Biotechnology Advances 34 (2016) 827–844

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Biotechnology Advances

journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Review of Levan polysaccharide: From a century of past experiences to

future prospects
Ebru Toksoy Öner a, Lázaro Hernández b, Joan Combie c,⁎
a
IBSB, Department of Bioengineering, Marmara University, Istanbul, Turkey
b
Enzyme Technology Group, Center for Genetic Engineering and Biotechnology (CIGB), Ave 31 e/ 150 and 190, PO Box 6162, Habana 10600, Cuba
c
Montana Biopolymers Inc., 119 Cathcart Circle, Winnsboro, SC 29180, USA,

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

Article history: Levan is a fascinating β-(2,6)-linked fructose polymer with an unusual combination of properties characterized
Received 7 January 2016 in this review. In nature, levan is synthesized from sucrose by a wide range of microorganisms and a few plant
Received in revised form 1 May 2016 species. Bacterial levans often have molecular weights over 500,000 Da, are commonly branched, and form com-
Accepted 4 May 2016
pact nanospheres offering a broad spectrum of applications. The most relevant genetic, biochemical and structur-
Available online 10 May 2016
al aspects of the biosynthetic enzyme levansucrase are detailed. Optimization of parameters for levan production
Keywords:
by intact bacteria and by the isolated enzyme is surveyed. The diversity of current and potential applications of
Levan levan is illustrated by a discussion of uses ranging from personal care and aquaculture to the medical and food
Polysaccharide industries.
Fructan © 2016 Elsevier Inc. All rights reserved.
Levansucrase

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 828
2. Levan in nature and its function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 828
3. Biosynthesis of levan-type fructans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 828
3.1. Classification of bacterial fructosyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829
3.2. Levansucrase gene expression and protein secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829
3.3. The levansucrase reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829
3.4. The synthesis of hetero-oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830
3.5. Structure-function relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 831
4. Microbial levan production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
4.1. Levan production by gram-positive bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
4.1.1. Genera Bacillus, Paenibacillus, Geobacillus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
4.1.2. Other gram-positive species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
4.2. Levan production by gram-negative bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
4.2.1. Acetic acid bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
4.2.2. The halophile Halomonas sp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834
4.2.3. Other gram-negative species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834
4.3. Recombinant levan production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 835
4.4. Microbial co-production of levan and non-fructan products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 835
4.5. Commercial production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836
5. Applications of levan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836
5.1. Personal care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836
5.1.1. Safety and activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836
5.1.2. Hair care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836
5.1.3. Whitener . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836
5.2. Medical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836

⁎ Corresponding author.
E-mail addresses: ebru.toksoy@marmara.edu.tr (E.T. Öner), lazaro.hernandez@cigb.edu.cu (L. Hernández), joan@polysaccharides.us (J. Combie).

http://dx.doi.org/10.1016/j.biotechadv.2016.05.002
0734-9750/© 2016 Elsevier Inc. All rights reserved.
828 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844

5.2.1. Healing wounds and burned tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836

5.2.2. Anti-irritant, anti-oxidant and anti-inflammatory activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
5.2.3. Weight loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
5.2.4. Lower cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
5.3. Prebiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
5.4. Aquaculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
5.5. Films for packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838
5.6. Levan in food. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838
5.7. Additional applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838
5.7.1. Future perspectives - demand, availability, inventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
6. Declaration of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839

1. Introduction 2. Levan in nature and its function

Levan is an unusual non-structural polysaccharide present in several
microorganisms and a few plant species. Consisting almost solely of
fructosyl residues linked via the β-2,6 carbons, this fructan molecule is
packed into nano-sized, spherical forms, providing it with a remarkably
low intrinsic viscosity (Arvidson et al., 2006) and somewhat greater sta-
bility than that of linear counterparts. Produced by either intact microor-
ganisms in submerged cultures or by the isolated enzyme, levan is usually
synthesized from sucrose, syrups or molasses. With some variability
caused by production conditions, the microbial source of the levansucrase
adds more differences to the molecular weight, degree of branching
(Jakob et al., 2013; Runyon et al., 2014), diameter, intrinsic viscosity, sta-
bility and functionalities such as immunogenic activity and adhesive
strength.
The levan story begins with natto, a traditional Japanese food that
was considered to promote long life and good health. As one component
of natto, levan has long attracted the interest of researchers looking for
natural products with health benefits.
Lippmann is often credited with first identifying levan since he used
the term “lävulan” to describe the gum recovered from molasses in the
sugar beet industry (v. Lippmann, 1881). But it was the Australian bacte-
riologist Greig-Smith who proposed the term “levan” for the levorotation
of polarized light and properties analogous to dextran in 1901 (Greig-
Smith, 1901).
For more than a hundred years numerous uses have been identified
for levan polysaccharide but only a few have been commercially realized
simply because it has not been widely available in meaningful quanti-
ties. While levan production in the laboratory is straightforward, bottle-
necks in the downstream process have hampered scale up. Technical
issues associated with handling large volumes of alcohol, impracticality
of dialysis on a large scale and unavailability of ultrafiltration equip-
ment for multi-ton operations have complicated purification. Moreover,
without prompt enzyme inhibition at the end of the fermentation step,
depolymerization quickly decimates levan. Drying of the sticky product
is another hurdle. Lyophilization and vacuum drying commonly
employed in the lab (Han, 1990; Kazak Sarilmiser et al., 2015) are not
readily scaled. Spray drying is widely used in industry but the adhesive
strength of levan requires use of a carrier resulting in decreased product
purity.
This review opens with a sketch of the functional roles of levan in na-
ture. It then moves on to a comprehensive discussion of levan biosyn-
thesis, detailing the most relevant genetic, biochemical and structural
aspects of levansucrase enzymes. Optimization of parameters for levan
production by intact bacteria and by the isolated enzyme is surveyed.
The diversity of current and potential applications of levan is illustrated
by consideration of uses ranging from personal care and aquaculture to
the medical and food industries. The review closes with suggestions of
innovative approaches to increasing productivity by levan producers
and suggests heretofore untested sectors for its use.
“Fructans: beneficial for plants and humans”. This title of a publica-
tion by Ritsema and Smeekens (2003) captures the spirit of the diverse
levan roles in nature. This paper, along with an earlier publication that
included bacteria (Vijn and Smeekens, 1999), highlight the value of
the best known fructans, levan and inulin. Vereyken et al. (2003) looked
at possible mechanisms for the protective effects of fructans in natural
settings. They hypothesized the 5-membered ring fructans interacted
with cell membranes in such a way as to stabilize them and thus seem-
ingly protect them from damage. They found the small, flexible head
groups of levan and inulin penetrated membrane surrogates unlike a
6-membered ring. It may be that this interaction provides improved
membrane stability and, in turn, a higher cell survival. Researchers
have identified further valuable functions described in the applications
section of this review running the gamut from anti-inflammatory and
anti-viral activity to films for food packaging.
Levan, often together with other exopolysaccharides, has been found
to be a structural component of some biofilms in different habitats.
When present in the biofilm of soil species such as Bacillus subtilis,
levan shields the microorganisms from desiccation as the water level
changes, helps glue cells in a favorable environment and protects the
community from predatory organisms (Dogsa et al., 2013). Sucrose uti-
lization by secreted levansucrase appears to be a fitness factor for the in
planta life of several bacterial species. Levan as a biofilm component con-
tributes to the virulence of the plant pathogens Erwinia amylovora and
Pseudomonas syringae (Koczan et al., 2009; Mehmood et al., 2015). In
the endophyte Gluconacetobacter diazotrophicus, levan in the biofilm
acts as an oxygen difussional barrier helping create the microaerobic
conditions required for nitrogen fixation (Hernández et al., 2000;
Velázquez-Hernández et al., 2011). As a secondary but no less important
role, levan in biofilms constitutes an extracellular nutrient reservoir that
can be used as an energy source by bacteria under starvation conditions.
3. Biosynthesis of levan-type fructans
In nature, fructans are synthesized from sucrose by a restricted
number of plant species and many microorganisms, which include Ar-
chaea, fungi and a wide range of bacteria. Plants synthesize fructans in
the vacuole by the concerted action of various fructosyltransferases,
each with its own preferential donor and acceptor substrates.
Sucrose:fructan 6-fructosyltransferase (6-SFT; EC 2.4.1.10), also recog-
nized as sucrose:sucrose 6-fructosyltransferase (6-SST) by its dual
ability to use a fructan molecule or sucrose as the acceptor substrate,
is responsible for the formation of β(2–6) fructosyl-fructose linkages
in plant fructans. Alone, 6-SFT synthesizes linear levan chains direct-
ly from sucrose via the trisaccharide intermediate 6-kestose. Other
natural fructosyl acceptors of this enzyme are 1-kestose and 6G-
kestotriose (Lasseur et al., 2011; and references therein). The production
of levan neoseries or mixed levan (graminan)-type fructans requires
 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844
the combined action of 6-SFT with a second, third, or even fourth
fructosyltransferase, depending on the plant species (Vijn and
Smeekens, 1999). 6-SFT is a key enzyme in fructan monocot biosynthe-
sis. The evergreen frost-hardy species Pachysandra terminalis (family
Buxaceae) is the only dicot plant reported so far to contain a functional
6-SST/6-SFT enzyme (Van den Ende et al., 2011).
Plant fructans generally have a low degree of polymerization
(DP b 101 to 102). Bacterial inulin and levan reach much higher molec-
ular masses (103 to N 104 fructosyl units), offering a broader spectrum of
biotechnological applications. As bacterial levan is the polysaccharide
addressed in this review, the current section will be focused on the bio-
synthetic enzyme levansucrase.
3.1. Classification of bacterial fructosyltransferases
Bacterial fructosyltransferases are multifunctional enzymes ca-
pable of directly converting sucrose [α-D-glucopyranosyl-(1,2)-β-
D -fructofuranoside] into highly polymerized fructans. The enzyme is
named inulosucrase (EC 2.4.1.9) when it synthesizes inulin, and
levansucrase (EC 2.4.1.10) when the polymerization product is levan.
Levansucrases can also form β-(2,1) fructosyl-fructose linkages either
to synthesize inulin-type fructooligosaccharides (FOS) or to create
branching points that connect the β-(2,6) linked chains of the polymer.
The presence of inulosucrase has been reported only in gram-positive
bacteria, specifically in species from three genera (Streptococcus,
Leuconostoc and Lactobacillus) which may additionally contain
levansucrases (Olvera et al., 2007; Anwar et al., 2010). The occurrence
of levansucrase is wide spread in gram-positive and gram-negative
bacteria.
3.2. Levansucrase gene expression and protein secretion
A simple search in databases reveals the occurrence of putative
levansucrase genes in bacteria with different habitats. Most of the spe-
cies contain a unique levansucrase gene which often constitutes the
first component of a chromosomal operon. Exceptionally, the plant
pathogen Pseudomonas syringae contains three levansucrase genes
(two in the chromosome and one in a plasmid) but only two of them
are functionally expressed (Li and Ullrich, 2001). In the soil bacterium
B. subtilis, the levansucrase gene initiates an operon which is tightly con-
trolled by a sucrose-inducible antitermination mechanism (Crutz et al.,
1990). In general, sucrose is required to induce levansucrase transcription
in the genera Bacillus, Paenibacillus and Geobacillus (Choi et al., 2004;
Homann et al., 2007; Inthanavong et al., 2013). By contrast, constitutive
expression of the enzyme is a common feature in bacteria that reside
in sucrose-containing habitats, for instance, Streptococcus salivarius
(Rathsam and Jacques, 1998), Actinomyces naeslundii (Bergeron et al.,
2000); Zymomonas mobilis (Senthilkumar et al., 2009); Erwinia amylovora
(Geier and Geider, 1993) and Gluconacetobacter diazotrophicus
(Hernández et al., 2000). In the latter species, the first gene of the
single-copy levansucrase-exolevanase operon is in fact expressed consti-
tutively, but the exolevanase transcription is induced at low fructose con-
centrations and repressed by glucose, which is the first released product
of the levansucrase reaction (Menéndez et al., 2009). Several bacteria
contain both levansucrase and endo- or exo-type levanase genes forming
either an operon or located in separate loci. Gene expression studies and
evolutionary analysis indicate that levan synthesis and degradation are
exclusive but also complementary habitat-related processes in a bacterial
host. Fructose, the degradation product of levan and inulin, activates
fructanase expression under cell-starvation conditions. Fructan hydroly-
sis is otherwise transcriptionally repressed by glucose, the immediate en-
ergy source generated during sucrose transfructosylation (Bergeron and
Burne, 2001; Martin-Verstraete et al., 1999; Menéndez et al., 2009;
Zeng et al., 2006). The strictly regulated control of levanase transcription
829
guarantees the prevalence of levan synthesis over degradation when the
substrate sucrose is available.
Levansucrases are released to the external medium or are attached
to the cell surface. Variations in the enzyme secretion routes have
been described for different hosts. Levansucrase secretion in gram-
positive bacteria involves cleavage of signal-peptide containing precur-
sors (Rathsam et al., 1993; Steinmetz et al., 1985; van Hijum et al., 2004;
Waldherr et al., 2008). The vast majority of gram-negatives secrete
levansucrase by a signal-peptide-independent pathway (Geier and
Geider, 1993; Jakob et al., 2012; Li and Ullrich, 2001; Song et al., 1993;
Song et al., 1998), with the most remarkable exception being
G. diazotrophicus. This endophytic bacterium is equipped with type-II
secretory machinery for translocation of the mature and fully folded
levansucrase across the outer membrane. The precursor enzyme is syn-
thesized with a 30-amino acid N-terminal signal peptide which is
cleaved off during transport to the periplasm (Arrieta et al., 2004;
Hernández et al., 1999a).
3.3. The levansucrase reaction
All levansucrases catalyze the transfer of fructosyl residues from su-
crose to a variety of acceptor molecules, such as water (release of glu-
cose and fructose), sucrose (synthesis of 1-kestose or 6-kestose), the
synthesized β-(2,6) linked oligofructans (formation and elongation of
levan), and the released monosaccharides glucose (synthesis of sucrose
or blastose) and fructose (synthesis of inulobiose or levanbiose). De-
pending on the enzyme origin, there are remarkable differences in the
affinity for the fructosyl acceptors emerging during the reaction. In gen-
eral, levansucrases from gram-positive bacteria (for instance, Bacillus
subtilis, Bacillus megaterium and Streptococcus salivarius) produce
the polymer without accumulating the intermediate oligofructans
(Chambert et al., 1974; Homann et al., 2007; Song and Jacques, 1999),
whereas the enzymes from gram-negative species (for instance,
Gluconacetobacter diazotrophicus, Zymomonas mobilis, Erwinia amylovora,
and Pseudomonas syringae) render high levels of oligofructans with a
consequent lower yield of levan (Bekers et al., 2002; Caputi et al., 2013;
Hernández et al., 1995; Visnapuu et al., 2015). The accumulated
oligofructans are successively released from the enzyme after each
fructosyl transfer in the so-called non-processive reaction. Little of the
G. diazotrophicus levansucrase products 1-kestose and nystose were con-
sumed either as a donor or acceptor substrate (Hernández et al., 1995),
explaining their constant high concentration even after sucrose deple-
tion. All levansucrases yield polysaccharides of high molecular weight,
which may differ in their size distribution. The high polymerization de-
gree of bacterial levans can be explained by the fact that the growing
fructan chain remains bound to the enzyme as it is progressively elongat-
ed through a processive mechanism (Ozimek et al., 2006). When the
original substrate sucrose is depleted, the synthesized polymer acts as
a fructosyl donor. Then, levansucrase cleaves the β-(2,6) linkages of the
levan chain in an exo-type manner causing the consecutive release of
the terminal fructose until the reaction stops at a branching point
(Chambert et al., 1974; Méndez-Lorenzo et al., 2015). In this sense, the
β-(2,1) fructosyl-fructose linkages appear to prevent branched levans
from extensive hydrolysis by its own synthesizing enzyme. Fig. 1 illus-
trates the structure of both linear and branched levans.
Levansucrase exhibits Michaelis-Menten kinetic properties for
both the hydrolase and transferase activities (Chambert et al., 1974;
Hernández et al., 1995; Song and Jacques, 1999). By contrast, inulosucrase
does not follow substrate-saturation kinetics (van Hijum et al., 2003), be-
having in this sense analogously to plant fructosyltransferases (Ritsema
et al., 2006). The ratio of hydrolysis versus transfructosylation activities
of both levansucrases and inulosucrases is highly dependent on the initial
reaction conditions, but it varies during the course of the reaction. Su-
crose hydrolysis occurs optimally at about 50–60 °C, but fructan (FOS
and/or levan) formation is favored at lower temperatures (10–40 °C)
and with the increase of initial sucrose concentrations to 300 mM or
830 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844
Fig. 1. Schematic representation of linear (A) and branched (B) levan.
more (Chambert and Gonzy-Treboul, 1976; Támbara et al., 1999; Vigants
et al., 2013; Visnapuu et al., 2015). As sucrose nears depletion, hydrolysis
tends to become the predominant reaction. The addition of NaCl and
organic solvents in the reaction mixture generates water-restricted envi-
ronments promoting the reactions of transfructosylation over hydrolysis
(Castillo and López-Munguía, 2004; Chambert and Petit-Glatron, 1989;
Trujillo et al., 2004). Levansucrases perform best in the pH range 5–7
and in most cases pH variations do not affect the rate of hydrolase/
transferase activities (Caputi et al., 2013; Hernández et al., 1995;
Homann et al., 2007; Kim et al., 2003; Visnapuu et al., 2015). Interestingly,
levansucrase from Zymomonas mobilis catalyzes almost exclusively the
synthesis of high DP levan at pHs below 6 in its insoluble microfibril
form, but behaves mostly as a hydrolase at pHs above 7 in its soluble
dimer form (Goldman et al., 2008). Levan polymerization by the enzyme
from other bacteria has not been associated with the formation of
microfibrils.
The reaction conditions may influence not only the output of
transfructosylation but also the average size and number of branches
of the product levan. The synthesis of a bimodal levan molecular weight
distribution is a particular property of levansucrase from Bacillus
species. Relevant reaction factors that modulate the polymer molecular
weight distribution include: enzyme concentration, temperature, ionic
strength, and the presence of water-miscible organic solvents. A low
concentration of B. subtilis levansucrase (0.1 U/mL) favored the synthe-
sis of high-molecular-weight (HMW) levan (average size 2300 kDa) by
promoting the processive elongation of the fructan chain, with negligi-
ble effect of the initial substrate concentration (Porras-Domínguez et al.,
2015; Raga-Carbajal et al., 2016). In the nineteen eighties two papers
also referring to the B. subtilis enzyme reported that the presence of eth-
anol, polyethylene glycol or acetonitrile favored the synthesis of HMW
levan, while an increase in the ionic force resulted in a shift from the bi-
modal type to a single low-molecular-weight (LMW) levan (7.2 kDa)
(Chambert and Petit-Glatron, 1989; Tanaka et al., 1980). At high tem-
perature (50 °C), levansucrase from Bacillus licheniformis RN-01 synthe-
sized HMW levan (612 kDa) as a major product, but when the
temperature was decreased to 30 °C or NaCl was added to the reaction,
LMW levan (11 kDa) was mainly synthesized (Nakapong et al., 2013).
3.4. The synthesis of hetero-oligosaccharides
Sucrose is the natural and preferred substrate of levansucrases. The
enzyme can also react on raffinose [α-D-galactopyranosyl-(1,6)-α-D-
glucopyranosyl-(1,2)-β-D-fructofuranoside] and probably other oligo-
saccharides containing a terminal sucrose with the free fructose unit.
The KM values for sucrose (10–50 mM) are lower than those for raffi-
nose in all kinetically characterized levansucrases (Alamäe et al., 2012;
Caputi et al., 2013 and references therein; Olvera et al., 2007). Two
decades ago, levansucrase from Pseudomonas syringae pv. phaseolicola
was reported to have a relatively high KM for sucrose (160 mM) and
was unable to react on raffinose (Hettwer et al., 1995). More recently,
in recombinant gene expression experiments, Alamäe et al. (2012) de-
termined that all three levansucrases (Lsc1, Lsc2 and Lsc3) of P. syringae
pv. tomato are capable of synthesizing levan from raffinose as the sole
substrate but the affinity for raffinose was much lower than for sucrose.
The same authors also demonstrated that at least one levansucrase
(LscA) of P. chlororaphis subsp. aurantiaca uses raffinose as a fructosyl
donor.
Levansucrase shows a wide spectrum of non-conventional fructosyl
acceptors, including mono and disaccharides, aromatic and aliphatic
alcohols (Beine et al., 2008; Han et al., 2009; Mena-Arizmendi et al.,
 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844 831

2011; Seibel et al., 2006; Visnapuu et al., 2011). Their combination with
the appropriate fructosyl donor allows the production of different hetero-
oligosaccharides. As the main example of biotechnological interest,
levansucrase can transfer the fructosyl residue of sucrose to the acceptor
lactose [β-D-galactopyranosyl-(1,4)-D-glucopyranose] to produce
lactosucrose [β-D-galactopyranosyl-(1,4)-α-D-glucopyranosyl-(1,2)-β-D-
fructofuranoside], a sweetener with a prebiotic effect as reviewed in Mu
et al. (2013).
3.5. Structure-function relationship
Bacterial fructosyltransferases (levansucrase and inulosucrase) belong
to family 68 of glycoside hydrolases (GH), and together with the family
GH32 members (eukaryotic fructosyltransferases, invertases, sucrose-6P
hydrolases and fructanases) comprise the β-fructofuranosidase clan GH-
J as defined in the CAZy database (http://www.cazy.org/). All these en-
zymes operate via a double-displacement mechanism (also known as
ping-pong mechanism) that involves the formation and subsequent hy-
drolysis of a covalent fructosyl-enzyme intermediate. The reaction occurs
with an overall retention of the anomeric configuration of the fructosyl
residue.
The crystal structure of the levan-producing enzyme 6-SST/6-SFT
from the eudicot species Pachysandra terminalis was determined in the
apo form and complexed with its acceptor substrate 6-kestose
(Lammens et al., 2012). To date it is still the only 3D structure of a
plant FT available in databases. As other GH32 enzymes, 6-SST/6-SFT
displays a bimodular fold consisting of a five-bladed β-propeller catalyt-
ic domain at the N-terminal part of the protein connected to a shorter C-
terminal β-sandwich domain which is important for maintaining struc-
tural stability (Fig. 2).
The GH68 enzymes structurally characterized so far are levansucrases
from B. subtilis (Meng and Fütterer, 2003), Gluconacetobacter
diazotrophicus (Martinez-Fleites et al., 2005), Bacillus megaterium
(Strube et al., 2011) and Erwinia amylovora (Wuerges et al., 2015),
inulosucrase from Lactobacillus johnsonii (Pijning et al., 2011), and
lactosucrose-producing β-fructofuranosidase from Microbacterium
saccharophilum K-1 (formerly known as Arthrobacter sp. K-1)
(Tonozuka et al., 2012), all of which display a single domain with the
conserved five-bladed β-propeller architecture (Fig. 2). The amino acid
residues involved in binding and cleavage of the fructosyl-donor sub-
strate are perfectly superimposable in a deep funnel-shaped central cav-
ity. Co-crystallization of catalytically inactive mutants with natural
ligands has revealed that sucrose is accommodated similarly at the active
site of all GH68 enzymes (Meng and Fütterer, 2003; Meng and Fütterer,
2008; Pijning et al., 2011). Three fully conserved carboxylic residues
(the catalytic triad) located at the bottom of the active site pocket cleave

Fig. 2. Overall structures of levan-synthesizing enzymes from different sources. (A) 6-SST/6-SFT from the plant species Pachysandra terminalis (PDB: 3UGF); (B) Levansucrase from the
gram-positive bacterium Bacillus subtilis (PDB: 1OYG); (C) Levansucrase from the gram-negative bacterium Gluconacetobacter diazotrophicus (PDB: 1W18). The plant 6-SST/6-SFT
(family GH32) displays a bimodular fold consisting of a five-bladed β-propeller catalytic domain and a C-terminal β-sandwich domain. The bacterial levansucrases (family GH68) display a
single domain with the conserved five-bladed β-propeller architecture. The blades 1 to 5 are colored in orange, blue, green, yellow and red, respectively. The three catalytic residues
(catalytic triad) of each enzyme are shown in ball-and-stick representation. The figures were prepared with Molecular Operating Environment (MOE), available at http://www.chemcomp.
com/MOE-Molecular_Operating_Environment.htm.
832 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844

the glycosidic bond by functioning as the nucleophile (Asp), the acid/base
catalyst (Glu) and the transition-state stabilizer (Asp) (Meng and
Fütterer, 2003; Pons et al., 2004). Extensive mutagenesis experiments
support the idea of an invariant mechanism for the hydrolase activity of
levansucrases and inulosucrases. On the contrary, the lack of conservation
in regions lining the surface of the active site cavity and presumably in-
volved in binding the fructosyl acceptors appears to determine the differ-
ences in the linkage type and chain length of the transfructosylation
products observed among GH68 enzymes (Anwar et al., 2012; Homann
et al., 2007; Mardo et al., 2014; Strube et al., 2011).
Fructosyltransferases from several gram-positive species require
calcium ions for activity (van Hijum et al., 2006 and references therein).
B. subtilis levansucrase and L. johnsonii inulosucrase contain a calcium-
binding site at a similar position (Meng and Fütterer, 2003; Pijning
et al., 2011), where Ca2+ can stabilize a region of important connections
between active site residues. In G. diazotrophicus levansucrase, which is
fully active in the absence of Ca2 +, a similar fold-stabilizing role is
accomplished by a disulfide bridge (Martinez-Fleites et al., 2005).
These structural variations are not directly responsible for the different
product specificity of the three enzymes.
4. Microbial levan production
Levan is produced extracellularly from sucrose-based substrates by a
variety of bacteria, including the genera Acetobacter, Bacillus, Erwinia,
Gluconobacter, Halomonas, Microbacterium, Pseudomonas, Streptococcus
and Zymomonas (Table 1). Like other microbial extracellular polymeric
substances (EPS), levan production is significantly affected by fermenta-
tion conditions such as temperature, pH, oxygen concentration, bioreac-
tor configuration and culture medium. Generally, the polymer
production is associated with microorganism growth. Upon the enzy-
matic cleavage of sucrose, the fructose moiety is added to the growing
levan chain. Part of the released glucose is metabolized for growth
and the rest is accumulated in the fermentation medium. Development
of strategies for the effective utilization of this excess glucose by co-
production of levan with other high value products has been a part of
many microbial levan production processes. For the production, both
free and immobilized cells may be used and the fermentations may be
operated in batch, fed-batch and continuous modes.
Syrups and molasses are low cost substitutes for sucrose and they
have been widely used as substrates for fermentative production of
microbial polysaccharides (Özcan and Toksoy Öner, 2015). As such,
levan production by Bacillus lentus V8 cultures on sugarcane molasses
(Abou-taleb et al., 2015), by Halomonas smyrnensis AAD6T cultures
on sugar beet and starch molasses (Küçükaşık et al., 2011), by
Microbacterium laevaniformans PTCC 1406 cultures on date syrup
(Moosavi-Nasab et al., 2010), by Paenibacillus polymyxa NRRL B-18475
cultures on sugar beet molasses and sugarcane syrup (Han and Watson,
1992) and by Zymomonas mobilis ATCC 31821 cultures on sugarcane
molasses and syrup (de Oliveira et al., 2007) have been reported. Besides
the crude form (Abou-taleb et al., 2015; Moosavi-Nasab et al., 2010)
pretreated forms of syrups and molasses were also used as substrate.
Levan yields comparable to sucrose containing media were recovered
from P. polymyxa cultures when filter clarified sugarcane syrup was sup-
plemented with peptone or when beet molasses was desalted by passing
it through a gel filtration column (Han and Watson, 1992). Other
pretreatment methods included various combinations of acid treatment,
pH adjustment, tricalcium phosphate and activated carbon treatments
(Küçükaşık et al., 2011) and centrifugation followed by filtration (de
Oliveira et al., 2007).
4.1. Levan production by gram-positive bacteria
4.1.1. Genera Bacillus, Paenibacillus, Geobacillus
The commercial natto starter Bacillus subtilis (natto) Takahashi has
long been investigated for high-level levan production. When the type
and concentration of sugars, initial pH, cultivation temperature and ag-
itation conditions were optimized, shaking cultures produced 49.4 g/L
levan within 21 h and GPC analysis of levan samples produced two
clear peaks of 1794 kDa and 11.9 kDa molecular weight (Shih et al.,
2005). Yields obtained by submerged cultures were further improved
by using alginate-immobilized B. subtilis (natto) Takahashi cells reaching
70.6 g/L levan in 72 h (Shih et al., 2010). In this study, sucrose concentra-
tion, medium pH, metal ions and agitation speed were optimized to
maintain the integrity of the beads. N70% of the biocatalytic activity
was retained even after nine successive 72 hours long reaction cycles.
The presence of the two peaks corresponding to high and low molecular
weights of the produced levan was found to be independent of metal ion
addition (Shih et al., 2010). With the same microbial system, a recent
study demonstrated high yield levan production under controlled
bioreactor conditions (Wu et al., 2013). In this study, a 10-L stirred
tank bioreactor was used and levan yields of 61 g/L and 100 g/L were re-
covered from the batch and fed-batch suspension cultures, respectively.

Table 1
Levan production by various native bacteria exposed to sucrose-containing substrates.

Bacterial species and strain Substrate Yield (Time) Reference

Acetobacter xylinum NCIM 2526 Suc (60 g/L) 13.25 g/L (60 h) Srikanth et al. (2015)
Bacillus amyloliquefaciens NK-ΔLP Suc (250 g/L) 22.6 g/L (48 h) Feng et al. (2015a)
B. amyloliquefaciens NK-Q-7 Suc (250 g/L) 31.1 g/L (48 h) Feng et al. (2015b)
Suc (250 g/L) 57.95 g/L (60 h)
Bacillus lentus V8 Abou-taleb et al. (2015)
Scane molasses (250 g/L) 49.86 g/L (60 h)
Suc (196.8 g/L) 47.8 g/L (96 h)
Bacillus licheniformis NS032 Kekez et al. (2015)
Suc (397.6 g/L) 99.2 g/L (96 h)
Bacillus methylotrophicus SK 21.002 Suc (300 g/L) 100 g/L (16 h) Zhang et al. (2014)
Suc (250 g/L) 61.46 g/L (24 h)
Bacillus subtilis (natto) Takahashi Wu et al. (2013)
Suc (250 g/L and pulse feeding at 30 mg/mL) 100 g/L (120 h)
B. subtilis (natto) CCT7712 Suc (400 g/L) 111.6 g/L (16 h) dos Santos et al. (2013)
Sbeet molasses (30 g/L) 12.4 g/L (210 h) Küçükaşık et al. (2011)
Halomonas smyrnensis AAD6 Suc (50 g/L) + mannitol (30 g/L) 6.60 g/L (160 h) Ateş et al. (2013)
Suc (50 g/L) + 50 mM boric acid 8.84 g/L (160 h) Kazak Sarilmiser et al. (2015)
Suc (200 g/L) 48.9 g/L (48 h)
Microbacterium laevaniformans PTCC 1406 Moosavi-Nasab et al. (2010)
Date syrup (250 g/L) 10.48 g/L (48 h)
Paenibacillus polymyxa EJS-3 Suc (188 g/L) 35.26 g/L (60 h) Liu et al. (2010)
Pseudomonas fluorescens NCIM 2059 Suc (60 g/L) 15.42 g/L (6 days) Jathore et al. (2012)
Serratia levanicum NN Suc (200 g/L) 50 g/L (178 h) Kikuchi et al. (2010)
Zymomonas mobilis CCT 4494 (Immobilized cells) Suc (300 g/L) 112.53 g/L (18 h) Lorenzetti et al. (2015)
Z. mobilis NRRL B-14023 Suc (299 g/L) 40.2 g/L (42.3 h)
Silbir et al. (2014)
Z. mobilis NRRL B-14023 (Immobilized cells) Suc (299 g/L) 31.8 g/L (D = 0.14 h−1)

Suc: sucrose, Scane: sugarcane, Sbeet: sugarbeet.

 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844
Another important aspect of this study is that it showed the possibil-
ity of controlling the molecular weight of levan by means of fermenta-
tion conditions. The total levan produced was the same regardless of
conditions but the ratios of high to low molecular weight versions var-
ied. At low agitation speeds and high sucrose concentrations, more
low molecular weight levan was produced than high molecular weight
but the ratio was reversed at a higher speed and low sucrose concentra-
tion (Wu et al., 2013). A lower ratio of enzyme to sucrose in the
submerged culture favors the initial production of more 6-kestose pre-
cursors which bind the enzyme and are elongated to form levan chains.
Thus, in the presence of excess sucrose, more levan chains are formed
but they are shorter. Less is known about the effects of the agitation
speed but it is possible that high agitation rates may simply increase
the number of effective contacts between the enzyme and sucrose,
thus increasing the polymerization rate.
In another study, B. subtilis CCT 7712 isolated from natto produced
111.6 g/L levan from 400 g/L of sucrose in 16 h. When the produced poly-
mers were analyzed by molecular exclusion chromatography, two frac-
tions were obtained with molecular weights of 568 kDa and 50 kDa
with abundances of 13.39% and 86.61%, respectively (dos Santos et al.,
2013). Hence the dominance of low molecular weight levan at high
sucrose concentrations was also observed with this strain.
Bacillus methylotrophicus SK 21.002 isolated from soil samples collect-
ed from beet and sugarcane gardens in China generated such a high yield
that it was considered economically viable for industrial levan produc-
tion. After optimization of production conditions, up to 100 g/L, low mo-
lecular weight levan could be recovered from cultures grown on 300 g/L
sucrose (Zhang et al., 2014).
Optimization of initial pH, incubation temperature, shaking speed
and inoculum size of Bacillus lentus V8 fermentations achieved yields
of 57.95 g/L or 49.86 g/L when cultures were grown on medium supple-
mented with sucrose or sugar cane molasses, respectively (Abou-taleb
et al., 2015). The levansucrase reaction is optimal under slightly acidic
conditions (pH range 5–7) and this is supported by numerous studies.
For example, a study investigating the effects of sucrose and ammonium
chloride concentrations and initial pH of the medium on levan produc-
tion in low (60–200 g/L) and high (300–400 g/L) sucrose systems by a
Bacillus licheniformis NS032 strain isolated from a petroleum sludge
sample pointed to the decisive role of the culture pH at the start of the
production (Kekez et al., 2015). The central and crucial role of the pH
optima 6.97 and 7.37 for low and high sucrose systems was attributed
to the need to increase the initial pH at high sucrose concentrations
that in turn suggested a correlation between the levan production and
the amounts of organic acids produced as metabolic end products.
The genus Paenibacillus is another microbial system that has
attracted great interest due to the high biotechnological potential
of the EPS that include levan and curdlan (Liang and Wang, 2015).
Levan production by Paenibacillus polymyxa (formerly known as Bacillus
polymyxa) was first reported in 1943 (Hestrin et al., 1943). Then in 1990,
production conditions and characterization of levan by P. polymyxa
NRRL B-18475 strain were reported for cultures grown on sucrose
(Han and Clarke, 1990). Levan produced by strain NRRL B-18475 had a
molecular weight of 2 × 106 Da and 71% β(2–6) linkages, 13% terminal
groups and 12% branching at the C1 position with a β(1–2) linkage.
When cultures were grown on low-cost fermentation substrates like
sugar cane syrup and sugar beet molasses, the low levan yields were im-
proved by supplementing the cane syrup with peptone and desalting
the beet molasses (Han and Watson, 1992). The observed strong antiox-
idant activity of the EPS produced by another strain, P. polymyxa EJS-3
(Liu et al., 2009), encouraged further investigation. Purification of the
crude EPS produced two fractions, EPS-1 and EPS-2 with respective mo-
lecular weights of 1.22 × 106 Da and 0.869 × 106 Da. Detailed structural
characterization revealed that EPS-1 and EPS-2 were composed of 79%
and 72% β(2–6) linkages, 11% and 15% terminal groups and 10% and
13% branching, respectively. Whereas both fractions were identified to
be levan type, EPS-2 had (2–1) linked mannose residues as branches
833
in addition to the fructose branches observed in both structures. Fur-
thermore, statistical optimization of medium composition pointed to
the importance of sucrose, yeast extract and CaCl2 content of the medium
and resulted in 35.26 g/L of levan produced by P. polymyxa EJS-3 cultures
grown in sucrose containing complex medium (Liu et al., 2010).
4.1.2. Other gram-positive species
Microbacterium laevaniformans is another levan producer (Fuchs
et al., 1985). Detailed chemical characterization of the levan as well as
its antitumor activity (Oh et al., 2004) and rheological properties (Bae
et al., 2008) were reported for the M. laevaniformans KCTC 9732. Chemical
characterization revealed that levan was made of 70.2% β(2–6) linkages,
17% terminal groups and 12.9% branching at the C1, C2 or C6 positions
with a β(1–2) linkage (Oh et al., 2004). The first systematic study on
the levan production by this bacterium was reported by Moosavi-Nasab
et al. (2010). In this study, when M. laevaniformans PTCC 1406 cultures
were grown under optimized fermentation conditions, more than four
times higher levan yields could be reached from sucrose as compared to
date syrup.
Lactic acid bacteria of the genera Streptococcus, Leuconostoc and
Lactobacillus are also known to produce levan (Olvera et al., 2007;
Rathsam and Jacques, 1998; Van Geel-Schutten et al., 1999). However,
most of the studies in the literature are mainly focused on the enzyme
production and characterization rather than levan production by the in-
tact bacterium in culture conditions.
4.2. Levan production by gram-negative bacteria
4.2.1. Acetic acid bacteria
Though the Acetobacter genus was known for producing levan
(Han, 1990), the first comprehensive study on levan biosynthesis and
levansucrase enzyme was reported on the endophytic nitrogen-fixing
bacterium Gluconacetobacter diazotrophicus (formerly known as
Acetobacter diazotrophicus) (Hernández et al., 1995). The authors found
that G. diazotrophicus strain SRT4 converted sucrose into a branched
levan with the molecular weight above 2 × 106 Da (Hernández et al.,
1995). In another study, the G. diazotrophicus type strain PAl 5 was re-
ported to produce 24.7 g/L levan in batch cultures growing on 100 g/L su-
crose and under biological N2-fixation (BNF) conditions which in turn
suggested the high potential of this strain as a microbial producer since
it does not require N-source supplementation of the production medium
(Molinari and Boiardi, 2013). For the same strain, enhanced levan forma-
tion was reported when G. diazotrophicus PAl 5 cells were cultivated on a
solid medium containing a high concentration of phosphate. However
this finding could not be verified with submerged cultures (Idogawa
et al., 2014). The gram-negative aerobe Gluconacetobacter xylinus
(formerly Acetobacter xylinum), well known for its ability to accumulate
high levels of cellulose from glucose, has also been shown to produce a
water soluble polymer when grown on sucrose. The presence of a single
fructose peak in the HPLC pattern of the acidic hydrolysate of the polymer
by A. xylinum NCI 1005 as well as comparison of the NMR spectra with
that of commercial levan of Erwinia herbicola confirmed the levan-type
structure of the polymer (Tajima et al., 1997). Another Gluconacetobacter
xylinus I-2281 strain isolated from vinegar fermentations was known to
produce a gluconacetan EPS composed of rhamnose, glucose, mannose,
and glucuronic acid monomers. To investigate the effect of nutritional fac-
tors on the nature, yield, and composition of EPS, cultures were grown on
glucose, fructose and sucrose. Based on enzymatic assays and the molar
ratio of monosaccharides of EPS hydrolysates, 75% gluconacetan and
25% levan (0.07 C-mol levan/C-mol sucrose yield) were found to be
produced in the presence of sucrose (Kornmann et al., 2003). Recently,
microbial levan production by Acetobacter xylinum NCIM 2526
(reclassified as Gluconacetobacter swingsii) cultures was investigated
in a sucrose rich medium and by using both one-factor-at-a-time and
factorial design. More than a 20-fold increase in levan yields could be
achieved (Srikanth et al., 2015). In this study, the branched levan
834 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844
structure was confirmed by sugar analysis, TGA, FTIR and NMR. Besides
the levan producing acetic acid bacteria species like Gluconobacter
oxydans, Gluconacetobacter xylinus and Gluconacetobacter
diazotrophicus, Jakob et al. (2013) reported Gluconobacter frateurii
TMW 2.767, Gluconobacter cerinus DSM 9533T, Neoasaia chiangmaiensis
NBRC 101099 and Kozakia baliensis DSM 14400 as high level levan pro-
ducing strains of acetic acid bacteria. Levan from K. baliensis stood out
with an exceptionally high MW of 2466 MDa.
4.2.2. The halophile Halomonas sp.
The gram-negative, aerobic bacterium Halomonas sp. was reported as
the first halophilic levan producer microorganism by Poli et al. (2009).
The recovered levan was an unbranched linear molecule and had a mo-
lecular weight of N1 × 106 Da when analyzed by density gradient centri-
fugation. The producer strain was identified as a novel species of the
genus Halomonas and named Halomonas smyrnensis AAD6T (Poli et al.,
2013). Since the strain combines the advantages of osmoadaptation
and halophilicity that enable unsterile production under high salinity, op-
timization studies were initiated to improve the yields in semi-chemical
media. For this, effects of initial carbon and nitrogen concentrations, pro-
duction pH, NaCl and nitrogen pulses, nitrogen and phosphorous limita-
tions, trace elements and thiamine content of the basal production
medium were systematically investigated. Supplementing the medium
with boric acid stood out as the most effective stimulator by improving
the sucrose utilization of the cultures and increasing the levan yields up
to five-fold (Kazak Sarilmiser et al., 2015). When the molar-mass and
molar-mass distributions of levan samples were analyzed by GPC-LS,
the linear relationship between RMS radius and molar-mass confirmed
the linear structure. The molecular weights of 9.861 × 106 Da and
1.483 × 106 Da were determined for levan produced in the absence and
presence of boric acid, respectively.
In another study, levan production by H. smyrnensis AAD6T cultures
was investigated in the presence of starch and sugar beet molasses
which had been subjected to different pretreatment methods like clar-
ification, pH adjustment, sulfuric acid, tricalcium phosphate, and acti-
vated carbon treatments as well as different combinations (Küçükaşık
et al., 2011). In this study, the highest levan yields were reached in
pretreated sugar beet molasses. Removal of heavy metals and increases
in iron concentration were found to result in diminished maintenance
of the cellular integrity and lower yields.
To transform microbial hosts into highly efficient cellular factories,
metabolic systems engineering is being used to redirect microbial me-
tabolism for the overproduction of chemicals of interest. In fact, an in-
creasing number of bio-based chemicals are being produced profitably
at industrial scale by use of engineered strains (Dai and Nielsen, 2015).
In the field of metabolic systems engineering, in silico genome-scale
models are valuable tools for deciphering the molecular mechanisms
and identification of targets for strain improvement. Such a genome-
scale model was constructed for H. smyrnensis AAD6T with the aim to
improve the productivity levels by elucidation of the interrelations be-
tween metabolic pathways and levan biosynthesis mechanisms (Ateş
et al., 2013). In this study, the in silico metabolic model was systematical-
ly analyzed to identify critical network elements (i.e., enzymes, biochem-
ical transformations, and metabolites) related to the levan biosynthesis
mechanism. Results pointed out the significant relationship between
levan biosynthesis and three metabolic pathways, namely the glycolysis,
pentose-phosphate pathway, and fructose-mannose metabolism where
the critical reactions were concentrated around three branch points,
ribulose 5-phosphate, glucose 6-phosphate, and mannitol. Supplementa-
tion with mannitol of the sucrose-based fermentation medium resulted
in a twofold increase in levan production in parallel with increased
sucrose depletion rate, accumulated extracellular glucose, and decreased
fructose uptake rate. When the levan fractions obtained in the presence
of mannitol were analyzed by GPC-LS, they were found to be
6.633 × 105 Da, which was shorter than previous studies with this mi-
crobial system and suggested a link to the levansucrase activity (Ateş
et al., 2013). Similar results have been reported for other microbial sys-
tems. The levansucrase activity in G. diazotrophicus cultures was in-
creased when using mannitol or sorbitol as the carbon source for cells
growth. This increase of levansucrase activity could be associated with
an enhancement of gene expression which positively influenced the en-
zyme secretion rate (Hernández et al., 1999b). On the other hand, the
use of high doses of the isolated B. subtilis levansucrase resulted in a sig-
nificant reduction of the levan size (Raga-Carbajal et al., 2016). Ongoing
studies on the Halomonas levansucrase enzyme will shed more light
onto the effect of reaction conditions on the structural features of the pro-
duced levan (Toksoy Öner, personal communication).
For such systems-based approaches, Next Generation Sequencing
(NGS) technologies play a central role by enabling high-throughput
genomic data at very high speed with a relatively low cost. Hence to
gain more insight into the genomic organization of this halophilic
levan producer and to elucidate its biotechnological and industrial po-
tential, the whole genome of H. smyrnensis AAD6T was obtained by NGS.
Moreover, this microbial strain was found to have many poten-
tial applications in biotechnology. In addition to being a levan pro-
ducer, H. smyrnensis AAD6T also has the capacity to produce
cationic exopolysaccharides consisting of partially acetylated N-
acetylgalactosamine and N-acetylglucosamine residues known to be in-
volved in the formation of a pellicle at the air-liquid interface of static
cultures (Diken et al., 2015). Recent studies point to the important
role of Pel in cellular adhesion by maintaining the structural stability
of biofilms as well as to the advantages provided by the ability to pro-
duce multiple exopolysaccharides with different charges in protecting
the cells from diverse antimicrobials (Jennings et al., 2015). Besides
levan and Pel, the H. smyrnensis AAD6T strain also has the genetic capac-
ity to accumulate polyhydroxyalkanoates (PHAs) and osmoprotectants
and to exhibit arsenic resistance (Diken et al., 2015).
4.2.3. Other gram-negative species
Levan production by Zymomonas mobilis was first reported in 1966
when levan biosynthesis was found to be the reason behind the low bio-
mass yields observed in the presence of sucrose (Dawes and Ribbons,
1966). In this study, a branched levan structure was confirmed by
periodate oxidation, partial hydrolysis and methylation analysis. Prom-
ising levels of antitumor and immunomodulatory activities of levans
produced by Z. mobilis cultures (Calazans et al., 1997) were shown to
be associated with the chain length with maximum inhibition observed
with samples of 0.457 × 106 Da (Calazans et al., 2000). These findings
led to more systematic studies on optimization of production conditions
to achieve high titers (Bekers et al., 2003). By statistical optimization in
the presence of sucrose, levan yields of 14.67 g/L and 21.69 g/L were re-
ported for the ZAG-12 (Melo et al., 2007) and ATCC 31821 (de Oliveira
et al., 2007) strains, respectively. In another study, disruption of the ex-
tracellular Z. mobilis sucrase gene (sacC) resulted in high levan yields,
most probably due to the three-fold increase in levansucrase (SacB) ac-
tivities in Z. mobilis CT2 mutant cultures (Senthilkumar et al., 2004).
When Z. mobilis CCT 4494 wild type cells were chemically mutated by
N-methyl N-nitro N-nitrosoguanidine (NTG) treatment, the ZW mutant
increased the 30.3 g/L levan yields of the wild type cultures up to
42.76 g/L in 24 h. Moreover, this study also showed the necessity of
maintaining anaerobic conditions and the influences of carbon/inorganic
nitrogen and organic/inorganic nitrogen ratios on the levan yields (Alegre
et al., 2005). Whereas the use of low-cost substrates like sugar beet juice
and syrup (Bekers et al., 2001) and sugar cane molasses (de Oliveira et al.,
2007) resulted in low levan yields, Z. mobilis ATCC 31821 cultures grow-
ing on sugar cane syrup were found to reach 15.5 g/L levan concentra-
tions comparable to the 21.69 g/L levan yields in commercial sucrose
(de Oliveira et al., 2007). In another systematic study, initial substrate
concentration, incubation time and initial pH of Z. mobilis B-14023
batch cultures were optimized by statistical design and 40.2 g/L was re-
ported as the maximum levan concentration under optimum conditions
(Silbir et al., 2014). Moreover, continuous levan production in a packed
 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844
bed bioreactor was also demonstrated in this study where Ca-alginate
immobilized Z. mobilis B-14023 cells were used. When Z. mobilis CCT
4494 cells were immobilized by entrapment in alginate and PVA
beads, levan concentrations in the range of 18.84 to 112.53 g/L were re-
ported for different pH, sucrose concentration and fermentation periods
(Lorenzetti et al., 2015).
The gram-negative, nitrate-reducing bacterium Pseudomonas
fluorescens is known to produce levan under anaerobic conditions
(Fuchs, 1956). When the levan production profile of P. fluorescens
NCIM 2059 was investigated under agitating and static culture condi-
tions, higher levan titers were achieved under static conditions. Medium
optimization studies resulted in the production of 15.42 g/L levan from
60 g/L sucrose (Jathore et al., 2012). Comparison of the FTIR spectra
as well as 13C and 1H NMR spectra of levan from P. fluorescens with
those from Z. mobilis and P. polymyxa suggested a branched structure
due to the high similarity of the peaks.
Serratia sp. bacteria isolated from soil samples were identified as
levan producers for the first time by Kojima et al. (1993). The high
molecular weight polymer (4.4 × 106 Da) was characterized by a low
degree of branching. Large scale levan production by the same Serratia
levanicum NN bacteria in a 200 L jar fermenter resulted in 39 g/L levan
production within the first 10 h. Then by controlling the pH of the me-
dium and lowering the production temperature down to 4 °C, the levan
level reached 50 g/L after seven days of cultivation (Kikuchi et al., 2010).
Levan production by Erwinia herbicola SRI403 isolated from a
crushing mill at a sugar factory was investigated by Blake et al. (1982).
The levan concentration in batch cultures growing on 156 g/L sucrose
reached 13.2 g/L after two days of incubation. One interesting feature
of levan from Erwinia herbicola SRI403 was its very low degree of
branching occurring at every 20 residues on average (Blake et al.,
1982). In another study, concentrations of medium components includ-
ing the nitrogen, carbon and phosphorous sources as well as fermenta-
tion parameters like pH and incubation time were optimized for
levan production by Erwinia herbicola ATCC 15552 batch cultures.
Under optimized conditions, cultures converted 19% of sucrose to
levan when grown in a continuous production mode (Keith et al.,
1991). This study also pointed to the importance of analysis methods
for measuring the molecular weight of levan. An order of magnitude
lower values (1.1–1.6 × 106 Da) were obtained with Gel Permeation
Chromatography (GPC) as compared to Low-angle Laser Light Scattering
(LALLS) method.
4.3. Recombinant levan production
The bacterium Escherichia coli has been the predominant host used
for the production of heterologous levansucrases. Strong induced pro-
moters (for instance, T7 RNA polymerase promoter) allows high expres-
sion levels of the transgene but the recombinant enzyme often forms
insoluble inclusion bodies. The use of constitutive promoters appears
to be a better choice to achieve the production of soluble and properly
folded levansucrases although at lower yields (Alamäe et al., 2012;
Hernández et al., unpublished results). E. coli is unable to secrete pro-
teins beyond its periplasm into the medium, which is a major inconve-
nience for the downstream purification process in scaled fermentations.
On the other hand, intact cells containing active levansucrase in the
periplasm have been permeabilized using toluene and successfully
reacted with sucrose to directly produce levan (Jang et al., 2001; Kang
et al., 2004b). Toluene-treated cells would require several water
washings before usage in the food and feed industry, an approach not
recommended to establish a large-scale industrial production process.
Contrary to E. coli, the yeasts Saccharomyces cerevisiae and Pichia
pastoris have both the GRAS (generally recognized as safe) status and
the ability to efficiently secrete recombinant proteins to the culture me-
dium. There are only a few reports in the literature about using yeast for
microbial levan production. Scotti et al. (1996) reported the cytoplasmic
accumulation of B. subtilis levansucrase in S. cerevisiae cells when
835
expressed under its own signal peptide or fused to the S. cerevisiae
acid phosphatase signal sequence. The first successful study on levan
producing strains of the yeast S. cerevisiae was reported only recently
where either the sucrose synthase (SuSy) gene from potato or the
spinach sucrose transporter (SUT) gene were integrated into the ge-
nome of an invertase deficient S. cerevisiae strain. When these integrand
strains were transformed with the levansucrase gene (M1FT) from
Leuconostoc mesenteroides, both sucrose accumulating strains were
found to produce levan. When grown in rich medium containing
sucrose, the strain co-expressing the levansucrase and SUT accumulated
7.75 g/L branched and short chain (polymerization degree of 280 equiv-
alent to an approximate molecular weight of 45 kDa) levan both intra
and extracellularly (Franken et al., 2013). The methylotrophic yeast
Hansenula polymorpha was used to express Z. mobilis levansucrase
enzyme (Park et al., 2004). Similarly, P. pastoris was also used to excrete
G. diazotrophicus (Trujillo et al., 2001) and L. mesenteroides (Kang et al.,
2011) levansucrase. However these yeast systems are rather useful for
high-level enzyme production for in vitro levan production purposes.
Transgenic plants have been approached as alternative hosts for the
production of levan with different degrees of polymerization. The idea
of in vivo conversion of the plant sucrose into a high DP levan was
first explored in tobacco and potato plants expressing the levansucrase
gene from B. subtilis (Ebskamp et al., 1994; Van der Meer et al., 1994).
Since then, other crops that do not produce fructans naturally such as
maize and sugar beets have been genetically modified to produce
bacterial type levan using different gene sources (see Banguela and
Hernández, 2006 for review). When levansucrase from G. diazotrophicus
was targeted to the vacuoles of transgenic tobacco, levan accumulated
to levels ranging from 30 to 70% (w/w) of the total dry weight of mature
leaves, the highest yield reported so far for a plant grown in soil (Banguela
et al., 2011). In general, plants expressing bacterial fructosyltransferase
genes exhibit aberrant phenotypes such as stunting and leaf bleaching,
so they are not good candidates for biotechnological applications. The
production of low DP levans by foreign plant 6-SFT appears to a more fea-
sible approach to achieve healthy transgenic plants. Transgenic sugar beet
roots expressing a timothy 6-SFT gene accumulated linear levan of short
chains (DP 3 to N 40) to amounts that reached 20 to 75 mg per gram
(fresh weight), without causing visible alterations of the plant phenotype
(Matsuhira et al., 2014).
4.4. Microbial co-production of levan and non-fructan products
Microbial exopolysaccharide producers usually have the genetic ca-
pacity to produce a mixture of different polymers rather than just one
type of EPS as the product (Özcan and Toksoy Öner, 2015). Simulta-
neous production of multiple microbial products is a desired biorefinery
approach but the processes are highly limited by the diversity of the mi-
crobial systems' metabolic requirements for each product (Sukan et al.,
2015). For example, the commercial natto starter Bacillus subtilis (natto)
Takahashi cultures were found to produce poly(gamma-glutamic acid)
(PGA) in medium containing L-glutamic acid without sucrose whereas
levan was the sole product when bacteria were cultivated in medium
containing sucrose without L-glutamate (Shih et al., 2005). By optimiz-
ing the sucrose and glutamic acid concentrations of the medium, copro-
duction of levan and PGA was achieved in a single-batch fermentation
(Shih and Yu, 2005). The same group investigated the sequential
production of levan with poly-ε-lysine (ε-PL) and ethanol where the
levan and B. subtilis (natto) cells were selectively removed from the
first fermentation cultures by ultrafiltration and the remnants contain-
ing the accumulated glucose were fed to the second stage for the ethanol
production by Z. mobilis cultures (Shih et al., 2010) or ε-PL production by
Streptomyces albulus cultures (Shih et al., 2011).
Co-production of levan and PGA was also reported for Bacillus
amyloliquefaciens NK-1. In a recent study, to obtain high purity levan,
the pgs cluster encoding for PGA biosynthesis genes was deleted. After
optimization of culture conditions with the newly constructed NK-ΔLP
836 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844
strain, up to a ten-fold increase in levan yields was reported (Feng et al.,
2015a). This strain was further improved by disrupting six extracellular
protease genes and the tasA gene encoding the major biofilm matrix
protein. Mutant NK-Q-7 shaking cultures reached 31.1 g/L levan con-
centrations with a 14.4% increase in yields (Feng et al., 2015b).
Similarly, coproduction of levan together with sorbitol (Viikari,
1984) and ethanol (Bekers et al., 1990; Bekers et al., 1999) were report-
ed for Z. mobilis cultures. Lactic acid bacteria are also known to produce
a mixture of dextran and levan. In order to produce pure dextran, levan
production has been suppressed by changing the pH in Lactobacillus
reuteri cultures (Van Geel-Schutten et al., 1999) or by UV radiation of
Leuconostoc mesenteroides cultures (Siddiqui et al., 2014).
4.5. Commercial production
Perhaps the earliest commercial scale production of levan was at
Economics Laboratory Inc., St. Paul, MN, USA in the early 1970s. The
company did not sell the levan directly but rather used it in-house to
produce levan hydrolase sold as a slime control agent for paper mills
(Combie, personal communication). IGI Biotechnology Inc. produced
levan for a short time and patented a use for levan in food applications
(Mays and Dally, 1989).
Today levan is produced commercially by a number of companies,
including Natural Polymers Inc., Bainbridge, GA, USA using Bacillus
subtilis (Combie, personal communication), Real Biotech Co., Ltd.,
Chungnam, Korea, which has produced levan with Zymomonas mobilis
(Kim et al., 2005a) and Advance Co., Ltd., Tokyo, Japan, a long time
user of Streptococcus salivarius (Takahashi et al., 1994). At the
moment, Rahn AG sells the most widely distributed, finished products
containing levan in the person care sector, Proteolea® to rejuvenate
the appearance of skin and Slimexir® which is touted as a silhouette
refiner (Rahn, 2012; Rahn, 2013).
5. Applications of levan
The unique combination of properties that distinguish levan from
most other polysaccharides has long attracted attention. While com-
mercial use has been limited, the increasing availability with the addi-
tion of more companies to the producer list is anticipated to bring
more levan-based items into the market. A few applications are de-
scribed here with a few more listed in a table at the end of this section.
5.1. Personal care
The focus on safety and green products by the personal care and
cosmetics arena is a particularly attractive venue for levan.
5.1.1. Safety and activity
Levan excels at meeting all safety criteria. The Human Repeated
Insult Patch Test (HRIPT) and Chorioallantoic Membrane Vascular
Assay (CAMVA) have shown no skin or eye irritation nor allergic contact
sensitization and no cytotoxicity in an Agar Diffusion Test (Montana
Polysaccharides, 2015). The bonus is the range of functionalities for
levan. This biopolymer is an effective moisturizer, with a Transepidermal
Water Loss (TEWL) nearly identical to hyaluronic acid (Kim et al., 2005a).
It functions as an anti-irritant, counteracts inflammation, lightens hyper-
pigmented patches on the skin (Kang et al., 2009; Kim et al., 2004; Kim
et al., 2005b; Masayo and Takayuki, 2006), stimulates the breakdown of
fat (Rahn, 2013) and forms a tightening film on the skin (Rahn, 2012,
2013).
5.1.2. Hair care
The main use for levan in hair care products is as a film-forming, hair
holding ingredient. Levan finds many uses in hair fixative products.
Simply adding it to existing hair holding gels, mousses, muds and anti-
frizz products to increase their hair holding properties is fairly straight
forward because the low intrinsic viscosity of aqueous levan solutions
does not greatly impact the physical properties of finished products.
Since levan is a nonionic polymer, it has broad chemical compatibility
with other ingredients and polymers. One use for levan is as a hair
spray resin. Because of its low viscosity, water and water/ethanol solu-
tions can be sprayed using existing pump spray and aerosol technology
(Carson, 2015).
Rhodia Inc. developed a levan-based surfactant with cationic
substituents for a conditioning shampoo (Gunn et al., 2009). A
carboxymethylated levan was found to increase the tensile strength of
chemically damaged hair. Scanning electron micrographs of hair dam-
aged by bleaching showed a decline in the lifting of cuticle layers caused
by bleaching after the hair had been treated with derivatized levan (Kim
et al., 2012).
5.1.3. Whitener
Hyperpigmentation is a darkening of the skin caused by an excess of
dark brown melanin. The production pathway starts with the oxidation
of tyrosine, first to L-DOPA and then to L-dopaquinone, followed by po-
lymerization. Tyrosinase catalyzes the rate-limiting, first two steps in
melanogenesis so researchers have been seeking tyrosinase inhibitors
as a means of damping overproduction of melanin. A number of com-
pounds do lighten hyper-pigmented areas on the skin but issues with
poor stability in water, oxidation by light and skin irritation have led
to searches for alternatives.
A 2006 patent application claims the use of levan to inhibit melanin
production by significantly slowing tyrosinase activity, resulting in a
“beautifully whitening effect” (Masayo and Takayuki, 2006). Use of an
interesting combination of levan and ascorbic acid is the subject of an-
other patent application. It is claimed that linking the fructosyl moiety
to ascorbic acid makes a compound with improved stability to oxidation
(Kim et al., 2005b).
5.2. Medical applications
5.2.1. Healing wounds and burned tissue
Some of the most exciting research with medical applications has
been on levan-based thin films for healing damaged tissue. Compared
with other polymers, what advantage does levan bring to these films?
One intriguing idea comes from the work of Sturzoiu et al. (2011).
They reported that levan has a role in metalloproteinase activation, a
key step in the healing of tissues that have been burned or mechanically
damaged.
Costa et al. (2013) assembled levan-phosphonate and chitosan layer-
by-layer to form films on glass slides. The fabrication was driven by elec-
trostatic self-assembly. Phosphated levan-chitosan films on glass slides
had remarkable lap shear strength of 2500 ± 300 kPa. They also investi-
gated the density of cells adhering to chitosan-derivatized levan films
compared with chitosan-alginate films prepared in a similar manner.
Cells adhered in far greater numbers to the levan-based films as com-
pared to those where the levan was replaced with chitosan (110 cells
per mm2 for levan versus 20 cells per mm2 for chitosan).
In another study, comprehensive characterization of cast films of
levan blended with chitosan and poly(ethylene oxide) (PEO) revealed
that stability, flexibility, and transparency of the ternary blends could
be enhanced by varying the ratios of the components. Levan enhanced
biocompatibility was shown by the increased cell attachment and
proliferation (Bostan et al., 2014).
Bone regeneration is hindered by poor adhesion or development of a
fibrous capsule. Methacrylated levan beads with glass nanoparticles
showed superior adhesive strength to similar beads prepared with
alginate (Leite et al., 2014).
Moreover, levan was also found to be a suitable polymer for laser-
based technologies. Sima et al. (2012) utilized matrix-assisted pulsed
laser evaporation (MAPLE) to make films for drug delivery, microsensors
and tissue regeneration. They prepared films with a gradient of levan and
 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844
oxidized levan. Cellular proliferation was highest on oxidized levan with
a substantial amount of growth observed around the boundary area be-
tween the oxidized levan and the native levan (Axente et al., 2014;
Sima et al., 2011).
5.2.2. Anti-irritant, anti-oxidant and anti-inflammatory activities
“All chronic diseases have two common components - oxidative
stress and inflammation” (Kaimal, 2009). At its most basic level, inflam-
mation is a good thing; it is the manifestation of the immune system ini-
tiating the healing process in response to an irritant or the toxicity of
reactive oxidant species. Too much inflammation, however, can be pain-
ful and injurious. For formation of an inflammatory lesion, circulating
cells must adhere to the lumen of the blood vessels. Sedgwick et al.
(1984) found that levan significantly reduced leucocyte adhesion and
the effect was seen whether they used vascular endothelial cells or
glass leading them to deduce that levan affects the leucocyte itself and
not the blood vessel cell. While allowing some effect due to counter
irritant properties, they concluded that levan may act by directly reduc-
ing processes involved in stimulating leucocytes to adhere to blood
vessel walls.
Working with levan and sulfated levan produced by B. subtilis, it was
found that two fractions were particularly potent free radical scavengers
when tested against 1,1-diphenyl-2-pycryl-hydrazyl (DPPH) radicals.
The fraction of DPPH bleaching required to reach the half maximal scav-
enging concentration (SC50) was as low as 1.5 μg per mL (Abdel-Fattah
et al., 2012). This compared favorably with the SC50 for ascorbic acid of
8.0 μg per mL (Ghareeb et al., 2013). The antioxidant activity of levan
from Acetobacter xylinum was 81% of ascorbic acid (Srikanth et al., 2015).
5.2.3. Weight loss
Kang et al. (2004a) found adding levan to the diets of laboratory rats
suppressed obesity and hyperlipidemia. Even on a high fat diet, the daily
weight gain was reduced from 2.58 ± 0.29 g/day for the controls to
1.61 ± 0.21 g/day for rats on a diet supplemented with 10% levan.
There was also a significant reduction in white fat development, adipo-
cyte hypertrophy, and the development of hyperinsulinemia and hyper-
lipidemia in a dose-dependent manner. Serum triglycerides, free fatty
acids and serum cholesterol were also reduced in rats receiving levan.
It was suggested that upregulated uncoupling protein (UCP) mRNA ex-
pression may have helped suppress the development of obesity through
increased energy expenditure.
Building on the known ability of some polysaccharides to have a
beneficial effect in weight control and the widespread use of ginseng
as an herbal medicine, Oh et al. (2014) looked at levan, fermented
ginseng and a combination of the two. Levan powder (100 mg/kg/day),
dried fermented ginseng (150 mg/kg/day) or a combination of levan
and ginseng were added to the high fat diet of mice for 11 weeks.
Mice on a high fat diet supplemented with the mix of levan and
fermented ginseng had a final total body weight of 11.7 ± 0.8 g, signif-
icantly less than the 16.1 ± 1.1 g for the control group. The ginseng plus
levan diet also resulted in a reduction of white adipose tissue weight,
fasting blood glucose, insulin resistance index and leptin compared
with controls.
5.2.4. Lower cholesterol
An extensive study on the effects of high molecular weight levan on
cholesterol levels showed benefits of the approach. Levan was added to
the cholesterol-free diet of rats at a rate of 1 or 5% (w/w) over the course
of one month. Serum cholesterol in the animals was significantly
decreased by both doses of levan. It was noted that fecal excretions of
sterols and lipids were higher in rats receiving levan than in the control
animals, so one theory of the mechanism was that levan prevented
sterol absorption (Yamamoto et al., 1999). Ishihara (1996) found
levan-based agents suppressed blood lipids and body fat even when
high-calorie foods were eaten. A later patent application claimed
levan as part of a treatment for hypercholesterolemia, hyperlipidemia
837
or as an arteriosclerosis reducing agent (Haber et al., 2006). Belghith
et al. (2012) tested the effect of levan on rats fed a cholesterol-rich
diet. Test animals were treated with 5% (w/w) levan dissolved in their
drinking water for 2 months. The levan treated rats had increased levels
of HDL cholesterol and lower levels of LDL cholesterol, demonstrating
the beneficial effect.
5.3. Prebiotics
Prebiotics promote proliferation of beneficial bacteria in the colon
and inhibit harmful microorganisms. Several studies have looked at var-
ious aspects of the role levan or levan-type fructooligosaccharides have
on probiotic bacteria and complex intestinal microbiota (Adamberg
et al., 2014; Adamberg et al., 2015; Arrizon et al., 2014; Marx et al.,
2000; Porras-Domínguez et al., 2014; Sonnenburg et al., 2010; Visnapuu
et al., 2015 and references therein). For example, Adamberg et al.
(2015) studied how levan reshapes microbiota in human fecal samples
using metagenomic and metabolomic methods. They showed levan
enriched Bacteroides, Escherichia, Streptococcus and Faecalibacterium
and provided a metabolic scheme to describe conversion of levan in
cooperative metabolism of gut bacteria.
Beneficial effects of levan on the intestinal microbial community in
the gut of farmed animals have also been studied. One series of experi-
ments on swine looked at levan as a potential alternative to prophylactic
use of antibiotics. Growing pigs were given 700 kDa levan at a rate of 0,
0.05, 0.1 and 0.2% of the diet over a 42 day period. Average daily gain,
the gain to feed ratio and the total tract digestibility optimally increased
with the 0.1% rate of levan addition (Li and Kim, 2013). A trial on older,
finishing pigs gave similar results. Levan had a positive impact on
weight gain over the 42 day period, Lactobacillus counts were increased
and E. coli levels decreased (Zhao et al., 2013a). The value of levan in the
diet of chickens was also investigated. Levan added to the feed im-
proved the gain to feed ratio, increased the weight of the breast meat,
decreased the amount of ammonia excreted, increased the levels of
beneficial bacteria (Lactobacillus and Bifidobacteria) in the cecum and
decreased the harmful bacteria (E. coli and Clostridium perfringens) in
the cecum (Zhao et al., 2013b).
5.4. Aquaculture
As for other farmed animals, there is interest in replacing prophylac-
tic antibiotics with safe alternatives to build up non-specific immunity
of aquacultured fish. Early studies using intraperitoneally administered
levan had limited success (Wang and Wang, 1997), but in light of the
beneficial immunological effect by levan on non-aquatic species, the
role of the administration route was considered. In a 75 day trial on
Cyprinus carpio (common carp), levan was added to the feed. Survival
rates were determined 10 days after the fish were challenged with an
intraperitoneal dose of Aeromonas hydrophila. 100% of the fish on the
0.5% levan diet survived (Rairakhwada et al., 2007), suggesting the pos-
sibility that the benefits of levan were conferred by the interaction with
the microbial population of the gastrointestinal tract. The Central Insti-
tute of Fisheries Education, Mumbai, followed up with a series of studies
on a different species of carp, Labeo rohita, finding only a 60% survival
rate but still an improvement over controls (Gupta et al., 2008).
Elevated temperatures are stressful for some fish species. Levan was
added to the feed of Labeo rohita acclimated at 26 °C. Selected fish were
transferred to thermostatic aquaria where the water temperature was
raised at a constant rate of 0.3 °C/min. When the fish exhibited a loss
of equilibrium, the temperature was designated the critical thermal
maximum. The temperature increase was continued until fish death
was recorded as the lethal thermal maximum. Fish on the diet including
1.25% levan had significantly higher critical thermal maxima and lethal
thermal maxima. High levels of heat shock protein (HSP) were found
in fish raised on a diet with levan while no HSP was found in controls.
The authors suggested that the enhanced tolerance to temperature
838 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844

stress was due to the stimulation of a nonspecific defense mechanism
(Gupta et al., 2010).
Huang et al. (2014) studied the value of levan in orange-spotted
grouper (Epinephelus coioides H.). Levan was added in pellet feed for
84 days. The grouper were then challenged with a common pathogen
for salt water fish, Vibrio harveyi. The group of fish receiving the 2.5%
dose of levan had the largest increase in growth and a 90% survival
rate (compared with 50% survival of the controls).
properties was useful. When levan was included in a wheat bread
recipe, as little as 1% levan produced bread that was 18–26% softer
than the control.
Even our favorite treats have been subjected to the addition of levan.
A patent has been obtained for a confectionery based on fructans, in-
cluding levan, to make a healthy candy with heart and digestive
benefits (Cordero, 2010).

5.5. Films for packaging 5.7. Additional applications

Bio-based food packaging materials are needed for safer and healthier In addition to the abovementioned applications, many additional
packaging and to reduce accumulation in the environment (Vijayendra uses have been reported in the literature. Some of those are listed in
and Shamala, 2014). Without long, flexible moieties in levan, films the following table. (See Table 2.)
made from this polymer are too brittle for practical use but addition of
clay or other plasticizers overcomes this issue.
A meticulous analysis of the interaction between exfoliated mont-
morillonite clay and levan provided insight into the interacting forces Table 2
Additional applications.
that result in levan-based films that are clear, flexible and tough.
Sodium montmorillonite was exfoliated and blended at 1, 5 or 10 wt% Application Description References
with a solution of B. subtilis levan. Following ultrasonication, water Anti-cancer Mice tumor free after 5–8 month Sinai et al. (1976)
was partially evaporated to produce viscous suspensions. The thickened activity treatment.
liquids were coated onto Mylar sheets and dried. Adding only 1% mont- Molecular weight/branching critical. Calazans et al.
morillonite to the formulation increased thermal stability but did not (2000), Yoo et al.
(2004)
significantly enhance mechanical properties of the cast films. In fact,
Sulfated levan raised caspase activity, Abdel-Fattah et al.
the slight negative effect suggested that the clay disrupted the levan effective radical scavenger. (2012), Bayoumi
structure but the clay platelets were too far apart to be bridged by et al.
levan. Ten percent montmorillonite made films with a tensile modulus (2012), Bayoumi
424% greater than that of pure levan and with 485% greater toughness. et al. (2013)
Quarternization enhanced anti-cancer Esawy et al. (2013)
It was theorized that at 10% montmorillonite, the clay platelets were activity.
aligned and close enough to be bridged by uncoiled levan molecules. Anti-bacterial Decreased foodborne pathogens. OD600 Byun et al. (2014)
Working in parallel with the molecular network of levan, the montmo- activity reduced from 1.9 to as low as 1.0 at 48 h.
rillonite added significant mechanical reinforcement. FTIR spectra sup- Potential to prevent and treat Donald et al. (2014)
gram-positive infections.
plied evidence that the interaction was via hydrogen bonding between
Anti-viral EC50 0.35 (μg/mL) against Herpes simplex. Koichi and Masahiro
the hydroxyl groups on the levan and oxygen molecules on the surface activity (2002)
of the montmorillonite (Chen et al., 2014). Bacillus subtilis levan activity against Ahmed et al. (2010),
One property of levan-based films that makes them particularly avian influenza H5N1 virus and enteric Esawy et al.
attractive for food packaging is the fact that they form a good oxygen adenovirus type 40. (2011), Esawy et al.
(2012)
barrier. The oxygen permeability of levan films formulated with 10% Diabetes Decreased plasma glucose in diabetic Dahech et al. (2011)
montmorillonite and 5% polyethylene glycol was b 0.05 cm3/m2 − day rats; no effect on normal rats.
(Montana Polysaccharides, 2015). Enhanced Difructose anhydride IV produced from Saito and Tomita
calcium levan stimulated calcium absorption (2000)
absorption from
5.6. Levan in food
rat small intestine.
Pharmaceuticals Conjugate allergenic drugs with levan. Moreno (1979)
Levan phosphate as a fat substitute was suggested by Roberts and Timed release drugs. Sezer et al. (2015)
Garegg (1998). Another patent application covers the use of the Open matrix network for orally Gupta et al. (2015)
smooth, fat-like properties of fructans to improve the mouthfeel, disintegrating tablets.
Decrease Sulfated levan protective against lethal Higginbotham
taste and spreadability of dairy products (Booten et al., 1998). toxicity doses of the drugs 48/80, polymyxin B (1959)
Other advantages of levan as a fat replacement include the fact that and stilbamidine.
the high molecular weight levan is too large to be detected by taste LD50 for shrimp to antiviral Poli et al. (2009)
sensors and the volatility is too low to emit detectable odors. A re- sesquiterpene hydroquinone (avarol)
decreased over 50-fold.
cent patent application describes how to make yogurt containing
Decreased acute toxicity of copper for Kekez et al. (2015)
levan to be consumed as a functional food (Xiao et al., 2014). Daphnia from 0.14 to 0.44 mg per liter.
Exploiting the texture levan can bring to a food, Vincent et al. Increased survival of carp fry to Gupta et al. (2013)
(2005) suggested inclusion of levan in fermented cereal, milk, and insecticide, fipronil.
juices. Mays and Dally (1989) developed a colloid system stabilized Adhesive Tensile strength on bare aluminum 500 Montana
to 1500 psi. Polysaccharides
by levan in the form of an emulsion, aerosol or foam. (2015)
Effective delivery of essential dietary micronutrients was demon- Electrospinning Levan electrospun from distilled water. Manandhar et al.
strated by coating selenium, cobalt and iron nanoparticles with levan. (2009)
The approach rendered the elements safer for intestinal and microbial Inhibition of Among most effective polysaccharides Finkenstadt et al.
metal tested as corrosion inhibitors. (2011)
cells and enhanced the stability of the preparation. The 2 products in 1
corrosion
approach offered all the benefits of nontoxic versions of vital elements Renewable Chemical conversion of fructose to Román-Leshkov
while simultaneously transporting a functional prebiotic to the intestinal transportation 2,5-dimethylfuran (DMF), potential et al. (2006)
microbes (Bondarenko et al., 2016). fuel liquid
Studying methods to increase the shelf life of bread, Jakob et al. biofuel with greater energy content than
bioethanol.
(2012) found the ability of levan to form a microgel with hydrocolloid
 E.T. Öner et al. / Biotechnology Advances 34 (2016) 827–844
5.7.1. Future perspectives - demand, availability, inventions
Various economic, political and social drivers call for more environ-
mentally responsible products. In turn, this has prompted manufac-
turers to seek renewable raw materials with a good safety profile. An
unusual combination of properties allows levan to stand apart from
competitors. The low intrinsic viscosity, high adhesive strength and
the ability to gel alcohol is not found in any other water soluble, film
forming, biodegradable polymer. The well-established safety and numer-
ous studies on health benefits are bonuses.
The issue has been producing levan at an economically viable price.
Research to improve levan yields has been mainly limited to conven-
tional approaches like optimization of production conditions. On the
other hand, systems based approaches that rely heavily on high-
throughput omics technologies have gained increasing attention in
various areas of industrial biotechnology with many success stories
(Dai and Nielsen, 2015). However, there are very few omics data avail-
able from levan producers. This data is confined to the genome level and
from a diverse family of bacterial levan producers. Whole genome
data of bacteria proven to produce levan has been reported for Bacillus
subtilis (Kunst et al., 1997) Gluconacetobacter diazotrophicus PAl 5
(Bertalan et al., 2009), Halomonas smyrnensis AAD6T (Sogutcu et al.,
2012) and Zymomonas mobilis strains ATCC 31821 (Seo et al., 2005)
and NRRL B-14023 (Kouvelis et al., 2014). But, genome data need to
be enriched by integrating with transcriptome, proteome or metabolome
data in order to gain a holistic view of the microbial system rather than
focusing on individual factors. Hence integrating systems biology to cur-
rent levan research will provide a deeper understanding of levan biosyn-
thesis and associated mechanisms. In the microbial system, the holistic
approach to improvement of the amount of levan produced from a
given amount of substrate must incorporate consideration of the total
levansucrase activity present and the catalytic features of this enzyme.
Structural diversity and use of different in vivo and in vitro bioactiv-
ity assays in structure-function analysis studies prevents making gener-
alizations in relation to the chemical structure and conformation to the
observed biological activity of levan. Unfortunately, there are very few
in silico studies on levan. Hence there is a clear need for analyzing the
3D structure of levan by using molecular dynamics simulations. Such
studies could provide valuable information on the conformational
preferences, intramolecular hydrogen-bonding patterns, torsional dy-
namics, and configurational entropies of levan-based polymers under
different conditions.
This review covers applications supported by research. The upcom-
ing availability of reasonably priced levan in commercial quantities will
allow consideration of heretofore untested arenas such as 3D printing
and ecofriendly pest control.
6. Declaration of interest statement
The authors of this article declare that they have no conflicts of
interest of any kind.
Acknowledgements
The authors wish to thank Emma Carpenter and Alexis Musacchio
for their help in the preparation of Fig. 2. The authors also thank the
reviewers whose comments contributed to the improvement of the
manuscript quality. E. Toksoy Öner gratefully acknowledges the financial
support provided by the Scientific and Technological Research Council of
Turkey (TUBITAK projects 114M239 and 115O495) for levan research by
IBSB.
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