Gluco-oligomers initially formed by the reuteransucrase enzyme of Lactobacillus reuteri 121 incubated with sucrose and malto-oligosaccharides

Article abstract of DOI:10.1093/glycob/cwt048

The probiotic bacterium Lactobacillus reuteri 121 produces a complex, branched (1 → 4, 1 → 6)-α-D-glucan as extracellular polysaccharide (reuteran) from sucrose (Suc), using a single glucansucrase/glucosyltransferase (GTFA) enzyme (reuteransucrase). To gain insight into the reaction/product specificity of the GTFA enzyme and the mechanism of reuteran formation, incubations with Suc and/or a series of malto-oligosaccharides (MOSs) (degree of polymerization (DP2-DP6)) were followed in time. The structures of the initially formed products, isolated via high-performance anion-exchange chromatography, were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and 1D/2D ¹H/¹³C NMR spectroscopy. Incubations with Suc only, acting as both donor and acceptor, resulted in elongation of Suc with glucose (Glc) units via alternating (α1 → 4) and (α1 → 6) linkages, yielding linear gluco-oligosaccharides up to at least DP ~ 12. Simultaneously with the ensemble of oligosaccharides, polymeric material was formed early on, suggesting that alternan fragments longer than DP ~ 12 have higher affinity with the GTFA enzyme and are quickly extended, yielding high-molecular-mass branched reuteran (4 × 10⁷ Da). MOSs (DP2-DP6) in the absence of Suc turned out to be poor substrates. Incubations of GTFA with Suc plus MOSs as substrates resulted in preferential elongation of MOSs (acceptors) with Glc units from Suc (donor). This apparently reflects the higher affinity of GTFA for MOSs compared with Suc. In accordance with the GTFA specificity, most prominent products were oligosaccharides with an (α1 → 4)/(α1 → 6) alternating structure. The Author 2013. Published by Oxford University Press.

Full text of DOI:10.1093/glycob/cwt048

Glycobiology vol. 23 no. 9 pp. 1084–1096, 2013
doi:10.1093/glycob/cwt048
Advance Access publication on June 25, 2013
Gluco-oligomers initially formed by the reuteransucrase
enzyme of Lactobacillus reuteri 121 incubated with sucrose
and malto-oligosaccharides
Justyna M Dobruchowska2, Xiangfeng Meng2,
Keywords: α-D-glucans / glucanotransferase / glucansucrase /
Lactobacillus reuteri / structural analysis
Hans Leemhuis2, Gerrit J Gerwig2,3
,
Lubbert Dijkhuizen1,2, and Johannis P Kamerling2,3
2Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology
Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen,
The Netherlands and 3NMR Spectroscopy, Bijvoet Center for Biomolecular
Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Introduction
In an earlier screening of a large collection of probiotic
Lactobacillus strains for their possible exopolysaccharide (EPS)
production, we have shown that Lactobacillus reuteri strain 121
cells produce both a glucan (EPS121) and a fructan on sucrose
(Suc) (α-D-Glcp-(1 ↔ 2)-β-D-Fruf) (Van Geel-Schutten et al.
1998). The water-soluble glucan was identified as a high-
molecular-mass (3.5 × 106 Da) branched α-D-glucan with terminal,
4-substituted, 6-substituted and 4,6-disubstituted Glc units. The
fructan turned out to be a low-molecular-mass (1.5 × 105 Da)
(2 → 6)-β-D-fructofuranan or levan (Van Geel-Schutten et al.
1999). Interestingly, from chemostat cultures of L. reuteri strain
121, a spontaneous mutant L. reuteri strain 35-5 was isolated
that lacked all levansucrase activity, but still possessed glucan-
sucrase (glucosyltransferase (GTFA)/GTF; EC 2.4.1.5) activity
and produced the wild-type α-D-glucan (EPS35-5 = EPS121) in
amounts up to 10 g/L (Van Geel-Schutten et al. 1999).
Further studies with L. reuteri strain 121 resulted in isolation
of the gtfA and gtfB genes, encoding the GTFA and GTFB
enzymes, respectively (Kralj et al. 2002, 2011). The GTFA
enzyme is responsible for α-D-glucan biosynthesis from Suc
(Kralj et al. 2002). It belongs to glycoside hydrolase family 70
(GH70), a group of bacterial glucansucrase or GTFA enzymes
(Cantarel et al. 2009). Glucansucrase enzymes differ in reac-
tion/product specificity, synthesizing high-molecular-mass
α-D-glucans with different (ratios of) glycosidic linkages and
degree of branching, depending on the bacterial source of the
enzyme (Kralj, van Geel-Schutten, Dondorff et al. 2004). The
mechanistic and structural features determining these differ-
ences in glucansucrase reaction/product specificity are still not
fully understood, and little is known about the products synthe-
sized early in time (Monchois et al. 1999; Moulis et al. 2006;
Leemhuis, Pijning et al. 2013; Leemhuis, Dijkman et al. 2013).
Also the GTFB type of enzymes is classified in family
GH70. Recently, we have reported that the GTFB enzyme of L.
reuteri strain 121 (Kralj et al. 2011; Dobruchowska et al. 2012)
is inactive with Suc, but instead has hydrolysis/transferase ac-
tivity on malto-oligosaccharides (MOSs) and starch-like poly-
saccharides (Dijkhuizen et al. 2010). The elongated linear
gluco-oligomers formed contain besides (α1 → 4) a high
Received on May 10, 2013; revised on June 20, 2013; accepted on June 20,
2013
The probiotic bacterium Lactobacillus reuteri 121 produces a
complex, branched (1 → 4, 1 → 6)-α-D-glucan as extracellu-
lar polysaccharide (reuteran) from sucrose (Suc), using a
single glucansucrase/glucosyltransferase (GTFA) enzyme
(reuteransucrase). To gain insight into the reaction/product
specificity of the GTFA enzyme and the mechanism of reu-
teran formation, incubations with Suc and/or a series of
malto-oligosaccharides (MOSs) (degree of polymerization
(DP2–DP6)) were followed in time. The structures of the ini-
tially formed products, isolated via high-performance anion-
exchange chromatography, were analyzed by matrix-assisted
laser-desorption ionization time-of-flight mass spectrometry
and 1D/2D 1H/13C NMR spectroscopy. Incubations with Suc
only, acting as both donor and acceptor, resulted in elong-
ation of Suc with glucose (Glc) units via alternating (α1 → 4)
and (α1 → 6) linkages, yielding linear gluco-oligosaccharides
up to at least DP ꢀ 12. Simultaneously with the ensemble of
oligosaccharides, polymeric material was formed early on,
suggesting that alternan fragments longer than DP ꢀ 12
have higher affinity with the GTFA enzyme and are quickly
extended, yielding high-molecular-mass branched reuteran
(4 × 107 Da). MOSs (DP2–DP6) in the absence of Suc turned
out to be poor substrates. Incubations of GTFAwith Suc plus
MOSs as substrates resulted in preferential elongation of
MOSs (acceptors) with Glc units from Suc (donor). This ap-
parently reflects the higher affinity of GTFA for MOSs com-
pared with Suc. In accordance with the GTFA specificity,
most prominent products were oligosaccharides with an
(α1 → 4)/(α1 → 6) alternating structure.
1To whom correspondence should be addressed: Tel: +31-503632150;
Fax: +31-503632154; e-mail: l.dijkhuizen@rug.nl
© The Author 2013. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
1084

Synthesis of gluco-oligomers by GTFA from L. reuteri 121
percentage of (α1 → 6) glycosidic linkages. In view of its reac-
tion/product specificity, the GTFB enzyme was classified as a
4,6-α-glucanotransferase, the first example of such an enzyme
activity in family GH70, representing a new subfamily (Kralj
et al. 2011).
(HPAEC), matrix-assisted laser-desorption ionization time-of-
flight mass spectrometry (MALDI-TOF-MS) and 1D/2D 1H/13C
nuclear magnetic resonance (NMR) spectroscopy, total correl-
ation spectroscopy (TOCSY), H detected heteronuclear single
quantum coherence spectroscopy (HSQC) and rotating-frame
nuclear Overhauser enhancement spectroscopy (ROESY)).
1
Recombinant GTFA purified from Escherichia coli, and the
culture supernatants of L. reuteri strain 35-5, synthesized virtual
identical α-D-glucan polymers (reuteran = EPS35-5 = EPS121)
when incubated overnight with Suc (according to 1H NMR ana-
lysis). Their molecular masses were determined to be 8 × 107
and 4 × 107 Da, respectively. Additionally, a detailed molecular
(construction of site-directed and deletion mutants) and bio-
chemical (main reactions catalyzed by wild-type and mutant
enzymes) analysis of the GTFA enzyme, called reuteransucrase,
has been reported (Kralj, van Geel-Schutten, van der Maarel
et al. 2004). Recently, also the crystal structure of functional
GTFA-ΔN, a 118-kDa fragment of GTFA comprising residues
745–1763 and including the catalytic domain, was determined at
3.6 Å resolution by molecular replacement (Pijning et al. 2012).
A detailed structural analysis of the reuteran product formed
from Suc by the recombinant glucansucrase GTFA enzyme
yielded a (1 → 4, 1 → 6)-α-D-glucan, with no repeating units
present. The constructed composite model is presented in
Figure 1. It should be noted that the reuteran structure showed
a large number of alternating (α1 → 4)/(α1 → 6) linkages
(Van Leeuwen, Kralj et al. 2008).
When exploring the activity of the recombinant GTFA
enzyme with the MOS series DP2–DP7, only DP4 (maltote-
traose) gave some clear higher-molecular-mass bands on thin-
layer chromatography (TLC) (S.K., unpublished data). However,
recombinant GTFA enzyme incubated with equimolar mixtures
of Suc and maltose for 60 h resulted in formation of panose
(major), maltotriose (minor) and two unknown products (minor)
(demonstrated by high-performance anion-exchange chromatog-
raphy on CarboPac PA-1). When using equimolar amounts of
Suc and isomaltose, low amounts of isomaltotriose and isomalto-
tetraose were seen, together with two unknown products (Kralj,
van Geel-Schutten, van der Maarel et al. 2004).
Results
Gluco-oligosaccharides initially formed by GTFA from
sucrose
To identify the initially formed oligosaccharides, precursors of
reuteran, 50 mM Suc was incubated at 37°C with 50 nM re-
combinant GTFA in 25 mM sodium acetate buffer/1 mM
CaCl2, pH 4.7. The progress of the reaction in time was fol-
lowed by TLC (data not shown). The formation of lower- and
higher-molecular-mass products than the substrate Suc indi-
cated hydrolysis/transferase activity, demonstrating that the
GTFA enzyme is active in the absence of an exogenous primer.
The HPAEC profiles of the oligosaccharide reaction products
formed from Suc at t = 0, 1, 3 and 6 h are depicted in Figure 2.
Evaluation of these profiles clearly indicated that the α1 ↔ 2β
linkage of Suc as donor was split, yielding fructose (Fru) and
Glc. Fru accumulated during the reaction; however, Glc
remained low, because it was used mainly for the transfer to
Suc and its formed gluco-elongations as acceptors, yielding an
ensemble of nonreducing gluco-oligosaccharides of increasing
DP (as determined by MALDI-TOF-MS analysis) with Fru at
the end. Complementary analysis performed by size-exclusion
chromatography revealed the presence of two major popula-
tions of products, increasing simultaneously during the reac-
tion. A peak with polymeric material eluted from 20 to 24 min
and a peak corresponding to oligosaccharides eluted from 30 to
32 min. The polymer formed after 15 min of incubation was
estimated to be already larger than 2 × 107 Da (data not shown).
To get insight into the structures of the formed oligosacchar-
ides in the reaction mixtures, the HPAEC fractions 2–11 were
isolated and studied by MALDI-TOF-MS and NMR spectros-
copy (1H, TOCSY, 1H–13C HSQC and ROESY). In the assign-
ment of the various NMR signals, our earlier developed
structural-reporter-group concept for α-D-glucans played an im-
portant role (Van Leeuwen, Kralj et al. 2008; Van Leeuwen,
Leeflang et al. 2008). Analysis of the peaks after a 1-h incuba-
tion showed that Glc is used to elongate Suc with an (α1 → 4)-
(Compound 3) or an (α1 → 6)- (Compound 2) linked Glc unit.
Later in time (t = 3 h, 6 h), more products are formed by elong-
ation of Suc-containing oligosaccharides, whereby the system-
atic build-up of the structures is impressive. Successive (1 → 4)
elongation could be observed only up to DP4 (Compounds 3
and 5), but otherwise the products contained alternating
(1 → 6)/(1 → 4) linkages (Compounds 2 → 4 → 5′ → 6 → 7 →
8 → 9 → 10 → 11). Due to the decrease in PAD response
with increasing DP of products, together with their low abun-
dance, it was not possible to detect the formation of higher-
molecular-mass material by HPAEC. The minor amounts of
two non-Fru-containing compounds (the tri- and tetrasacchar-
ide marked with an asterisk in Figure 2, t = 6 h) most likely are
hydrolysis products of initially formed larger oligosaccharides.
It should be noted that Suc was not chemically hydrolyzed in
To gain a better understanding of the activity of the recombin-
ant GTFA enzyme, especially during the first hours of in vitro in-
cubation with Suc, the present study focused specifically on the
structural analysis of the initially formed oligosaccharides en
route to the polysaccharide reuteran. Furthermore, product mix-
tures from incubation of MOS with GTFA in the absence and
presence of sucrose were analyzed. In all cases, the glycan pro-
ducts were analyzed by high-pH anion-exchange chromatography
Fig. 1. Composite structure of the exopolysaccharide reuteran (EPS35-5/
EPS121) of L. reuteri 121 obtained by incubation of Suc with recombinant
GTFA enzyme (Van Leeuwen, Kralj et al. 2008).
1085

JM Dobruchowska et al.
Fig. 2. HPAEC-PAD profile (0–500 mM NaOAc gradient in 100 mM NaOH) on CarboPac PA-1 of the oligosaccharide mixture generated from Suc by incubation
with the GTFA enzyme at t = 0, 1, 3 and 6 h (pH 4.7, 37°C). Established oligosaccharide structures for isolated fractions are included. G = glucose; F = fructose.
Two non-Fru-containing compounds are marked with an asterisk.
the incubation buffer after 6 h at 37°C. Also, enzymatic
cleavage of the glycosidic bond in the Suc fragment of higher
oligosaccharides is highly improbable. Therefore, the presence
of these products indicates that GTFA has a minor endo-
(α1 → 4)-glycosidase activity, although it cannot be completely
excluded that Glc units are transferred for a very low percentage
onto free Glc as an acceptor. Finally, only a minor amount of
leucrose (α-D-Glcp-(1 → 5)-D-Fru; <2%) was found, indicating
that Fru is not a good acceptor substrate for GTFA.
As an example of the rationalization behind the interpretation
1
of the various H and 13C NMR assignments, the TOCSY,
ROESY and HSQC spectra of one of the reaction products,
Glc5Suc (Compound 7; m/z 1175.3, [M + Na]+ according to
MALDI-TOF-MS), are shown in Figure 4. Typical chemical
shift values for the Suc fragment (Glc, g; Fru, f) are δ 5.427,
stemming from Glc g H-1 and δ 4.220, stemming from Fru f
1
H-3. The H chemical shifts of the anomeric signals around δ
5.37 and 4.95 indicate the presence of (α1 → 4) and (α1 → 6)
linkages, respectively, between Glc residues (Van Leeuwen,
Leeflang et al. 2008). Starting from the anomeric signals of the
1
The 1D H NMR spectra of Compounds 1–11, including
some exact chemical shift values, are depicted in Figure 3. For
further NMR data, see Table I. An adequate denotation system
for the constituent residues was necessary (see Scheme I),
since there are small variations of some of the chemical shift
values of the different Glc residues, depending on their position
in the oligosaccharide structure.
1
Glc residues AX, AI, BI, BII and E in the 2D H–1H TOCSY
spectra (20, 50, 100 and 200 ms), and additional data from the
HSQC and ROESYexperiments, all chemical shifts of the non-
anomeric protons of the differently substituted Glc residues
could be determined (Table I). Although the anomeric signals
1086

Synthesis of gluco-oligomers by GTFA from L. reuteri 121
Fig. 3. 1H NMR spectra (D2O, 300 K) of Compounds 1–11 (Figure 2) found in the oligosaccharide mixture after incubation of Suc with the GTFA enzyme for 6 h
(pH 4.7, 37°C).
1087

JM Dobruchowska et al.
Table I. 1H and 13C chemical shiftsa (D2O, 300 K) of saccharide residues present in linear oligomers (DP ≥ 4) formed by incubation of Suc with the GTFA enzyme
of L. reuteri 121
Residue
H-1a
C-1
H-1b
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6a
C-6
H-6b
4.02
3.76
g
5.427
92.9
–
–
3.57
71.8
3.74
73.4
3.52
70.5
4.05
72.3
3.69
66.9
-(1 → 6)-α-D-Glcp-
(1 ↔ 2)-β-D-Fruf
g′
-(1 → 4)-α-D-Glcp-
(1 ↔ 2)-β-D-Fruf
f [g → f]
5.402
93.1
3.58
72.2
4.02
75.1
3.69
78.0
3.96
72.1
3.85
61.5
3.67
62.5
3.66
62.5
3.67
3.66
–
-
4.220
77.3
4.20
77.3
3.69
74.3
4.01
74.4
3.67
73.8
3.74
72.8
4.05
74.9
4.02
3.90
82.3
3.89
82.3
3.94
72.4
3.85
71.3
3.73
73.7
3.75
72.3
3.85
63.5
3.86
3.78
-(1 ↔ 2)-β-D-Fruf
f [g′ → f]
104.5
-
3.80
-(1 ↔ 2)-β-D-Fruf
104.5
3.59
72.4
3.60
72.5
3.59
72.7
3.56
72.4
74.9
63.5
AX,I,II
5.38X/37I/36IIb
100.7
3.49X/46I,II
70.5
3.73X/77I,II
66.5
3.98X/94I,II
3.83
-(1 → 6)-α-D-Glcp-(1 → 4)-
BI,II,X
-(1 → 4)-α-D-Glcp-(1 → 6)-
D
α-D-Glcp-(1 → 4)-
E
α-D-Glcp-(1 → 6)-
4.96I/95II,Xb
98.9
–
3.63I/65II,X
78.5
3.87
61.5
3.84
61.5
3.85
61.5
5.40/39b
100.7
–
3.417
70.3
3.426
70.5
3.75
4.954
98.8
–
3.77
aIn ppm relative to the signal of internal acetone (δ 2.225 for 1H and δ 31.07 for 13C).
bAverage chemical shifts values of the anomeric protons (see exact values: 1H NMR spectra, Figure 3).
Scheme I. Monosaccharide coding system for the discussed oligosaccharides
The set of chemical shifts of g H-2, H-3, H-4, H-5 and H-6a/b
at δ 3.57, 3.74, 3.52, 4.05 and 3.69/4.02, respectively, corre-
sponds with a 6-O-substituted g residue [-(1 → 6)-α-D-Glcp-
(1 ↔ 2)-β-D-Fruf]. The TOCSY f H-3 track (δ 4.220) revealed
cross-peaks with f H-4, f H-5 and f H-6a/b at δ 4.05, 3.90 and
3.85/3.78, respectively, in agreement with literature data
(Timmermans et al. 1993).
The sequence of the five Glc residues followed directly from
the ROESY experiments, revealing inter-residual cross-peaks
between E H-1 and AX H-6a (E1 → 6AX), AX H-1 and BII H-4
(AX1 → 4BII), BII H-1 and AI H-6a (BII1 → 6AI), AI H-1 and
BI H-4 (AI1 → 4BI) and BI H-1 and g H-6a (BI1 → 6g). The 4-
and 6-substitutions of the residues BI/BII and AX/AI/g, respect-
ively, were further supported by their downfield C-4 and C-6
chemical shifts, compared with terminal Glc (deduced from
13C–1H HSQC measurements): BI/BII C-4 at δ 78.5 and AX/
AI/g C-6 at δ 66.5/66.9.
In conclusion, the NMR chemical shift data, together with
the peak areas of the anomeric signals and the molecular mass
(1152 Da), revealed the complete structure of the oligosacchar-
ide, being a linear heptasaccharide consisting of Suc elongated
by five Glc residues with alternating (α1 → 6)/(α1 → 4) glyco-
sidic linkages: α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 4)-α-D-Glcp-
(1 → 6)-α-D-Glcp-(1 → 4)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 2)-
β-D-Fruf.
Residue
Position in the oligosaccharide sequence
g-f
Suc of which Glc (g) is substituted at O-6
g′-f
Suc of which Glc (g′) is substituted at O-4
AI
-(1 → 6)-α-D-Glcp-(1 → 4)- A residue, most close to Suc
More remote -(1 → 6)-α-D-Glcp-(1 → 4)- A residues
Sub-terminal -(1 → 6)-α-D-Glcp-(1 → 4)- A residue
-(1 → 4)-α-D-Glcp-(1 → 6)- B residue, most close to Suc
More remote -(1 → 4)-α-D-Glcp-(1 → 6)- B residues
Sub-terminal -(1 → 6)-α-D-Glcp-(1 → 4)- B residue
Terminal α-D-Glcp-(1→4)- residue
AII,III
AX
BI
BII,III
BX
D
E
Terminal α-D-Glcp-(1→6)- residue
of E (H-1, δ 4.954) and BI/BII (H-1, δ 4.963/4.954) strongly
overlap, the differences in chemical shift of their H-3, H-4,
H-5, H-6 signals could be deduced from the TOCSY built-up
series of mixing times (20, 50, 100 and 200 ms; Figure 4,
200 ms). The set of chemical shifts of E H-2, H-3, H-4, H-5
and H-6a/b at δ 3.56, 3.74, 3.426, 3.75 and 3.85/3.77, respect-
ively, corresponds with that of a terminal α-D-Glcp-(1 → 6)-
unit. The set of chemical shifts of BI/BII H-2, H-3, H-4I/H-4II,
H-5 and H6a/b at δ 3.60, 4.01, 3.63I/3.65II, 3.85 and 3.87/3.83,
respectively, corresponds with that of internal -(1 → 4)-α-D-
Glcp-(1 → 6)- units. The small differences in chemical shift
between BI H-1 and BII H-1 and between BI H-4 and BII H-4
are due to the proximity of BI to the Suc fragment (ROESY
data, see below). The sets of chemical shifts of AX and AI
reflect the presence of internal -(1 → 6)-α-D-Glcp-(1 → 4)-
units (AX/AI: H-1, δ 5.380/5.367; H-2, δ 3.59; H-3, δ 3.69;
H-4X/H-4I, δ 3.49/3.46; H-5, δ 3.94; H6aX/bX and H6aI/bI, δ
3.73X/3.98X and δ 3.77I/3.94I). In this case, the small differ-
ences in chemical shift for H-1, H-4 and H6a/b are due to the
proximity of AI to the Suc fragment (ROESY data, see below).
The data show that during the first 6 h of incubation with
Suc, GTFA mainly produces linear oligosaccharides with alter-
nating (1 → 6)/(1 → 4) linkages; no branched oligosaccharides
were formed. However, the consecutively formed polymeric
material starting from DP14 on clearly contained → 4,6)-
1
branched Glc residues, as indicated by H-NMR signals at δ
5.33. The degree of branching may increase during the further
growth of the glucan chain. Up to DP20, the presence of the
Suc fragment in the structures could be observed in the NMR
1088

Synthesis of gluco-oligomers by GTFA from L. reuteri 121
Fig. 5. 1H NMR spectrum of the void-volume Bio-Gel P-2 polysaccharide
fraction, obtained after 72 h incubation of Suc with the GTFA enzyme. The
spectrum is identical to the 1H NMR spectrum of the native polysaccharide
produced by L. reuteri 121 (Van Leeuwen, Kralj et al. 2008).
containing 14% of →4,6)-branching, for which the composite
model is depicted in Figure 1 (Van Leeuwen, Kralj et al. 2008).
Note that the NMR signals of Suc/Fru are no longer observable
in the NMR spectrum of the polysaccharide.
Products of the GTFA enzyme from malto-oligosaccharide
substrates in the absence of Suc
To generate more information about the earlier observed activ-
ity of recombinant GTFAwith MOSs, a detailed structural ana-
lysis of products formed was carried out. To this end, 100 mM
solutions of MOS (DP2–DP6) were individually incubated for
different times up to 72 h at 37°C with 50 nM recombinant
GTFA in 25 mM sodium acetate buffer/1 mM CaCl2, pH 4.7.
TLC analysis (data not shown) of the various incubation mix-
tures revealed that only for MOS DP > 3, the GTFA enzyme
clearly possessed hydrolysis/transferase activity. GTFA con-
verted only minor amounts of maltose and maltotriose. For
MOS DP4–DP6, the yield of product formation was higher but
still less than observed during the incubation of Suc together
with maltose (see below) under the same conditions and incu-
bation time. GTFA thus has relatively minor activity with these
MOS substrates.
Incubation of maltose (MOS DP2) with the GTFA enzyme
for 24 h on a semi-preparative scale, followed by HPAEC pro-
filing, product isolation, MALDI-TOF-MS and NMR analysis,
showed only minimal formation of Glc and panose [α-D-Glcp-
(1 → 6)-α-D-Glcp-(1 → 4)-D-Glcp] (Compound 3) (Figure 6), in-
dicating that GTFA is poorly hydrolyzing maltose. HPAEC-PAD
profiling of the incubations of GTFA with MOS DP3–DP6 over
periods up to 30 h gave more insight into the type of products
formed. The chromatograms of the incubations of DP3 and DP4 at
relevant incubation times are shown in Supplementary data,
Figure S1 and Figure 7, respectively. Most of the fractions isolated
were analyzed by MALDI-TOF-MS and 1D/2D NMR spectros-
Fig. 4. 1D 1H NMR, TOCSY (200 ms), ROESY (200 ms) and HSQC spectra
of α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 4)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 4)-
α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 2)-β-D-Fruf (Compound 7 in Figure 2).
1
copy (including TOCSY, H–13C HSQC and ROESY experi-
ments). For general 1H and 13C NMR data, see Tables II and III.
The established structures are included in the various figures.
Next to the hydrolysis products Glc (Compound 2) and
maltose (Compound 3), the transfer product (Compound 4)
formed from maltotriose (MOS DP3) at t = 6 h is maltotetraose
spectra by the small signal at δ 4.220 stemming from Fruf H-3.
The polysaccharide material at 72 h of incubation was isolated
via size-exclusion chromatography on Bio-Gel P2 by collecting
the void-volume fraction. The 1D 1H NMR spectrum (Figure 5)
of this fraction was identical to the spectrum of reuteran,
1089

JM Dobruchowska et al.
To illustrate the structure determination of oligosaccharides
with a (1 → 2)-linked α-D-Glcp residue in a reducing end → 2,4)-
D-Glcp situation, the NMR analysis of Compound 13 (Figure 7)
(tetrasaccharide; MALDI-TOF-MS m/z 689.3, [M + Na]+) is pre-
sented here in more detail. The 1D 1H NMR spectrum of
Compound 13, together with the TOCSY (200 ms), ROESY
(200 ms) and HSQC spectra, is depicted in Figure 8, and the
various 1H and 13C NMR data are presented in Table II.
The 1D 1H NMR spectrum of Compound 13 showed 6
anomeric signals, which were used as starting points for the as-
signment of the chemical shifts of all nonanomeric protons in the
2D TOCSY spectrum (TOCSY spectra with spin-lock times 20,
50, 100 and 200 ms were used to acquire all assignments). The
H-1 signals at δ 5.441 (Rα1) and 4.809 (Rβ1) are in agreement
with a reducing -(1 → 2,4)-D-Glcp R unit (http://www.casper.
organ.su.se/casper/; Jansson et al. 2006). The splitting of the G
H-1 doublet into two doublets (Gα H-1, δ 5.099 and Gβ H-1, δ
5.387) corresponds with the long-range effect induced by the α-
and β-anomeric forms of residue R (Van Leeuwen, Leeflang
et al. 2008). The anomeric signals at δ 5.433 and 4.958 reflect
the presence of an internal -(1 → 6)-α-D-Glcp-(1 → 4)- unit and
a terminal α-D-Glcp-(1 → 6)- unit, respectively (Dobruchowska
et al. 2012). In the 2D ROESY spectrum, strong inter-residual
couplings were observed between Gα H-1 and Rα H-2, Gβ H-1
and Rβ H-2, A H-1 and Rα H-4, A H-1 and Rβ H-4 and E H-1
and A H-6a/b, in accordance with the structure α-D-Glcp-(1 → 6)-
α-D-Glcp-(1 → 4)-[α-D-Glcp-(1 → 2)-]D-Glcp. The various substi-
tution patterns are confirmed by their 13C NMR data, deduced
from the 13C–1H HSQC spectrum: Rα (C-2, δ 76.5; C-4, δ 77.5),
Rβ (C-2, δ 79.5; C-4, δ 77.5), A (C-6, δ 66.7) (Bock
and Thøgersen 1982; Van Leeuwen, Leeflang et al. 2008;
Dobruchowska et al. 2012).
Incubation of maltohexaose (MOS DP6) with GTFA yielded,
after 30 h, 23 isolated products (Figure 9) (MALDI-TOF-MS:
DP2–DP9; [M + Na]+, m/z 365.3–1661.2), which were sub-
jected to a detailed structural analysis. The major compounds
comprised structures 2 (Glc), 8 (maltose), 7 (panose), 6 (malto-
triose), 3 (maltopentaose), 5 (maltopentaose elongated with an
(1 → 6)-linked α-D-Glcp unit), 1 (maltohexaose) and 4 (malto-
hexaose elongated with an (1 → 6)-linked α-D-Glcp unit), indi-
cating hydrolysis activity and an (α1 → 4) → (α1 → 6) linkage
transfer of a single α-D-Glcp unit. Besides structures similar to
those seen for MOS DP4, including relatively minor products
with an (1 → 2)-linked α-D-Glcp unit (Compounds 10, 11 and
13), a minor 4,6-branched structure was identified (Compound
22). Interestingly, in this case also minor structures with alter-
nating (α1 → 6)/(α1 → 4) linkages were detected (for NMR
details, see Table III). Linear sequences of (1 → 6)-linked
α-D-Glcp units, as seen in the incubation of MOS DP4 with
GTFA, were not observed with MOS DP6. 1D 1H NMR spectra
of Compounds 19, 21, 22 and 23 (Figure 9), including some
exact chemical shift values, are depicted in Supplementary
data, Figure S2.
Fig. 6. HPAEC-PAD profile (0–500 mM NaOAc gradient in 100 mM NaOH)
on CarboPac PA-1 of the oligosaccharide mixture generated from maltose
(MOS DP2) with the GTFA enzyme at t = 0 and 24 h (pH 4.7, 37°C).
Established oligosaccharide structures for isolated fractions are included.
(MOS DP4), the result of an (α1 → 4) → (α1 → 4) transfer of a
single α-D-Glcp unit (Supplementary data, Figure S1). After 24
h, an (α1 → 4) → (α1 → 6) linkage transfer of a single α-D-Glcp
unit to the nonreducing end and an (α1 → 4) → (α1 → 2)
linkage transfer of a single α-D-Glcp unit to the reducing end of
maltotriose was observed. Furthermore, panose (Compound 5)
was detected, showing that formed maltose also acts as an ac-
ceptor for an (α1 → 6) elongation, in agreement with the direct
incubation of maltose with GTFA.
Incubation of maltotetraose (MOS DP4) with GTFA yielded,
after 30 h, an interesting ensemble of products (MALDI-TOF-
MS showed DP2–DP6; [M + Na]+, m/z 365.2–1661.1); the
major higher-molecular-mass product at t = 12 h comprised an
(α1 → 4) → (α1 → 6) linkage transfer of a single α-D-Glcp
unit to the nonreducing end of maltotetraose (Compound 4 in
Figure 7). As can be seen from the sixteen isolated products char-
acterized (Figure 7), GTFA displays hydrolysis (Compounds 2, 3
and 9), as well as disproportionation activity, namely elongations
with α-D-Glcp-(1 → 6) (Compounds 4, 6, 7, 10 and 11) and
α-D-Glcp-(1 → 4) (Compound 5) units at the nonreducing end,
as well as elongations with a single α-D-Glcp-(1 → 2) unit at the
reducing end (Compounds 8, 12 and 13). After prolonged incuba-
tion, small amounts of higher-molecular-mass isomalto-/
malto-oligomers were detected (Compounds 14, 15 and 16), a
type of structure that has been found earlier as products from
incubations of MOS with GTFB, although with longer sequences
of (1 → 6)-linked α-D-Glcp units (Dobruchowska et al. 2012).
GTFA, thus, also has (minor) 4,6-α-glucanotransferase activity.
Note that after 30 h, the original donor substrate maltotetraose
(Compound 1) had nearly disappeared. Higher polymeric mater-
ial could not be detected: Only oligosaccharide products (up to
DP10) were observed. The 1D 1H NMR spectra of Compounds 4
and 16 (Figure 7), including some exact chemical shift values, are
depicted in Supplementary data, Figure S2.
Products of the GTFA enzyme incubated with MOSs plus Suc
as substrates
Previously, it has been observed that the presence of both
maltose and Suc influences the GTFA-catalyzed spectrum of
generated oligosaccharides, compared with Suc alone as
1090

Synthesis of gluco-oligomers by GTFA from L. reuteri 121
Fig. 7. HPAEC-PAD profile (0–500 mM NaOAc gradient in 100 mM NaOH) on CarboPac PA-1 of the oligosaccharide mixture generated from maltotetraose (MOS
DP4) with the GTFA enzyme at t = 0, 12 and 30 h (pH 4.7, 37°C). Established oligosaccharide structures for isolated fractions are included.
substrate (Kralj, van Geel-Schutten, van der Maarel et al.
2004). For a more detailed structural analysis, solutions of 100
mM sucrose and 100 mM MOS (DP2–DP6) were each incu-
bated with 50 nM recombinant GTFA in 25 mM sodium
acetate buffer/1 mM CaCl2, pH 4.7, at 37°C. The reaction was
followed in time by TLC screening (data not shown).
Inspection of the HPAEC profiles of products formed after 1,
3 and 6 h incubation of sucrose and maltose with GTFA
(Figure 10) showed the formation of a series of products up
to DP7, and after 6 h, the complete disappearance of Suc.
Prolonged incubation resulted in an increase in molecular size of
the products, but the amounts were very low (according to TLC
and MALDI-TOF-MS screening), and these were not studied
in more detail. Most of the products formed within 6 h were
isolated and investigated by MALDI-TOF-MS and 1D/2D
NMR spectroscopy. Surprisingly, the majority of the initially
formed compounds did not stem from elongations of Suc, but
from extensions of maltose by Glc residues. Apparently, GTFA
cleaves Suc and the Glc units generated are preferentially used to
elongate maltose instead of Suc. Only traces of Suc elongations,
i.e., two trisaccharides (marked with an asterisk, Figure 10) were
detected. Fru and panose (Compound 3 in Figure 10) were found
as the most abundant products. The (α1 → 4)/(α1 → 6) alternat-
ing build-up of the structures of the minor oligosaccharides 5, 6,
11 and 12 in Figure 10 is similar to the Suc-containing structures
found for the Suc incubation with GTFA (Figure 2). In contrast
to the “sucrose-only” incubation, just a minor amount of
polymeric material (DP > 20 according to MALDI-TOF-MS)
was detected after prolonged incubations, and now a → 4,6)-
branched structure was already seen after 6 h of incubation
(structure 5′, Figure 10). Also in this case, the three structures
with (1 → 2)-linked α-D-Glcp, earlier found in the GTFA incuba-
tions with maltotriose (only one of the three), maltotetraose and
maltohexaose (Supplementary data, Figure S1; Figures 7 and 9),
were detected. Compound 9 (Figure 10) was also traced in the in-
cubation of maltotetraose with GTFA (Compound 14 in
Figure 7), showing the possibilities of synthesizing linear
sequences of (1 → 6)-linked α-D-Glcp units. For a survey of the
1091

JM Dobruchowska et al.
Table II. 1H and 13C chemical shiftsa (D2O, 310 K) of Glc residues present in -2,4)-substituted-oligomers formed by incubation of MOSs (with and without Suc)
with the GTFA enzyme of L. reuteri 121
Residue
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6a
C-6
H-6b
A
5.433
100.5
5.45/40b
100.6
4.958
99.0
5.432
100.0
5.44b
90.1
3.58
72.4
3.59
72.5
3.56
72.3
3.61
72.3
3.68
76.5
3.43
79.5
3.56
72.3
3.55
72.3
3.68
73.3
3.69
73.8
3.74
73.9
3.96
74.1
4.09
72.5
3.87
75.8
3.78
73.6
3.75
73.6
3.50
70.2
3.43
70.2
3.43
70.3
3.65
77.7
3.71
77.5
3.68
77.5
3.45
70.3
3.45
70.3
3.90
71.1
3.73
73.6
3.72
72.6
3.85
72.0
3.97
70.6
3.60
75.3
3.91
72.7
3.99
72.5
3.74
66.7
3.85
61.3
3.85
61.2
3.89
61.3
3.87
61.2
3.94
61.7
3.85
61.2
3.82
61.2
3.98
3.77
3.76
3.82
3.83
3.77
3.80
3.79
-(1 → 6)-α-D-Glcp-(1 → 4)-
D
α-D-Glcp-(1 → 4)-
E
α-D-Glcp-(1 → 6)-
F
-(1 → 4)-α-D-Glcp-(1 → 4)-
Rα
-(1 → 2,4)-α-D-Glcp
Rβ
-(1 → 2,4)-β-D-Glcp
Gα
α-D-Glcp-(1 → 2)-
Gβ
4.809
97.0
5.099
97.2
5.40/38b
98.6
α-D-Glcp-(1 → 2)-
aIn ppm relative to the signal of internal acetone (δ 2.225 for 1H and δ 31.07 for 13C).
bAverage chemical shifts values of the anomeric protons (see exact values: 1H NMR spectra, Supplementary data, Figure S4).
Table III. 1H and 13C chemical shiftsa (D2O, 300 K) of Glc residues present in oligomers (DP ≥ 4) formed by incubation of MOS (with and without Suc) with the
GTFA enzyme of L. reuteri 121
Residue
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6a
C-6
H-6b
AI,II,III,X
5.40X/38I,III/36IIb
101.0
3.59
72.7
3.60
72.2
3.65
-
3.59
72.5
3.56
72.5
3.62
72.5
3.58
72.3
3.56
72.3
3.27
75.0
3.69
74.1
4.00
74.4
3.97
-
3.67
73.9
3.75
72.7
3.96
74.3
3.70
74.3
3.97
74.2
3.77
77.2
3.49X/46I,II,III
70.4
3.65
78.5
3.74
-
3.42
70.5
3.43
70.5
3.66
77.8
3.51
70.6
3.61
78.5
3.62
78.5
3.94
72.4
3.85
71.4
4.01
-
3.74
73.9
3.75
72.4
3.84
72.2
3.91
71.2
3.95
71.0
3.61
75.6
3.73X/77I,II,III
66.9X/67.3I,II,III
3.89
61.5
3.83
3.98X/94I,II,III
66.9X/67.3I,II,III
3.82
-(1 → 6)-α-D-Glcp-(1 → 4)-
BII,X
4.98/95II,Xb
99.0
-(1 → 4)-α-D-Glcp-(1 → 6)-
C
-(1→4,6)-α-D-Glcp-(1→4)-
D
α-D-Glcp-(1 → 4)-
E
α-D-Glcp-(1 → 6)-
F
-(1 → 4)-α-D-Glcp-(1 → 4)-
J
-(1→6)-α-D-Glcp-(1→6)-
Rα
-(1 → 4)-α-D-Glcp
Rβ
-(1 → 4)-β-D-Glcp
5.36b
4.03
3.75
3.77
3.81
3.98
3.83
3.80
-
-
5.39/38/34b
99.0
3.84
61.5
3.85
61.5
3.88
61.5
3.76
66.7
3.88
61.5
3.94
61.9
4.99/95b
99.0
5.39b
100.7
4.955
98.8
5.22/23b
92.9
4.650
96.8
aIn ppm relative to the signal of internal acetone (δ 2.225 for 1H and δ 31.07 for 13C).
bAverage chemical shifts values of the anomeric protons (see exact values: 1H NMR spectra, Supplementary data, Figures S2 and S3).
1H and 13C NMR data of the various compounds identified, see
Table II [→2,4)-branched oligosaccharide products] and
Table III [alternating (α1 → 4) and (α1 → 6) linkages]. For a
survey of some typical 1D 1H NMR spectra (Compounds 5, 5′,
6, 7, 8, 10, 11 and 12 in Figure 10), including some exact chem-
ical shift values, see Supplementary data, Figures S3 and S4.
Incubation of GTFA with Suc/MOS DP3–DP6 resulted in
transferase activity in each case, yielding products similar (data
not shown) to those described for the Suc/MOS DP2 experi-
ment, confirming the GTFA preference for Glc transfer to MOS
acceptors above transfer to Suc. This clearly reflects a higher af-
finity of GTFA for MOS than for Suc as acceptor substrate.
Discussion
Detailed characterization of the initially formed oligosacchar-
ides in the biosynthesis of reuteran (EPS121, EPS35-5) from
Suc with the L. reuteri GTFA enzyme as catalyst clearly shows
that it possesses mainly transferase activity. Under the applied
conditions, GTFA transfers Glc residues from donor Suc to
acceptor Suc and to the growing glucan chain attached to a Suc.
Such a reaction was suggested earlier in a study of the mechan-
ism of a Leuconostoc mesenteroides NRRL B-512F dextranase
and a L. mesenteroides NRRL B-1355 alternansucrase, whereby
product oligosaccharides of unknown structure were found, of
1092

Synthesis of gluco-oligomers by GTFA from L. reuteri 121
Our ongoing studies aim to identify these different binding
possibilities. After growth of the oligomer up to DP14, →4,6)-
branching occurred and increased during further growth of
the polymer (DP > 20) to an ultimate 14% branching content.
Currently, experiments are in progress to investigate the effects
of different Suc concentrations on the synthesis of these oligo/
polysaccharides and to obtain further information about the
branching reaction occurring during prolonged incubation.
Also, the GTFA structural features determining the molecular
mass of its EPS formed remain to be elucidated.
Replacement of Suc by MOSs (DP2–DP6) in the incubation
with GTFA showed that the latter compounds were relatively
poor substrates, and very low amounts of only relatively short
oligosaccharides (<DP10) were generated in time; no polymer
formation was seen and the major reaction was hydrolysis. The
established structures showed besides (α1 → 4) linkages, elonga-
tions at the nonreducing end with (1 → 6)-linked α-D-Glcp and
at the reducing end with (1 → 2)-linked α-D-Glcp residues.
It should be noted that (α1 → 2) linkages have not been found
in the detailed structural analysis of reuteran (EPS121, EPS35-5)
(Van Leeuwen, Kralj et al. 2008). So this minor (α1 → 2)-
glucosylation activity of GTFA is only seen when it is incubated
with relatively high concentrations of MOS, containing a redu-
cing Glc residue. Recently, the 3D structure of an (α1 → 2)-
branching glucansucrase, isolated from a L. mesenteroides strain,
transferring Glc units from Suc onto gluco-oligosaccharides, has
been reported (Brison et al. 2012, 2013).
Incubations of GTFA with MOS plus equimolar amounts of
Suc revealed that the enzyme initially uses Suc as Glc donor
and MOS as acceptor substrates. Previous studies have shown
that maltose is the most effective acceptor for an alternansu-
crase enzyme of a L. mesenteroides strain (Côté et al. 2008).
The GTFA enzyme clearly forms novel and interesting (alter-
nan) oligosaccharide mixtures. These compounds will be further
evaluated for their prebiotic properties and ability to reduce the
blood Glc levels after food consumption (Dijkhuizen et al. 2010;
Monsan and Ouarné 2010; Monsan et al. 2010; Leemhuis,
Pijning et al. 2013).
Materials and methods
Fig. 8. 1D 1H NMR, TOCSY (200 ms), ROESY (200 ms) and HSQC spectra
of α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 4)-[α-D-Glcp-(1 → 2)]-α/β-D-Glcp
(Compound 13 in Figure 7).
Preparation and isolation of recombinant GTFA enzyme
The recombinant N-terminally truncated GTFA enzyme was
prepared and isolated as previously described (Kralj, van
Geel-Schutten, Dondorff et al. 2004).
which methylation analysis and NMR spectroscopy showed the
presence of Fru or Suc located at the oligosaccharide extremity
(Moulis et al. 2006).
Incubation of Suc and MOSs with GTFA
Before incubation, commercially available MOSs (DP2–DP6)
were purified via size-exclusion chromatography on Bio-Gel
P-2 (DP2–DP4) or via HPAEC on CarboPac PA-1 (DP5 and
DP6). Solutions of Suc (50 mM) and the purified MOSs (100
mM) were individually incubated with 50 nM GTFA in 25 mM
sodium acetate/1 mM CaCl2, pH 4.7, at 37°C. The progress of
the reactions was followed by analyzing aliquots of the incuba-
tion mixtures by TLC, MALDI-TOF-MS and HPAEC-PAD.
Furthermore, mixtures of malto-oligosacharides (DP2–DP6,
100 mM), individually together with Suc (100 mM), were incu-
bated in a similar way. Comparable incubations without the
addition of GTFA showed no product formation.
Incubation of Suc with the GTFA enzyme resulted
initially in synthesis of oligosaccharides that were all linear.
The transferred Glc residues were mainly attached in alternating
(α1 → 6)/(α1 → 4) linkages, forming a Suc-containing oligo-/
polysaccharide; no linear (α1 → 6) sequences were found. This
observation provides important new insights into the reaction
specificity of the GTFA reuteransucrase enzyme. Apparently,
the GTFA reaction mechanism seems to dictate alternate
binding of the growing linear oligosaccharide acceptor sub-
strates in two different ways in or near the active site, resulting
in alternate formation of (α1 → 6) and (α1 → 4) linkages.
1093

JM Dobruchowska et al.
Fig. 9. HPAEC-PAD profile (0–500 mM NaOAc gradient in 100 mM NaOH) on CarboPac PA-1 of the oligosaccharide mixture generated from maltohexaose (MOS
DP6) with the GTFA enzyme at t = 0, 12 and 30 h (pH 4.7, 37°C). Established oligosaccharide structures for isolated fractions are included.
Isolation and purification of product oligosaccharides
Matrix-assisted laser-desorption ionization time-of-flight
mass spectrometry
Product mixtures of oligosaccharides obtained after incubation
were fractionated by HPAEC on a Dionex DX500 workstation
(Dionex, Amsterdam, The Netherlands), equipped with a
CarboPac PA-1 column (250 × 9 mm; Dionex) and an ED40
pulsed amperometric detector (PAD), using a linear gradient of
0–500 mM sodium acetate in 100 mM NaOH (3 mL/min) or
isocratic conditions of 100 mM sodium acetate in 100 mM
NaOH (3 mL/min). Collected fractions were immediately neu-
tralized with 4 M acetic acid, desalted on CarboGraph SPE
columns (Alltech, Breda, The Netherlands) using acetonitrile:
water = 1:3 as eluent and lyophilized. Polysaccharide material
(DP > 14) was isolated in the void volume on a Bio-Gel P-2
column (90 × 1 cm), eluted with 10 mM NH4HCO3 at a flow
rate of 12 mL/h.
MALDI-TOF-MS experiments were performed on an Axima™
mass spectrometer (Shimadzu Kratos, Inc., Manchester, UK)
equipped with a nitrogen laser (337 nm, 3 ns pulse width).
Positive-ion mode spectra were recorded using the reflector
mode at a resolution of 5000 full width at half maximum and
delayed extraction (450 ns). The accelerating voltage was 19
kV with a grid voltage of 75.2%; the mirror voltage ratio was
1.12, and the acquisition mass range was 200–6000 Da.
Samples were prepared by mixing on the target 0.5 μL sample
solutions with 0.5 μL aqueous 10% 2,5-dihydroxybenzoic acid
as matrix solution.
NMR spectroscopy
Resolution-enhanced 1D/2D 500-MHz H NMR spectra were
1
recorded in D2O on a Bruker DRX-500 spectrometer (Bijvoet
Center, Department of NMR Spectroscopy, Utrecht University)
or a Varian Inova Spectrometer (NMR Center, University of
Groningen) at probe temperatures of 300/310 K. Before ana-
lysis, samples were exchanged twice in D2O (99.9 atom% D,
Cambridge Isotope Laboratories, Inc., Andover, MA) with
intermediate lyophilization, and then dissolved in 0.6 mL
Thin-layer chromatography
Samples were spotted in 1-cm lines on TLC sheets (Merck
Kieselgel 60 F254, 20 × 20 cm), which were developed with
n-butanol:acetic acid:water = 2:1:1. Bands were visualized by
orcinol/sulfuric acid staining and compared with a simultan-
eous run of standard oligosaccharides.
1094

Synthesis of gluco-oligomers by GTFA from L. reuteri 121
Fig. 10. HPAEC-PAD profile (0–500 mM NaOAc gradient in 100 mM NaOH) on CarboPac PA-1 of the oligosaccharide mixture generated from Suc + maltose
(MOS DP2) with the GTFA enzyme at t = 0, 1, 3 and 6 h (pH 4.7, 37°C). Established oligosaccharide structures for isolated fractions are included. G = glucose;
F = fructose. Two Fru-containing compounds are marked with an asterisk.
D2O. Suppression of the HOD signal was achieved by applying
a water eliminated Fourier transform pulse sequence for 1D
experiments and by a pre-saturation of 1 s during the relaxation
delay in 2D experiments. The 2D TOCSY spectra were
recorded using an MLEV-17 (composite pulse devised by
Levitt et al. (1982)) mixing sequence with spin-lock times of
20–200 ms. The 2D ROESY spectra were recorded using the
standard Bruker XWINNMR software with a mixing time of
200 ms. The carrier frequency was set at the downfield edge of
the spectrum in order to minimize TOCSY transfer during spin-
locking. Natural abundance 2D 13C–1H HSQC experiments
(1H frequency 500.0821 MHz, 13C frequency 125.7552 MHz)
were recorded without decoupling during acquisition of the 1H
free induction decay. Resolution enhancement of the spectra
was performed by a Lorentzian-to-Gaussian transformation for
1D spectra or by multiplication with a squared-bell function
phase shifted by π/(2.3) for 2D spectra, and when necessary, a
fifth-order polynomial baseline correction was performed.
Chemical shifts (δ) are expressed in ppm by reference to intern-
al acetone (δ 2.225 for 1H and δ 31.07 for 13C).
Supplementary data
Supplementary data for this article is available online at http://
glycob.oxfordjournals.org/.
Funding
This work was financially supported by grants from the
European Union (the European Regional Development Fund,
EFRO), the Dutch Ministry of Economic Affairs, Agriculture
1095

JM Dobruchowska et al.
Jansson P-E, Stenutz R, Widmalm G. 2006. Sequence determination of oligo-
saccharides and regular polysaccharides using NMR spectroscopy and a
novel web-based version of the computer program. Carbohydr Res. 341:
1003–1010.
Kralj S, Grijpstra P, van Leeuwen SS, Leemhuis H, Dobruchowska JM, van der
Kaaij RM, Malik A, Oetari A, Kamerling JP, Dijkhuizen L. 2011.
4,6-α-Glucanotransferase, a novel enzyme that structurally and functionally
provides an evolutionary link between glycoside hydrolase enzyme families
13 and 70. Appl Environ Microbiol. 77:8154–8163.
and Innovation (EL&I), the Northern Netherlands collaboration
initiative (SNN EZ/KOMPAS RM119) and the 7th Framework
EU project Amylomics (FP7 KBBE.2010.3.3-01—Food,
Agriculture and Fisheries, Biotechnology—Robust and Novel
Biocatalysts for Industrial Applications; grant agreement no:
265992).
Acknowledgements
Kralj S, van Geel-Schutten GH, Dondorff MMG, Kirsanovs S, van der Maarel
MJEC, Dijkhuizen L. 2004. Glucan synthesis in the genus Lactobacillus:
Isolation and characterization of glucansucrase genes, enzymes and glucan
products from six different strains. Microbiology. 150:3681–3690.
Kralj S, van Geel-Schutten GH, Rahaoui H, Leer RJ, Faber EJ, van der Maarel
MJEC, Dijkhuizen L. 2002. Molecular characterization of a novel glucosyl-
transferase from Lactobacillus reuteri strain 121 synthesizing a unique,
highly branched glucan with α-(1 → 4) and α-(1 → 6) glucosidic bonds.
Appl Environ Microbiol. 68:4283–4291.
We thank Professor Rolf Boelens (Bijvoet Center for
Biomolecular Research, Department of NMR Spectroscopy,
Utrecht University, The Netherlands) for providing us with
measuring time on the 500-MHz NMR instrument.
Conflict of interest
Kralj S, van Geel-Schutten GH, van der Maarel MJEC, Dijkhuizen L. 2004.
Biochemical and molecular characterization of Lactobacillus reuteri 121 reu-
teransucrase. Microbiology. 150:2099–2112.
None declared.
Leemhuis H, Dijkman WP, Dobruchowska JM, Pijning T, Grijpstra P, Kralj S,
Kamerling JP, Dijkhuizen L. 2013. 4,6-α-Glucanotransferase activity occurs
more widespread in Lactobacillus strains and constitutes a separate GH70
subfamily. Appl Microbiol Biotechnol. 97:181–193.
Leemhuis H, Pijning T, Dobruchowska JM, van Leeuwen SS, Kralj S, Dijkstra
BW, Dijkhuizen L. 2013. Glucansucrases: Three-dimensional structures,
reactions, mechanism, α-glucan analysis and their implications in biotech-
nology and food applications. J Biotechnol. 163:250–272.
Levitt MH, Freeman R, Frenkiel J. 1982. Broadband heteronuclear decoupling.
J Magn Reson. 47:328–330.
Monchois V, Willemot R-M, Monsan P. 1999. Glucansucrases: Mechanism
of action and structure-function relationships. FEMS Microbiol Rev. 23:
131–151.
Abbreviations
DP, degree of polymerization; EPS, exopolysaccharide; Fru,
fructose; GH70, glycoside hydrolase family 70; Glc, glucose;
GTFA, glucosyltransferase; HPAEC, high-pH anion-exchange
chromatography; HSQC, 1H detected heteronuclear single
quantum coherence spectroscopy; MALDI-TOF-MS, matrix-
assisted laser-desorption ionization time-of-flight mass spec-
trometry; MOSs, malto-oligosaccharides; NMR, nuclear mag-
netic resonance; PAD, pulsed amperometric detection; ROESY,
rotating-frame nuclear Overhauser enhancement spectroscopy;
Suc, sucrose; TLC, thin-layer chromatography; TOCSY, total
correlation spectroscopy.
Monsan P, Ouarné F. 2010. Oligosaccharides derived from sucrose. In:
Chalarampopoulos D, Rastall RA, editors. Prebiotics and Probiotics Science
and Technology. Springer. p. 293–336.
Monsan P, Remaud-Siméon M, André I. 2010. Transglucosidases as efficient
tools for oligosaccharide and glucoconjugate synthesis. Curr Opin Microbiol.
13:293–300.
References
Moulis C, Joucla G, Harrison D, Fabre E, Potocki-Veronese G, Monsan P,
Remaud-Siméon M. 2006. Understanding the polymerization mechanism of
glycoside-hydrolase family 70 glucansucrases. J Biol Chem. 281:31254–31267.
Pijning T, Vujicic-Zagar A, Kralj S, Dijkhuizen L, Dijkstra BW. 2012. Structure
of the α-1,6/α-1,4-specific glucansucrase GTFA from Lactobacillus reuteri
121. Acta Cryst. F68:1448–1454.
Bock K, Thøgersen H. 1982. Nuclear magnetic resonance spectroscopy in the
study of mono- and oligosaccharides. Annu Rep NMR Spectrosc. 13:1–57.
Brison Y, Laguerre S, Lefoulon F, Morel S, Monties N, Potocki-Véronèse G,
Monsan P, Remaud-Simeon M. 2013. Branching pattern of
gluco-oligosaccharides and 1.5 kDa dextran grafted by the α-1,2 branching
sucrase GBD-CD2. Carbohydr Polym. 94:567–576.
Timmermans JW, de Waard P, Tournois K, Leeflang BR, Vliegenthart JFG.
1993. NMR spectroscopy of nystose. Carbohydr Res. 243:379–384.
Van Geel-Schutten GH, Faber EJ, Smit E, Bonting K, Smith MR, ten Brink B,
Kamerling JP, Vliegenthart JFG, Dijkhuizen L. 1999. Biochemical and struc-
tural characterization of the glucan and fructan exopolysaccharides synthe-
sized by the Lactobacillus reuteri wild-type strain and by mutant strains.
Appl Environ Microbiol. 65:3008–3014.
Brison Y, Pijning T, Malbert Y, Fabre É, Mourey L, Morel S, Potocki-Véronèse G,
Monsan P, Tranier S, Remaud-Siméon M, et al. 2012. Functional and structural
characterization of α-(1 → 2) branching sucrase derived from DSR-E glucan-
sucrase. J Biol Chem. 287:7915–7924.
Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B.
2009. The Carbohydrate-Active EnZymes database (CAZy): An expert re-
source for glycogenomics. Nucleic Acids Res. 37:D233–D238.
Van Geel-Schutten GH, Flesch F, ten Brink B, Smith MR, Dijkhuizen L. 1998.
Screening and characterization of Lactobacillus strains producing large
amounts of exopolysaccharides. Appl Microbiol Biotechnol. 50:697–703.
Van Leeuwen SS, Kralj S, van Geel-Schutten IH, Gerwig GJ, Dijkhuizen L,
Kamerling JP. 2008. Structural analysis of the α-D-glucan (EPS35-5) pro-
duced by the Lactobacillus reuteri strain 35-5 glucansucrase GTFA enzyme.
Carbohydr Res. 343:1251–1265.
Van Leeuwen SS, Leeflang BR, Gerwig GJ, Kamerling JP. 2008. Development
of a 1H NMR structural-reporter-group concept for the primary structural
characterisation of α-D-glucans. Carbohydr Res. 343:1114–1119.
Côté G, Sheng S, Dunlap CA. 2008. Alternansucrase acceptor products. Biocat
Biotransform. 26:161–168.
Dijkhuizen L, van der Maarel MJEC, Kamerling JP, Leemhuis RJ, Kralj S,
Dobruchowska JM. 2010. Glucose-oligosaccharides comprising (α1 → 4)
and (α1 → 6) glycosidic bonds, use thereof, and methods for providing
them. Patent WO 2010/128859 A2.
Dobruchowska JM, Gerwig GJ, Kralj S, Grijpstra P, Leemhuis H, Dijkhuizen L,
Kamerling JP. 2012. Structural characterization of linear isomalto-/malto-
oligomer products synthesized by the novel GTFB 4,6-α-glucanotransferase
enzyme from Lactobacillus reuteri 121. Glycobiology. 22:517–528.
1096