly associ-
ated with each other (in the form of H-bonded dimers or small oligo-
mers), causing a different behavior of this specific sugar as compared
to its arabinose homolog. These differences in H-bonding strengths
can also influence the effective size and shape of the sugar molecules
once in aqueous solution, and hence affect their interaction with the rel-
evant protein surfaces.
An aspect that was not discussed above, mainly due to the limited
scope of this report, is the actual catalytic activity of the enzymes studied
here. Except for the very clear production of polymeric levan resulting
from the catalytic reaction promoted by LevanSR, it seems that most
of the changes observed in the current study are due to non-specific
interactions on the surface of the proteins rather than specific inter-
actions and/or reactions in the active site of the target enzymes. This
conclusion is supported by the fact that although AbnB was expected
to interact specifically with arabinose, and similarly XT6 with xylose,
no such specific interactions have been observed. Similar effects
were observed for AraP, expected to interact specifically with arabi-
nose, and GanP, expected to interact specifically with glucose and ga-
lactose. Nevertheless, such specific binding and catalysis can not be
completely ruled out, as suggested for other sugar-protein systems
[57]. Related changes in the spectra of glucose were also observed
in aqueous solution under the action of an inorganic catalyst, sug-
gested to reflect either ring-opening or another form of molecular
isomerization of the glucose [58].
5. Conclusion
Taken together, the spectra discussed above, together with those
available in the SI, suggest several interesting phenomena, which have
not received a proper attention before. It was pointed out here that
the formation of N-substituted glycosylamine, the first stage of a
Maillard reaction between sugars and proteins, may take place not
only at high temperatures, but also at room temperatures and normal
physiological conditions. It was also demonstrated that upon mixing
with proteins, the basic sugar frame may undergo significant changes,
probably some kind of ring opening and/or covalent oligomerization.
The changes observed in the Amide-II NH band of the LevanSR enzyme,
indicated that during the process of the enzymatic levan polymeriza-
tion, this protein underwent some significant changes in its main-
chain conformation, confirming earlier suggestions of such unique phe-
nomenon based on other experimental techniques. These three impor-
tant phenomena could be detected in the current study only through
the combined application of FTIR and NMR techniques, without which
they could have been easily overlooked. Although these phenomena
were demonstrated here only for specific proteins and specific sugars,
we feel that they are more general, and may be relevant and important
for the study of a wide range of sugars and proteins, a statement that ob-
viously requires a wider and more systematic research.
Acknowledgements
This work was supported by the Israel Science Foundation Grants
500/10, 152/11, 1072/14 and 1905/15, the I-CORE Program of the Plan-
ning and Budgeting Committee of the Council for Higher Education of Is-
rael, the Israeli Ministry of Environmental Protection, and The Israeli
Ministry of Science and Technology (MOST, 3-12484/15). The research
also received funding from the European Community's Seventh Frame-
work Programme (FP7/2007-2013) under BioStruct-X (grant agree-
ment N°283570). Y.S. acknowledges partial support by the Russell
Berrie Nanotechnology Institute, Technion-Israel Institute of Technol-
ogy and The Lorry I. Lokey Interdisciplinary Center for Life Sciences
and Engineering, Technion-Israel Institute of Technology. S.L. is grateful
to the Azrieli Foundation for the award of an Azrieli Fellowship.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2019.02.085.
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