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Fructansucrase enzymes and sucrose analogues: A new approach for the synthesis of unique fructo-oligosaccharides

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Fructansucrase enzymes of lactic acid bacteria use the cheap compound sucrose (Glc-Fru) to synthesize a variety of poly- and oligosaccharide products. Recently, it has been shown that a variety of sucrose analogues (e.g. Gal-Fru, Man-Fru) can be efficiently synthesized. This has exciting potential for the development of novel (fructo) oligosaccharide derivatives.
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ORIGINAL ARTICLE
Fructansucrase enzymes and sucrose analogues: A new approach for
the synthesis of unique fructo-oligosaccharides
S. KRALJ
1
, K. BUCHHOLZ
2
, L. DIJKHUIZEN
1
, & J. SEIBEL
2
1
Centre for Carbohydrate Bioprocessing, TNO-University of Groningen and Department of Microbiology, Groningen
Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands and
2
Technical
Chemistry, Department for Carbohydrate Technology, Technical University Braunschweig, Braunschweig, Germany
Abstract
Fructansucrase enzymes of lactic acid bacteria use the cheap compound sucrose (GlcFru) to synthesize a variety of poly-
and oligosaccharide products. Recently, it has been shown that a variety of sucrose analogues (e.g. GalFru, ManFru) can
be efficiently synthesized. This has exciting potential for the development of novel (fructo) oligosaccharide derivatives.
Introduction
Oligosaccharides (OSs) and polysaccharides (PSs)
are ubiquitous in nature as components of a broad
range of molecular structures. They function as
structural scaffolds for energy storage, and as key
components in many biological processes. OSs and
PSs are associated with cell-cell communication,
differentiation, development, bacterial and viral
infections, and they are diagnostic tools in certain
cancers (Kaur et al. 2002; Nakakuki 2002; Seibel
et al. 2006b; Wong 2005). Due to the difficulty in
producing sufficient quantities at reasonable prices,
OSs and PSs are not commonly used in the
pharmaceutical industry (Rastall et al. 2003). Su-
crose chemistry has elicited much interest since
sucrose (a-
D
-glucopyranosyl b-
D
-fructofuranoside)
is available on a large scale with high purity at low
cost. A large variety of OSs and PSs can be
synthesized enzymatically from sucrose using glu-
cansucrases (GSs; Monchois et al. 1999) and
fructansucrases (FSs; Cerning 1990). These include
glucans containing various glucosidic linkages, e.g.
dextran with an a-1,6-bound glucose (Glc) back-
bone and a-1,2 and a-1,3 side chains (Binder et al.
1983; Buchholz et al. 2003; Coˆte´ & Robyt 1982;
Hehre 1941; Monchois et al. 1997; Remaud-Simeon
et al. 1994), alternan (a-1,6- and a-1,3-bound Glc
units ; Coˆ te´ & Robyt 1982), reuteran (a-1,4 and
a-1,6-bound Glc units; Kralj et al. 2002, 2004), and
the fructans (Yun 1996) levan with b-2,6-bound
fructose (Fru) units (Chambert et al. 1974; van
Hijum et al. 2001) and inulin (b-2,1-bound Fru
units; van Hijum et al. 2002, 2006).
The challenge of synthesis
Carbohydrate biosynthesis and glycosylation are
topics of current interest in academia and industry,
e.g. for OS and PS use in food ingredients, neu-
traceuticals and pharmaceuticals, such as vaccines,
glycoprotein therapeutics and glycosylated drugs in
general (Wong 2005). Glycosylation is important
with respect to pharmacokinetics and pharmacody-
namics, immunogenicity and efficiency of drugs
(Werner et al. 2007). Furthermore, glycosylation
may improve such properties as specificity, solubi-
lity, toxic effects and bitterness of neutraceuticals,
isoflavones and aroma peptides.
The potential of carbohydrates as functional foods
and new therapeutics has highlighted the require-
ment for a general availability of larger amounts of
various carbohydrate structures (Nakakuki 2002;
Rastall & Hotchkiss 2003). Thus, the synthesis of
OSs, PSs and glycosylated substances is a major
challenge. In this sense, activated sugars are key
substrates for synthesis and glycosylation (Seibel
et al. 2006b).
Correspondence: J. Seibel, Technical Chemistry, Department for Carbohydrate Technology, Technical University Braunschweig, Hans-
Sommer Strasse 10, 38106 Braunschweig, Germany. E-mail: j.seibel@tu-bs.de
Biocatalysis and Biotransformation, JanuaryFebruary 2008; 26(12): 3241
ISSN 1024-2422 print/ISSN 1029-2446 online #2008 Informa UK Ltd
DOI: 10.1080/10242420701789478
Fructansucrase enzymes
Most bacterial FSs known are levansucrases (LSs;
EC 2.4.1.10), synthesizing fructan PSs composed of
b-2,6-linked fructose units (levan; Gross et al. 1992;
Steinmetz et al. 1985; van Hijum et al. 2004); only
limited information is available about bacterial
inulosucrases (ISs; EC 2.4.1.9) producing b-2,
1-linked fructan PSs (inulin; Baird et al. 1973;
Olivares-Illana et al. 2002; Rosell & Birkhed 1974;
van Hijum et al. 2002).
LSs are present in Gram-positive, as well as
Gram-negative bacteria. However, ISs have been
identified exclusively in lactic acid bacteria (LAB;
van Hijum et al. 2006). Recently, more FS genes
have become available due to elucidation of several
LAB genome sequences. Interestingly, unique chi-
meric LS and IS enzymes have also been character-
ized recently, carrying additional (glucan-binding)
domains also found in GSs (Morales-Arrieta et al.
2006; Olivares-Illana et al. 2002).
Bacterial FSs cleave the glycosidic bond of their
natural substrate sucrose and use the energy released
to synthesize fructan PSs. Besides this polymeriza-
tion reaction, the enzymes also perform a hydrolysis
reaction, and in the presence of suitable acceptors
OSs are synthesized (Chambert et al. 1974; Seibel
et al. 2006c; van Hijum et al. 2006).
Mechanistic insights into LSs and ISs
LSs and ISs belong to glycoside hydrolase family 68
(GH68) and together with GH32 (e.g. invertase,
inulinase) they comprise clan GH-J, according to
CAZy (CAZy 2006, www.cazy.org). Two different
LS X-ray structures have been elucidated, the first
from the gram-positive bacterium Bacillus subtilis
(SacB, also with bound sucrose; Meng & Fu
¨tterer
2003) and one from the gram-negative bacterium
Gluconobacter diazotrophicus (Martinez-Fleites et al.
2005). At present, no 3D structural information is
available for an IS protein. In addition, three 3D
structures of GH32 representatives are now avail-
able: invertase from Thermotoga maritima (also with
the trisaccharide raffinose in the active site), fructan
1-exohydrolase from Cichorum intybus and exo-
inulinase from Aspergillus niger (Alberto et al. 2004,
2006; Nagem et al. 2004; Verhaest et al. 2005). All
five proteins possess a five-bladed b-propeller fold
with a deep, negatively-charged, central cavity. Their
active sites are positioned at the end of this cavity
with a funnel-like opening towards the molecular
surface.
The GH68 B. subtilis SacB X-ray structure with
sucrose trapped in the active site clearly shows
binding of sucrose in subsites 1 (fructosyl residue)
and 1 (glucosyl residue; Meng et al. 2003; subsite
nomenclature according to Davies et al. 1997). The
geometry of its active site shows a salt bridge
between E342 and R246, further disrupting subsites
2 and 3 (Meng & Fu
¨tterer 2003; Ozimek et al.
2004). A similar situation exists in Neisseria poly-
saccharea amylosucrase (sucrose active enzyme, but
belonging to family GH13; Skov et al. 2002).
Experimental data, indicating that FS enzymes per-
form a disproportionation type of reaction, corrobo-
rated the presence of only one sugar binding donor
subsite 1 in FS enzymes (Ozimek et al. 2006b).
Amino acid sequence alignments of various LSs
and ISs, in combination with site-directed mutagen-
esis experiments and structural data of family
members, pointed towards various relevant func-
tional amino acid residues, and provided the first
insights into structure-function relationships (Meng
& Fu
¨tterer 2003; Martinez-Fleites et al. 2005;
Ozimek et al. 2006a; Homann et al. 2007). In LSs
and ISs from various bacteria Asp
95
, Glu
352
and
Asp
257
(Bacillus megaterium numbering) constitute
the catalytic triad (Ozimek et al. 2004; Homann
et al. 2007). Most likely, the acid/base catalyst
Glu
352
protonates the glycosidic bond of sucrose,
while the Asp
95
residue acts as a nucleophile
(Chambert & Gonzy-Treboul 1976), attacking the
glucopyranosyl residue of the sucrose, presumably
forming an enzyme-fructosyl intermediate with in-
version of the glycosidic bond in the intermediate
state. The residue Asp
257
may assist in coordinating
the hydrogen bonds of the fructofuranoside at
positions 3-OH and 4-OH.
Poly- versus oligosaccharide synthesis
Although LSs and ISs use a similar substrate, these
enzymes differ strongly in their reaction and product
specificities, i.e. in b-2,6 vs. b-2,1 glycosidic bond
specificity (resulting in levan and inulin synthesis,
respectively), and in the ratio of hydrolysis vs.
transglycosylation activities (FOSs; Ozimek et al.
2006b).
The ratio between PS and OS synthesis activities
differs significantly depending on the enzyme
source. The LS of B. subtilis catalyses the formation
of a high-molecular-mass levan without transient
accumulation of oligofructan molecules (Chambert
et al. 1974; Hernandez et al. 1995). A similar
reaction was observed for the LS of Lactobacillus
reuteri 121 (Ozimek et al. 2006b) and the LS of B.
megaterium (Homann et al. 2007). This suggested
that the growing polymer chain remained bound to
the enzyme and that fructan chain elongation
proceeded via a processive type of reaction. In
contrast, the LS of G. diazotrophicus,Zymomonas
Fructansucrase enzymes and sucrose analogues 33
mobilis, and Lactobacillus sanfranciscensis, and the IS
of L. reuteri 121, mainly synthesized short FOSs;
kestose and nystose) from sucrose (Doelle et al.
1993; Hernandez et al. 1995; Korakli et al. 2001,
2003; Ozimek et al. 2006b, 2006a, b). These
enzymes thus employ a non-processive type of
reaction, involving release of the fructan chain after
(virtually) each fructosyl transfer.
Active site architecture (subsites
1, 1
and 2)
Amino acid residues located at subsites 1 and 1 in
the FS active site directly interacting with sucrose
have been identified based on the 3D structure of B.
subtilis SacB levansucrase with bound sucrose (Meng
& Fu
¨tterer 2003), and by several FSs mutagenesis
studies (Chambert & Petit-Glatron 1991; Homann
et al. 2007; Ozimek et al. 2004; Yanase et al. 2002).
A total of eight residues involved in catalysis and/or
interacting with the bound fructose and glucose at
subsites 1 (W85, D86, R246, D247 and W163) and
1 (R360, E340, and R246; B. subtilis SacB
numbering) are conserved in FSs. The FS subsite
1 is highly specific for binding fructose units
(Chambert et al. 1974; Hernandez et al. 1995;
Song et al. 1999), whereas subsite 1 is more
flexible, exhibiting affinity for glucose in binding
sucrose and raffinose, affinity for fructose in binding
sucrose, as an acceptor substrate during transglyco-
sylation, and affinity for different glycopyranosides
(mannose, galactose, fucose and xylose) and dis-
accharides (maltose, lactose and melibiose; Seibel
et al. 2005, 2006c). Some first insights regarding
products formed have been obtained. Key amino
acid residues in LSs important for OS formation
have been identified. For instance, changes in Arg
360
from B. subtilis SacB (Chambert & Petit-Glatron
1991) at subsite 1, and the similar amino acid
residue Arg
370
of B. megaterium SacB, resulted
in time-dependent accumulation of different OSs
during their catalysis, and accumulation of neokes-
tose (b-Fruf-2,6-a-Glcp-1,2-b-Fruf) and blastose
(b-Fruf-2,6-a-Glcp; Homann et al. 2007).
Arg
423
(subsites 1 and 1) in IS is part of the
highly conserved ‘RDP motif ’ in clan GH-J. Clearly,
it is part of a complex network of interactions in the
active site of FS enzymes (Meng & Fu
¨tterer 2003).
Changes in L. reuteri 121 IS residues at acceptor
subsite 1, R423K (also interacting with a glucosyl
residue at subsite 1; Arg
246
residue in B. subtilis
SacB), or W271N (Trp
85
residue in B. subtilis SacB),
caused altered FOS product patterns with sucrose,
yielding much less OSs and significantly more PSs
(Ozimek et al. 2006a). This suggests that modifica-
tion of amino acid residues, or the glycosidic binding
mode, at subsite 1 is important in determining the
product profile, i.e. PS vs. OS synthesis. Interest-
ingly, mutation R423H in L. reuteri IS virtually
abolished all enzymatic activity (Ozimek et al.
2006a). It was shown that the corresponding argi-
nine residue (R188) in exo-inulinase of family GH32
participates in substrate binding, is important for
recognition of the sugar ring and might be respon-
sible for specificity of the enzyme towards the
fructopyranosyl residue (Nagem et al. 2004).
The mutations W340N in L. reuteri IS (W163 in
B. subtilis LS SacB) and W172A in B. megaterium LS
greatly reduced enzyme activity and resulted in
synthesis of only small amounts of 1-kestose (IS)
and neokestose (LS; Homann et al. 2007; Ozimek
et al. 2006a). This residue does not directly interact
with the substrate, but forms the active-site bound-
ary at subsite 1. Removal of the large Trp side chain
lining the sucrose-binding pocket will enlarge the
size of subsite 1. Therefore, a tight fit of the
fructose moiety of sucrose in this subsite is clearly
favourable for the enzyme reaction.
Mutation W271N in L. reuteri IS (W85 in B.
subtilis LS SacB), an amino acid residue present in
many GH32 members, located at the bottom of the
active site near subsite 1, forming a hydrogen bond
with the C6 hydroxyl group of fructose) resulted in a
drastic drop in enzyme activity and in a three-fold
reduction in affinity for sucrose. Interestingly,
W271N synthesized larger fructan products com-
pared with the wild-type enzyme (Ozimek et al.
2006a).
B. megaterium LS residue Asn
252
(subsite 2)
clearly plays an important role in transfructosylation
(Homann et al. 2007). Substitution of Asn
252
to Ala
or Gly completely abolished PS production without
significantly affecting K
m
and k
cat
values, but causing
these variants to switch from mainly PS synthesis to
hydrolysis. In contrast, mutation N252D reduced
PS synthesis without changing K
m
and k
cat
values
significantly. The X-ray structure of the B. subtilis
SacB (74% amino acid identity with B. megaterium
LS) provides insights into the function of this Asn
252
residue. Its position at subsite 2 may allow it to
stabilise the third fructosyl unit of the growing OS
chain and direct it as an acceptor substrate into
the optimal position for further transfructosylation
(Homann et al. 2007).
The mutation N252D in B. megaterium LS clearly
reduced the coordination of a fructosyl unit at
subsite 2; however, it still occurred, as indicated
by the residual formation of PS. These findings
agree with a proposed model of the sugar-binding
subsites in two L. reuteri 121 FSs (Ozimek et al.
2006b). For fructan synthesis, subsites 2 and 3
need high binding affinity for the growing fructan
34 S. Kralj et al.
polymer chain. No structural determinants in FS
enzymes have been reported for additional acceptor
subsites (2, 3) so far.
Residues Arg
370
and Asn
252
, which appear to be
crucial for polyfructan synthesis, are conserved in
LSs from Gram-positive bacteria. In contrast, the
endophytic Gram-negative bacterium G. diazotrophi-
cus SRT4 secretes a constitutively expressed LS
(LsdA, EC 2.4.1.10), which mainly converts sucrose
into FOSs. It contains His
419
instead of Arg
370
at the
equivalent position (Martinez-Fleites et al. 2005).
This His residue is strictly conserved in Gram-
negative LSs. Furthermore, Asn
252
is strictly con-
served in LSs of Gram-positive bacteria, while
Gram-negative bacteria possess a conserved Arg
residue in the equivalent position (Homann et al.
2007). This section of the 3D structure of LsdA is
almost perfectly superimposable with the equivalent
residues of B. subtilis SacB in the active site. In
contrast the region constituting subsite 2 shows
clear differences (data not shown, Figure 1).
Expansion of substrate and product specificity
Although the substrate sucrose offers the advan-
tage of a high-energy glycosidic bond, similar to
that of nucleotide-activated sugars, it limits transfer
to glucose and fructose. The Gibbs energy change
for sucrose hydrolysis is DG
0
A
26.5 kJ mol
1
(298
K, pH 5.65; Goldberg & Tewari 1989; Goldberg
et al. 1989). This represents a high energy value
compared to other disaccharides, whose DG
0
A
values
for hydrolysis are 7, 8.8, and 15.5 kJ mol
1
for
isomaltose, lactose and maltose, respectively (Tewari
et al. 1991). This difference in the DG
0
A
value of
sucrose hydrolysis is available for synthesis of OSs
and PSs by sucrases using sucrose as a substrate (see
above). Those pathways can be expanded to sucrose
Figure 1. Local identification of amino acid residues crucial for structure-function relationships in FSs. Superimposition showing an
overlay of the 3D structures of the LSs from B. subtilis (Bs, blue; Meng & Fu
¨tterer 2003; accession code: 1PT2) and G. diazotrophicus (Gd,
grey; Martinez-Fleites et al. 2005; accession code: 1W18) with sucrose in the active site. Indicated are residues experimentally shown to be
important in structure-function relationships (FOS vs. PS synthesis) in FSs (Chambert & Petit-Glatron 1991; Homann et al. 2007; Ozimek
et al. 2006a). The analogous amino acid residues of B. megaterium (Bm) LS and L. reuteri IS are given in parentheses. (The figure was
generated with pyMOL 0.99rc6, DeLano Scientific LLC).
Fructansucrase enzymes and sucrose analogues 35
analogues, i.e. b-
D
-fructofuranosyl-a-
D
-galactopyra-
noside, b-
D
-fructofuranosyl-a-
D
-fucopyranoside and
b-
D
-fructofuranosyl-a-
D
-xylopyranoside (GalFru,
D
-Fuc-Fru, Xyl-Fru etc.; Baciu et al. 2005; Seibel
et al. 2005, 2006c). These may be used as new
substrates for OS and PS synthesis:
.using FSs in pathway A for the synthesis of new
glycopyranosyloligofructosides:
.alternatively in pathway B using GSs for glyco-
pyranoside transfer (Figure 2; Seibel et al.
2006b).
The transfer of either fructose or the glycopyrano-
side to other sugars, or to different natural products
as acceptors, is a challenging perspective. Advan-
tages of this sucrase system compared to the
currently employed enzymes, such as glycosynthases
and glycosyltransferases (Hancock et al. 2006;
Trincone et al. 2006), are that industrially estab-
lished GSs and FSs, and their genetically engi-
neered, improved, variants, may be usable for
extended substrate and product spectra.
Synthesis of sucrose analogues
The functionality of sucrose, with eight nearly
equivalent hydroxyl groups, makes selective chemi-
cal synthesis of derivatives laborious and difficult.
Sucrose analogues are unnatural activated substrates
that may serve for the synthesis of new OSs and PSs.
They provide a convenient and low-cost access to
synthesis and glycosidation. So far three different
approaches to sucrose analogues have been devel-
oped: chemical, enzymatic and chemo-enzymatic
synthesis (Figure 3). Enzymatic synthesis of the
sucrose analogues has been recently established for
a variety of structures. The exo-fructosyltransferase
(EC 2.4.1.162) from B. subtilis NCIMB 11871
transfers the fructosyl residue of sucrose to mono-
saccharide
D
-glucopyranosyl acceptors (
D
-mannose,
D
-galactose, 2-deoxy-
D
-glucose,
D
-fucose,
D
-xylose)
to yield the b-
D
-fructofuranosyl-a-
D
-glycopyrano-
sides (
D
-ManFru,
D
-GalFru,
D
-2-deoxy-Glc
Fru,
D
-FucFru,
D
-XylFru; Baciu et al. 2005;
Seibel et al. 2006c). A range of these are formed
in good or high yields, with respect to the
Figure 2. Enzymatic synthesis concept for OSs using sucrose analogues as substrates and FSs and GSs in pathways A and B. In pathway A,
unique fructans and FOSs are synthesized with a terminal glucose analogue (e.g. mannose, allose and xylose, etc). In pathway B, novel
glycan PSs and OSs may be synthesized, different from the normal glucan- and gluco-OSs, e.g. mannan- allan and xylan-type PSs and OSs.
(Seibel et al. 2006b).
36 S. Kralj et al.
quasi-equilibrium, under kinetic control and optimal
conditions. Use of l-glycopyranosides as acceptor
substrates results in formation of b-
D
-fructofurano-
syl-a-l-glycopyranoside (l-GlcFru, l-GalFru, l-
FucFru, l-XylFru, RhaFru), sucrose analogues
with b-1,2-glycosidic linkages (Seibel et al. 2006c).
Those b-1,2-glycosidic linkages are found in nature
as nucleotide-activated l-sugars, i.e. uridine dipho-
sphate (UDP)-a-l-arabinose and the GDP-a-l deri-
vative of fucose (Fuc) and cytidine monophosphate
activated sialic acid (b-CMP-NeuAc).
In contrast, the chemically equivalent non-enzy-
matic organic synthesis of
D
-GalFru occurs in
six synthetic steps (Seibel et al. 2005). The synthe-
tic approach additionally gives access to further
deoxyglycopyranosyl derivatives like 2-deoxy-2-
Figure 3. Different routes to sucrose analogues. A) Enzymatic using the levansucrase from B. subtilis. B) Chemo-enzymatic using the
dehydrogenase enzyme from A. tumefaciens followed by chemical modifications of the keto group. C) Chemical synthesis (2-deoxy-2-
aminoacetyl-D-Glc-Frc). The red circles show modifications in the various sucrose analogues compared to sucrose.
Fructansucrase enzymes and sucrose analogues 37
acetamido-b-
D
-fructofuranosyl-a-
D
-glucopyrano-
side (Lichtenthaler & Mondel 1997).
In the chemoenzymatic approach a range of new
sucrose derivatives can be obtained from 3-ketosu-
crose in aqueous medium with few reaction steps.
Agrobacterium tumefaciens produces a dehydrogenase
that oxidizes sucrose specifically at the 3-position,
yielding a-
D
-ribo-hex-3-ulopyranosyl-b-
D
-fructofur-
anoside (3-ketosucrose) (Figure 3). The biotrans-
formation was optimized and scaled up to give 70%
yield of 3-ketosucrose (Stoppok et al. 1992). Sub-
sequent chemical steps provide access to a range of
products, including a-
D
-allopyranosyl b-
D
-fructo-
furanoside (allosucrose; Timme et al. 1998), the
corresponding cyanohydrin (Timme et al. 2001),
oximes, and 3-amino-3-deoxy-a-
D
-allopyranosyl
a-
D
-fructofuranoside via the classical route of re-
ductive amination (Pietsch et al. 1994). Aminoacyl
and peptide conjugates were obtained through con-
ventional peptide synthesis with activated and pro-
tected amino acids (Figure 3). Deprotection yielded
new glyco-derivatives having unconventional substi-
tution patterns, namely 3-(aminoacylamino) allosac-
charides. Furthermore, Grignard reactions gave
CC linked derivatives with alkyl, as well as allyl
substituents (Anders et al. 2006).
Proof of concept: Sucrose analogues as novel
substrates for fructansucrase
Recently, it has been shown that both LSs and ISs
can use a wide range of sucrose analogues, except for
allosucrose, producing interesting new OSs and PSs
and demonstrating the first step towards a new route
for OS synthesis (Table I). The results of these
studies with
D
-ManFru,
D
-GalFru,
D
-XylFru,
and
D
-FucFru donors established that these su-
crose analogues are substrates for FSs. These
analogues have a glycosidic bond energy similar to
that of sucrose and thus should drive synthetic
reactions by non-Leloir glycosyl transferases.
D
-
ManFru,
D
-GalFru,
D
-XylFru, and
D
-FucFru
(all a-1,2-b-linked) were accepted by different LSs
like the LS of B. subtilis (Seibel et al. 2006a) and the
LS of L. reuteri (Biedendieck et al. 2007), thus
presenting proof of principle for this strategy. The
kinetic studies of the different substrates provided
deeper insights into the LS mechanism (Seibel et al.
2006c). So far from the kinetic and docking studies
of such sucrose analogues it can be assumed that the
FS enzyme possesses one donor/acceptor site that is
identical to the substrate site (Figure 4). Very
recently the preparative synthesis of 1-kestose and
1-nystose analogues (ManFru
2,3
, GalFru
2,3
, Xyl-
Fru
2,3
) from sucrose analogues has been acheived
with a b-fructofuranosidase from A. niger (Zucarro
et al. 2007).
Conclusion
Structure-function relationships in bacterial FSs are
not yet fully understood. However, structural fea-
tures determining the ratio of FOS versus polymer
produced are beginning to be revealed. Questions
that remain are how the type (i.e. in b-2,6 vs. b-2,1
glycosidic bond specificity) and size of OSs or PSs,
respectively, are determined.
New efficient methods now exist for enzymatic
synthesis of sucrose analogues. Most of these can be
used efficiently by FSs, several with high reaction
rates and good yields. Novel FOS synthesized with
this technology may be of potential use as prebiotics
or dietary fibre in food and health products, as well
as synthons to synthesize glycosylated drugs or
pharmaceutical ingredients.
However, a more extensive effort is required to
overcome several bottlenecks: low activity with some
of these sucrose analogues, and thus low reaction
rates and low reaction yields due to unfavourable
equilibria in some cases. To improve the potential of
this technology, the synthesis of these sucrose
analogues needs to be further optimized particularly
with respect to downstream processing, which may
represent a major bottleneck. Furthermore, the
Table I. Catalytic reactions of different fructansucrase with various sucrose analogues (D and L pyranoses)
1
.
Sucrose
D
-ManFru
D
-AllFru
D
-GalFru
D
-XylFru
D
-FucFru
LS L. mes. PS n.t. n.t. PS, OS PS, OS n.t.
IS L. citreum PS n.t n.t. PS PS, OS n.t.
LS B. subtilis PS P, OS H
1
PS,OS PS, OS PS, OS
LS B. meg. PS,OS,H n.t. n.t. H, PS PS, OS n.t.
LS L. reuteri PS, OS OS n.t. OS,PS PS, OS PS, OS, H
FF A. niger OS OS n.t. OS OS n.t.
1
Allosucrose is a very poor substrate compared with sucrose.
LS, levansucrase; IS, inulosucrase; FF, b-fructofuranosidase; L. mes., Leuconostoc mesenteroides;L. citreum,Leuconostoc citreum;B. subtilis,
Bacillus subtilis;B. meg., Bacillus megaterium;L. reuteri, Lactobacillus reuteri;A. niger, Aspergillus niger; n.t., not tested; H, hydrolysis; OS,
oligosaccharide formation; PS, polysaccharide formation.
When a specific reaction is dominant it is indicated in bold face.
38 S. Kralj et al.
different sucrases need to be optimized by rational
design/random mutagenesis for these novel sub-
strates. Another exciting possibility may be to use
these analogues in combination with GS enzymes to
synthesize novel galactan, mannan, and allan PSs
and OSs.
In addition, new FOSs may be synthesized by
using sucrose analogues where, instead of glucose,
the fructose moiety of sucrose is modified. The
relatively recent elucidation of LS 3D information,
together with the synthesis of sucrose analogues,
suggests a promising future for the development of
unique tailor-made FOSs.
In the future, novel FSs may be discovered that
are able to use the other hydroxyl moieties present
on the fructose molecule (as is the case for GSs) so
that besides the known b-2,6 and b-2,1 linkage
types, other types of linkages and thus other
saccharides may be synthesized.
Acknowledgements
Financial support by the German Research Founda-
tion via Sonderforschungsbereich 578 ‘From Gene
to Product’ and by the EET programme of the
Dutch government (project number EETK01129) is
gratefully acknowledged.
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Fructansucrase enzymes and sucrose analogues 41
... Sucrose analogs are interesting starting materials for the synthetic production of valuable glycostructures with potential applications in pharmaceutical, cosmetic, food and feed industry (Daude et al., 2012;Kralj et al., 2008;Seibel et al., 2006). They can serve as non-natural Figueroa et al. (2013) activated substrates for a range of sucrase enzymes in oligo-and polysaccharide synthesis. ...
... They can serve as non-natural Figueroa et al. (2013) activated substrates for a range of sucrase enzymes in oligo-and polysaccharide synthesis. Chemical and chemo-enzymatic approaches, but also enzymatic routes for in vitro synthesis of sucrose analogs were described in literature (Daude et al., 2012;Hirano et al., 2012;Kralj et al., 2008;Lichtenthaler et al., 1995;Sato et al., 2012). Here we give a detailed overview on sucrose analog synthesis using SuSy. ...
... Sucrose analogs, which are not cleavable by invertases, such as 1′-deoxy-1′-fluoro-sucrose, [ 13 C 1 ]-β-D-fructofuranosyl-α-D-glucopyranoside, α-D-glucopyranosyl-α-D-lyxopyranoside, α-D-glucopyranosyl-α-L-sorbofuranoside, 2-acetamido-2-deoxy-α-D-glucopyranosyl-β-D-fructofuranoside (Card and Hitz, 1984;Card et al., 1986;Römer et al., 2001Römer et al., , 2003, are useful biochemical probes to study sugar signaling and sugar transport in plants (Sinha et al., 2002;Ward et al., 1997). Furthermore, fructansucrases and glucansucrases can use sucrose-like molecules as alternative glycosyl donors for synthesis of new oligo-and polysaccharides (Daude et al., 2012;Kralj et al., 2008;Seibel et al., 2006). While fructansucrases accept sucrose Notes to: analogs with a modified glucosyl unit, the glucansucrases are highly specific for the glucosyl moiety accepting only subtle chemical modifications. ...
Article
Sucrose synthase (SuSy, EC 2.4.1.13) is a glycosyltransferase (GT) long known from plants and more recently discovered in bacteria. The enzyme catalyzes the reversible transfer of a glucosyl moiety between fructose and a nucleoside diphosphate (NDP) (sucrose+NDP↔NDP-glucose+fructose). The equilibrium for sucrose conversion is pH dependent, and pH values between 5.5 and 7.5 promote NDP-glucose formation. The conversion of a bulk chemical to high-priced NDP-glucose in a one-step reaction provides the key aspect for industrial interest. NDP-sugars are important as such and as key intermediates for glycosylation reactions by highly selective Leloir GTs. SuSy has gained renewed interest as industrially attractive biocatalyst, due to substantial scientific progresses achieved in the last few years. These include biochemical characterization of bacterial SuSys, overproduction of recombinant SuSys, structural information useful for design of tailor-made catalysts, and development of one-pot SuSy-GT cascade reactions for production of several relevant glycosides. These advances could pave the way for the application of Leloir GTs to be used in cost-effective processes. This review provides a framework for application requirements, focusing on catalytic properties, heterologous enzyme production and reaction engineering. The potential of SuSy biocatalysis will be presented based on various biotechnological applications: NDP-sugar synthesis; sucrose analog synthesis; glycoside synthesis by SuSy-GT cascade reactions.
... Besides, inulosucrase and levansucrases have a tendency to use the same acceptor substrates such as sucrose, kestotriose, and raffinose suggesting the active site mimicry in between these enzymes. However, they discretely differ from each other in terms of glycosidic bond specificity (regioselectivity), preference for the synthesis of FOS over fructan polymers, and hydrolysis/transglycosylation ratio Kralj et al. 2008;Meng and Fütterer 2008;Visnapuu et al. 2011). Studies on substitutions of amino acid residues near or next to subsite −1 revealed specific substrate specificities in respective FTs indicating the presence of unidentified further substrate-binding subsites . ...
Chapter
Fructans are fructose-based oligo- and polysaccharides synthesized using sucrose as substrate. Depending on the glycosidic bonds in their structure, they are classified as inulin and levan types or a mixture of these, namely graminans and agavins. Fructans constitute one of the most widespread functional biomolecules in nature and they occur in microbes and plants and to a lesser extent in some fungi and certain algal species. The escalating number of evidence on their health-promoting effects made fructans an important class of platform chemicals. In fact, they have the largest market share among the natural functional additives in the food sector. Plants are the main resources of inulin-, graminan-, and agavin-type fructans but levan type of fructans are commercially produced by microorganisms. In microbes, levan and inulin are synthesized by extracellular fructosyltransferase (FT) enzymes named levansucrase (EC 2.4.1.10) and inulosucrase (EC 2.4.1.9), respectively. Although microbial levan producers are widespread in nature, microbial inulin production is only limited in few Gram-positive bacteria. This chapter first introduces fructans and its microbial and enzymatic production processes followed by the discussion on different classes and structure-functional features of FT enzymes.
... The longer DP FOS produced by this isolate are comparable to those reported for Bacillus amyloliquefacians and named as oligolevan by the authors (M. . It is likely that synthesis of wide range of FOS by GYC2-3 follows the non-processive mechanism, in which the products are released from the enzyme after each fructosyltransferase, resulting in the accumulation of intermediates in the medium (Kralj, Buchholz, Dijkhuizen, & Seibel, 2008;Ua-Arak, Jakob, & Vogel, 2017). It has also been reported that the fructan product size distribution could be modulated by the fermentation conditions (Ua-Arak et al., 2017). ...
... and fructosyltransferases (FTase) (EC 2.4.1.9) are used [5,6]. ...
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... In the former, the monomer units are successively incorporated to a single chain until the elongation process is finished when the enzyme releases the polymer into the solution while in the latter, the monomer units are added to molecules taken up from the solution according to their affinity for the enzyme. In the particular case in which the products are released from the enzyme after each fructosyl transfer, the intermediates accumulate and can be observed in the reaction medium as the synthesis takes place (Kralj et al. 2008). ...
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... Methods to synthesize naturally occurring and modified α-dextrans and β-fructans are being developed with the goal to obtain products with desired (e.g. prebiotic) properties (Homann & Seibel, 2009b;Kralj et al., 2008a;Maiorano et al., 2008). ...
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Response surface method (RSM) is a recent common method used to identify culture condition for optimal production of particular metabolite. In the present study, RSM is used to optimize a catalytic reaction in levan production by heterologous expression of lsbl-bk2 gene isolated from halophilic bacteria Bacillus licheniformis. Levan is a polyfructose polymer produced from sucrose by the action of extracellular levansucrase secreted by the microorganism. Three factors for levan production, namely sucrose concentration, pH, and temperature of reaction were optimized by full factorial and central composite designs in RSM. The result indicated that the optimum in vitro condition for levan production was achieved when the levansucrase catalytic reaction was performed at 32°C, pH 8, in 12% (w/v) sucrose solution. Levan produced by this procedure was verified by FTIR and NMR spectroscopies.
Article
Sucrose Synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate (NDP) into NDP-glucose and fructose. Biochemical characterization of several plant and bacterial SuSys has revealed that the eukaryotic enzymes preferentially use UDP whereas prokaryotic SuSys prefer ADP as acceptor. In this study, SuSy from the bacterium Acidithiobacillus caldus, which has a higher affinity for ADP as reflected by the 25-fold lower Km value compared to UDP, was used as a test case to scrutinize the effect of introducing plant residues at positions in a putative nucleotide binding motif surrounding the nucleobase ring of NDP. All eight single to sextuple mutants had similar activities as the wild-type enzyme but significantly reduced Km values for UDP (up to 60 times). In addition, we recognized that substrate inhibition by UDP is introduced by a methionine at position 637. The affinity for ADP also increased for all but one variant, although the improvement was much smaller compared to UDP. Further characterization of a double mutant also revealed more than 2-fold reduction in Km values for CDP and GDP. This demonstrates the general impact of the motif on nucleotide binding. Furthermore, this research also led to the establishment of a bacterial SuSy variant that is suitable for the recycling of UDP during glycosylation reactions. The latter was successfully demonstrated by combining this variant with a glycosyltransferase in a one-pot reaction for the production of the C-glucoside nothofagin, a health-promoting flavonoid naturally found in rooibos (tea).
Chapter
Carbohydrates are biomolecules that have an essential role in every form of life. The reservoir of naturally occurring glyco-structures is incredibly large and involves a tre-mendous number of carbohydrate-active enzymes (more than 280,000 released modules in the Carbohydrate Active enZymes database) for their synthesis and degradation. Nevertheless, natural enzymes do not necessarily present all the requested properties in terms of efficiency, specificity or stability when considering their usage for carbohydrate or glyco-derivative manufacturing. In addition, if existing, the identification of an enzyme perfectly adapted to a specific function from the natural diversity may be critical due to the lack of available biochemical data and may necessitate intensive screening efforts. To circumvent such limitations and provide optimized solutions, protein engineering has been considered. Leloir-type glycosyltransferases, for example, are mainly involved in the biosynthesis of glycoconjugates in Nature and they have been widely studied and en-gineered for this purpose. However, these enzymes are often found as membrane-bound proteins, what renders difficult their isolation and purification. In addition, their need of low-abundant activated sugars as glycosyl donors also impairs their usage. Alternatively, enzymes that use more abundant glycosyl donor directly issued from agro-ressources have been considered to access to new glyco-derivatives. This has promoted the use of glucansucrases (GS) that catalyze transglycosylation reactions from sucrose substrate. These enzymes are of particular interest for synthetic purpose and have found industrial interest for pharmaceutical and fine chemical applications. To diversify their applications, various approaches of engineering have been exploited to improve expression level, stability, or change substrate or product specificity of these enzymes. In particular, the range of molecules recognized and the osidic linkages formed by GS is broad but yet limited. Therefore, protein engineering methods have been applied to further increase the diversity of glycosylation reactions catalyzed by these enzymes. Sequence analysis and mutagenesis experiments have enabled the identification of key amino acid residues of glucansucrases either involved in catalysis or substrate specificity. Moreover, the de-termination of three-dimensional structures of glucansucrases from both families 13 and 70 of Glycoside-Hydrolases (GH) have provided powerful information for understanding the sequence-structure-function relationships and guiding structure-based rational and semi-rational engineering of these proteins. To assist these efforts, high-throughput screening and biomolecular methods have been developed for the directed evolution of these enzymes. Here are reported some of the successes in the bioengineering of glu-cansucrases from precursor work to latest results, as well as the methods developed for screening and developing efficient variant libraries. The major progresses and break-throughs in the field will be highlighted and further prospects will be considered and discussed.
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