International Journal of Biological Macromolecules 113 (2018) 29–37

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Biosynthesis of levan from sucrose using a thermostable levansucrase

from Lactobacillus reuteri LTH5448
Dawei Ni a, Wei Xu a, Yuxiang Bai a, Wenli Zhang a, Tao Zhang a,b, Wanmeng Mu a,b,⁎
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China
b
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu 214122, China

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

Article history: Levan, a versatile fructan, possesses promising prospects for application in the food and pharmaceutical indus-
Received 4 December 2017 tries and in many other fields due to the superior physicochemical properties and physiological functions of
Received in revised form 21 January 2018 this fructan. In this study, a thermostable levansucrase was identified from Lactobacillus reuteri LTH5448. This en-
Accepted 29 January 2018
zyme shared higher identity with reported inulosucrases than levansucrases but, unexpectedly, yielded a levan-
Available online 6 February 2018
type fructan. Structural analysis of the produced polysaccharide revealed that the glycosidic bonds were β-(2,
Keywords:
6) linkages, and the average molecular weight was 3.924 × 107 Da under optimized conditions. The optimal
Lactobacillus reuteri LTH5448 pH and temperature for levan formation were 6.0 and 35 °C, respectively. The total activity was enhanced to
Levansucrase 135% in the presence of Ca2+. After optimizing the production of levan, 13 U/g sucrose of levansucrase and
Thermostability 500 g/L sucrose were utilized to obtain 183 g/L levan after a 12-h reaction.
Identity © 2018 Elsevier B.V. All rights reserved.
Levan

1. Introduction
Levan is a type of fructan with a glucose residue at the reducing end
[1]. Unlike inulin, in which fructosyl residues are connected via β-(2,
1) glycosidic bonds, the fructosyl chain of levan is formed by β-(2, 6) gly-
cosidic bonds, and some microorganism-derived levans may possess
branched structures [2,3]. As a kind of functional polysaccharide, levan
has many potential applications in the fields of food, pharmaceutical,
and materials chemistry and personal care due to the variety of physio-
logical functions and physicochemical characteristics of levan. Due to
the favorable solubility and indigestibility of levan [4–6], this compound
is considered a promising dietary fiber with ideal prebiotic effects, such
as stimulation of the proliferation of beneficial bacteria such as
Bifidobacterium and Lactobacillus and inhibition of harmful bacteria
such as Escherichia coli and Clostridium perfringens in the cecum [7–9].
In addition to prebiotic roles, many studies have indicated that levan
also exhibits antitumor [10], antibacterial [11], antiviral [12], anti-
obesity [13], anti-diabetes [14], anti-oxidant [15], and hypolipidemic
[16] activities and promotes calcium absorption [17] and
immunostimulation [18]. In the food industry, levan can be used as
sweetener, emulsifier, stabilizer, and thickener and can improve the
shelf life of food [19,20]. Recent studies have shown that levan has po-
tential application in the field of materials chemistry as a composite
exhibiting unique performance [21,22].
⁎ Corresponding author at: State Key Laboratory of Food Science and Technology,
Jiangnan University, Wuxi, Jiangsu 214122, China.
E-mail address: wmmu@jiangnan.edu.cn (W. Mu).
Only a few plants can produce levan as a carbohydrate reservoir via a
single enzyme, sucrose:fructan 6-fructosyltransferase (6-SFT, EC
2.4.1.10) [23]; however, the quantities produced are not enough to
fulfill commercial requirements. Therefore, it has been necessary to
adopt a biological strategy to produce levan using microbial
fructosyltransferases. Fructosyltransferases include levansucrase and
inulosucrase, which synthesize the β-(2, 6) linkage of levan and the β-
(2, 1) linkage of inulin, respectively. Levansucrase (EC 2.4.1.10) belongs
to the glycosyl hydrolase family 68 (GH68) and it is found in many mi-
croorganisms, including gram-positive and gram-negative bacteria. The
biosynthesis of levan by microorganisms has been summarized in previ-
ous reviews [6,24,25] and has been recently identified in organisms
such as Leuconostoc mesenteroides NTM048 [26], Brenneria goodwinii
[27]. Bacillus licheniformis ANT 179 [28], Clostridium acetobutylicum
[29], and Clostridium arbusti SL206 [30]. Levan can be biosynthesized
from sucrose, syrup [31] and molasses [32] by native, recombinant
and immobilized levansucrase [24,25]. Levansucrase catalyzes three
types of reactions during levan formation, namely, hydrolysis (fructosyl
transfer to water), transglycosylation (fructosyl transfer to sucrose),
and polymerization (fructosyl transfer to the fructan polymer). In addi-
tion to these three reactions, Hernandez and coworkers [33] used 14C la-
beled sucrose and unlabeled glucose to demonstrate that levansucrase
could conduct an exchange reaction, transforming the fructosyl moiety
of sucrose to glucose.
Levansucrase exhibits extensive acceptor specificity. For example,
water, oligosaccharides (mono-, dis-, oligosaccharides and levan), and
alcohols (alkyl alcohols and aromatic alcohols) can accept the fructosyl
moiety of sucrose to form novel, potentially valuable saccharides or

https://doi.org/10.1016/j.ijbiomac.2018.01.187
0141-8130/© 2018 Elsevier B.V. All rights reserved.
30 D. Ni et al. / International Journal of Biological Macromolecules 113 (2018) 29–37

glycosides, such as lactosucrose (formed upon fructosyl transfer to lac- Healthcare, Sweden) and chelat-ing Sepharose Fast Flow resin column
tose) [34,35], methyl β-D-fructoside (formed upon fructosyl transfer (GE Healthcare) as described by Liu [27]. All the purification steps
to methanol) [36], and hydroquinone fructoside (formed upon fructosyl were carried out at 4 °C.
transfer to hydroquinone) [37,38]. The subsite −1 of levansucrase can
specifically identify and combine with fructosyl [39], so some saccha-
rides that possess fructosyl may acts as donors in the reaction. Even 2.2. Identification of purified levansucrase
though sucrose is usually used as the optimal donor, raffinose has also
been seen to function as a donor to produce melibiose [40]. In addition, The molecular weight of the levansucrase was determined by so-
lactulose, sucralose, stachyose and melezitose have also been studied, dium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
but free fructose was only detected in the reaction system that used The concentrations of stacking gel and separating gel were 5% and
stachyose as the substrate [41,42], which indicates that stachyose can 12%, respectively. Coomassie brilliant blue R250 was used for staining
serve as a fructosyl donor, but lactulose, sucralose and melezitose the protein. Then, the gel was destained until the protein bands were
cannot. clearly visible and the background became colorless. Protein concentra-
Different mechanisms for sucrose hydrolysis and levan elongation tion was determined by the Bradford method [46], using bovine serum
have been hypothesized to understand the reaction process of albumin as the standard.
levansucrase. Based on studies of Bacillus subtilis levansucrase, a ping-
pong mechanism was established for sucrose hydrolysis, which involves
2.3. Enzyme assay
the formation of a fructosyl-enzyme intermediate [43]. For the elonga-
tion of levan, processive and non-processive mechanisms have been
During the biosynthesis of levan from sucrose by levansucrase, three
proposed for the synthesis of high and low molecular weight levans, re-
types of enzymatic activities (hydrolysis, transglycosylation and total
spectively [44].
activity) are involved. Levansucrase activities were assayed using su-
In this study, we identified a novel levansucrase from Lactobacillus
crose as a substrate. The amount of glucose and fructose released were
reuteri LTH5448 and expressed this enzyme heterologously in E. coli.
indicative of total activity and hydrolytic activity, respectively. The
The structure of the biosynthesized polysaccharide was determined to
transglycosylation activity was calculated by subtracting the hydrolytic
be that of a levan-type fructan. The enzymatic properties of the purified
activity from the total activity. One milliliter of the reaction mixture
recombinant enzyme and the process of levan production were identi-
contained 10% (w/v) sucrose, 10 μg of purified levansucrase and
fied and optimized. The thermostability of the recombinant L. reuteri
50 mM acetate buffer (pH 6.0). The reaction was conducted at 35 °C
LTH5448 levansucrase was higher than that of other reported
for 20 min. Then, the samples were heated in a boiling water bath for
levansucrases. Sequence alignment showed that the sequence of the L.
10 min to deactivate the enzyme and stop the reaction. The concentra-
reuteri LTH5448 levansucrase shared higher identity with the sequences
tions of sucrose, glucose and fructose were detected by High Perfor-
of reported inulosucrases rather than those of levansucrases. We have
mance Liquid Chromatography (HPLC). Waters e2695 system (Waters
attempted to explain this observation based on evolution and sequence
Corporation, MA, USA), Waters 2414 RI detector and a Sugar-Pak I col-
alignment. This phenomenon is worthy of further research in order to
umn (6.5 mm × 300 mm, Waters, USA) were used to analyze. The sam-
explore the mechanism of formation of β-(2, 1) and β-(2, 6) linkages.
ples were eluted using ultrapure water as mobile phase at the constant
flow rate of 0.4 mL/min. One unit of total or hydrolytic activity was de-
2. Methods and materials
fined as the amount of enzyme that released 1 μmol of glucose or fruc-
tose per minute. In this article, enzyme activity refers to total activity
2.1. Cloning, expression and purification of L. reuteri levansucrase
unless otherwise stated.
Schwab [45] found that L. reuteri LTH5448 could produce levan;
however, information about the levansucrase required and the gene se- 2.4. Characterization of the recombinant levansucrase
quence encoding the enzyme remained unknown. Recently, a ftfA gene
coding for fructosyltransferase from L. reuteri LTH5448 was deposited in 2.4.1. Effect of pH
the GenBank database with the accession number AJ812736.2. The par- The effect of pH on the activity of levansucrase was studied using
tial sequence of the C terminus was discarded, and the core region of the three types of buffer with different pH ranges: acetate buffer
levansucrase-encoding gene was synthesized commercially by Shang- (4.0–6.0), sodium phosphate buffer (6.0–7.5) and Tris-HCl buffer
hai Generay Biotech Co., Ltd. (Shanghai, China) with a 6 × −histidine- (7.5–9.0); the concentration of all the buffers was 50 mM. The reactions
tag sequence at the 3′ terminus and NdeI and XhoI restriction sites at were performed at 35 °C for 20 min.
the 5′ and 3′ termini, respectively. The modified sequence was inte-
grated into the pET-22b(+) vector via the restriction sites, resulting in
a recombinant plasmid. 2.4.2. Effect of temperature
For levansucrase expression, the recombinant plasmid was trans- The effect of temperature was examined by incubating the samples
formed into E. coli BL21 (DE3) competent cells, and LB broth was used at various temperatures within a range of 20–75 °C at intervals of 5 °C.
to cultivate the E. coli BL21 (DE3) cells. The cells were grown in The reactions were performed at pH 6.0 for 20 min.
200 mL of LB broth containing ampicillin (100 μg/mL broth) to avoid
contamination by other bacteria at 37 °C. When the absorbance value
at 600 nm reached 0.6, 1 mM isopropyl β-D-1-thiogalactopyranoside
(IPTG) was added to induce recombinant levansucrase expression at
28 °C for an additional 6 h.
The cells were collected by centrifugation at 13000 ×g for 15 min.
Lysis buffer (50 mM acetate buffer, 100 mM NaCl, pH 6.5) was used
for suspension of the cells and the latter were disrupted on ice using a
Scientz-ІІ D ultrasonic cell disruptor (Ningbo Scientz Biotechnology Co
Fig. 1. Phylogenetic tree of fructosyltransferases from Lactobacillus. The phylogenetic tree
Ltd., China) for 15 min. Cellular debris was removed by centrifugation was constructed by MEGA 5.1. IS and LS are the abbreviations of inulosucrase and
at 16000 ×g for 25 min. The supernatant containing crude recombinant levansucrase, respectively. The content behind IS and LS are the accession numbers in
levansucrase was purified using an ÄKTA Purifier System (GE the GenBank database.
 D. Ni et al. / International Journal of Biological Macromolecules 113 (2018) 29–37 31

2.4.3. Effect of metal ions 2.5. Optimization of levan production

The effect of metal ions was probed at 35 °C and pH 6.0 for 20 min.
Seven kinds of divalent metal ions were added to the reaction system
in the form of CaCl2, CuSO4, FeSO4, MgSO4, MnSO4, NiSO4, ZnSO4 at a
final concentration of 1 mM. The enzymatic activity in the absence of
supplemental metal ion was regarded as 100%.
2.5.1. Optimization of enzyme dosage
The reaction containing 10% sucrose (w/v) was performed with the
addition of different dosages of purified levansucrase, ranging from 1 to
20 U/g sucrose; other conditions were the same as in the enzyme assay
described above.

2.4.4. Thermostability
To determine the thermostability of the recombinant levansucrase,
the purified enzyme was preincubated at different temperatures (35,
40, 45, 50, 55, 60, 65, 70 °C) for 12 h. The enzyme was extracted at cer-
tain time intervals to be used for the reaction, and the activity of the en-
zyme without preincubation was considered to be 100%.
2.5.2. Optimization of sucrose concentration
Various concentrations of sucrose, ranging from 10% to 100% (w/v),
were used to produce levan using an enzyme dosage of 13 U/g
sucrose; other conditions were the same as in the enzyme assay
described above.

Fig. 2. Multiple sequence alignment of fructosyltransferases from Lactobacillus. The alignment was conducted using ESPript software. The strictly conserved areas are shown with a red
background, and the highly conserved residues are shown using red words surrounded by a blue box.
32 D. Ni et al. / International Journal of Biological Macromolecules 113 (2018) 29–37
Fig. 3. Whole-cell SDS-PAGE. Lanes 1–4 represent cells induced by IPTG after 0, 2, 4 and
6 h. Lanes 5 and 6 represent the purified levansucrase and the protein marker with
standard enzymes with the following molecular weights: 97.4, 66.3, 42.7, 31.0, and
14.4 kDa.
2.5.3. Determination of reaction time
To determine the optimal reaction time for levan formation, the re-
action was carried out under optimized conditions for 12 h, and the
samples were extracted at 2-h intervals.
2.6. Structural analysis of the biosynthesized levan
2.6.1. Purification of biosynthesized levan
A reaction system containing 50% sucrose (w/v) was used to pro-
duce levan by adding 13 U/g sucrose of purified levansucrase at 35 °C
and pH 6.0 for 12 h. The protein was removed from the reaction mixture
using Sevag reagent (n-butanol: chloroform = 1:4, v/v) [47] to avoid
any effect of the protein on the structure of the biosynthesized polysac-
charide. The protein-free solution was treated with 3 volumes of anhy-
drous ethanol at 4 °C for 10 h to precipitate the polysaccharide. The
precipitate was redissolved in deionized water and reprecipitated by
anhydrous ethanol. This step was repeated until alcohol-soluble saccha-
rides (sucrose, glucose or fructose) could not be detected in the polysac-
charide solution by HPLC measurement. Then, the sample was freeze-
dried for 24 h to obtain purified polysaccharide.
2.6.2. Fourier-transform infrared (FTIR) analysis
Functional groups and chemical bonds of biosynthesized polysac-
charides can be determined by FTIR spectroscopy. The freeze-dried
sample was mixed with KBr at a ratio of 1:100, ground evenly and
Fig. 4. Effect of pH and metal ions on the activity of the recombinant levansucrase. (A) Effect of pH. (B) Effect of metal ions.
pressed into semi-transparent tablets. The FTIR spectrum was recorded
within the wavenumber region of 400–4000 cm−1 at a resolution of
4 cm−1 using a Thermo Nicolet NEXUS 470 FT-IR (Thermo Fisher Scien-
tific, USA).
2.6.3. Nuclear magnetic resonance (NMR) analysis
The type of fructosyl linkage in the biosynthesized fructan was iden-
tified by 13C and 1H NMR analysis. Thirty milligrams of freeze-dried
sample were dissolved in 1.5 mL of deuterium oxide (D2O) and heated
in an 80 °C water bath until the sample dissolved completely. 13C and
1
H NMR spectra were recorded using an AVANCE III NMR spectrometer
(Brucker Co., Billerica, MA, USA) at 70 °C. The chemical shifts (δ) were
obtained as ppm. 1, 4-Dioxan (δC = 66.50 ppm) and acetone (δH =
2.225 ppm) were used as internal reference standards for 13C and 1H
NMR spectra, respectively.
2.6.4. Molecular weight measurement
The molecular weight of the biosynthesized levan was measured by
High Pressure Size Exclusion Chromatography (HPSEC) by coupling two
detectors, an Optilab® T-rEX refractive-index (RI) detector and a DAWN
HELEOS-II multiangle laser light scattering (MALLS) detector (Wyatt
Technology, Santa Barbara, CA, USA) at the He-Ne light wavelength of
658 nm. A Shodex OH-pak SB-806 HQ column (Waters Corporation,
Milford, MA, USA) was used to measure the molecular weight at 25 °C.
The mobile phase was 0.1 M NaNO2 with a flow rate of 0.5 mL/min.
3. Results and discussions
3.1. Sequence analysis of L. reuteri LTH5448 levansucrase
In a previous study, L. reuteri LTH5448 was shown to express a glyco-
syltransferase, and sucrose played a positive role in promoting the ex-
pression of the enzyme [48]. Thereafter, Schwab and coworkers [45]
qualitatively detected levan in bread fermented with sorghum sour-
dough and L. reuteri LTH5448. These discoveries indicated that L. reuteri
LTH5448 might encode a levansucrase, which synthesizes levan from
sucrose. However, detailed information regarding the properties of the
enzyme and the product remained unknown.
Recently, a gene for fructosyltransferase from L. reuteri LTH5448
and the corresponding protein sequence of this gene were deposited
in the GenBank database under the accession numbers AJ812736.2
and CAH25436.2, respectively. However, the complete sequence of
798 amino acids contained dozens of non-essential residues at the
C-terminus, which could influence the expression and secretion of
the recombinant enzyme in E. coli. The C-terminus included 20
repeats of PXX residues (where P refers to proline and X refers to
any other amino acid) and a cytoderm-anchoring LPQTG motif
 D. Ni et al. / International Journal of Biological Macromolecules 113 (2018) 29–37
Fig. 5. Effect of temperature. (A) Effect of temperature on the activity of the recombinant levansucrase. (B) Effect of temperature on the stability of the recombinant levansucrase.
from gram-positive bacteria. In fact, the C-terminal sequence of
some fructosyltransferases, such as levansucrase and inulosucrase,
from gram-positive bacteria contain a cytoderm-anchoring motif
[49,50]. Therefore, C-terminal truncations are commonly used to
study fructosyltransferases expressed in E. coli. In this work, we
truncated the 100C-terminal amino acids of the original L. reuteri
LTH5448 levansucrase (GenBank: CAH25436.2). The remaining
gene sequence of levansucrase was synthesized and analyzed.
So far, many levansucrases have been identified and sequenced,
while only seven inulosucrases have been identified and sequenced,
most of which were from Lactobacillus (Table S2). Interestingly, upon
comparing the amino acid sequence of the L. reuteri LTH5448
levansucrase with other reported levansucrases (Table S1) and
inulosucrases (Table S2), we found that this levansucrase shared higher
identity with inulosucrases, but the biosynthesized polysaccharide was
a levan-type fructan. The L. reuteri LTH5448 levansucrase shared more
than 47% identity with all inulosucrases except one from Leci CW28
(38%). In contrast, this levansucrase shared less than 43% identity with
all levansucrases except two from Lactobacillus, Lare 121 (61%) and
Lasa TMW 1.392 (58%). The evolutionary relationship of the reported
fructosyltransferases from Lactobacillus is shown in Fig. 1. L. reuteri
LTH5448 levansucrase is more closely related with inulosucrases than
with levansucrases. Multiple sequence alignment of
fructosyltransferases from Lactobacillus (Fig. 2) shows that most of the
amino acids in the catalytic centers of levansucrases and inulosucrases
are strictly conserved. These conserved residues may play a small role
in controlling the formation of glycosidic bonds. In contrast, those
amino acids of L. reuteri LTH5448 which are conserved among
levansucrases but are different from inulosucrases may affect the occur-
rence of β-(2, 1) and β-(2, 6) linkages.
In fact, levansucrase and inulosucrase are similar in many ways, such
as in the three-dimensional structures of the enzymes, the conforma-
tions of catalytically active centers, the roles of the catalytic residues,
the mechanism of donor combination and the catalytic mechanism;
however, levansucrase and inulosucrase synthesize levan- and inulin-
type fructans, respectively. Mutation-based studies have been con-
ducted in an attempt to explain the different products and related resi-
dues involved in the formation of glycosidic bonds. However, these
mutants only changed the length of the fructan produced or the enzyme
activity without affecting the linkages [51,52]. Therefore, the formation
mechanism of the two types of glycosidic linkages has remained un-
clear. The levansucrase from L. reuteri LTH5448 shares high sequence
identity with inulosucrase, but the product synthesized by this enzyme
is levan. This phenomenon may provide a novel perspective from which
explore the mechanism of β-(2, 1) and β-(2, 6) linkage formation and
warrants further research.
33
3.2. Characteristics of purified recombinant L. reuteri LTH5448 levansucrase
The recombinant C-terminal L. reuteri LTH5448 levansucrase
contained 704 amino acids with a theoretical isoelectric point (pI) and
molecular weight (Mw) of 5.08 and 76,819 Da, respectively, as calcu-
lated by the ExPASy Compute pI/Mw tool. The recombinant
levansucrase was overexpressed and purified as described in
Section 2.1. The purified enzyme was used for SDS-PAGE. As shown in
Fig. 3, expression of the recombinant levansucrase was induced, and
the protein purified successfully, and an obvious band appeared on
the gel at approximately 75 kDa, which is consistent with the predicted
value.
3.3. Effect of pH and metal ions on the activities of the recombinant
levansucrase
The effect of pH on levansucrase activity was measured at 35 °C at
various pH values (4.0–9.0). As shown in Fig. 4A, the change in total, hy-
drolytic and transfructosylation activities exhibited the same trends.
The total and transfructosylation activities were highest in slightly
acidic environments, especially at pH 4.0–6.5, and were maximum at
pH 6.0. The transfructosylation activity was dominant under acidic con-
ditions, but did not exceed the hydrolytic activity at pH values greater
than 7.0. Compared to hydrolytic activity, the transfructosylation activ-
ity was more sensitive to pH change. The buffer systems greatly influ-
enced the transfructosylation activity, which improved approximately
12% in acetate buffer at pH 6.0 than in sodium phosphate buffer
(Fig. 4A). Levan was formed by transfructosylation; therefore, a high
proportion of transfructosylation to hydrolytic activity (T/H) was con-
ducive to the synthesis of levan. The effect of pH on the ratio of T/H is
shown in Fig. S1A; acetate buffer at pH 6.0 was optimal for the recombi-
nant levansucrase, and the T/H ratio reached 1.23.
Fig. 4B shows the effect of divalent metal ions on total levansucrase
activity. Ca2+, Mg2+ and Ni2+ improved total activity to different
degrees; notably, Ca2+ increased the total activity to 135%. The T/H
ratio increased markedly upon supplementation with Fe2+ and Zn2+
(Fig. S1B), but the total activity decreased to 39% and 51%, respectively.
Mg2+ inhibited the activity slightly, and the enzyme was inactivated in
the presence of 1 mM Cu2+. According to the crystal structure analysis
of levansucrase from Gluconacetobacter diazotrophicus, the Ca2+-
binding subsites of levansucrases from gram-negative bacteria are vari-
able, and the activity is not affected by metal ions, including Ca2+ [53].
However, the Ca2+-binding subsites of levansucrases from gram-
positive bacteria are relatively conserved and exhibit maximal activity
upon the addition of extra Ca2+. L. reuteri LTH5448 is a gram-positive
34 D. Ni et al. / International Journal of Biological Macromolecules 113 (2018) 29–37

bacterium, and the results of this study and other studies [33,41,54–56]
followed the regular trend.

3.4. Effect of temperature and thermostability

The effect of temperature on levansucrase activity was measured in

acetate buffer at pH 6.0 at various temperatures (20–75 °C). As shown in
Fig. 5A, the recombinant levansucrase showed the highest total activity
and transfructosylation activity at 35 °C, while the hydrolytic activity
was the highest at 45 °C. Many previous studies have shown that
levansucrase tends to produce levan by transfructosylation activity at
lower temperatures. In contrast, the enzyme prefers to hydrolyze su-
crose at higher temperatures. For example, levansucrase from Erwinia
amylovora showed optimal transfructosylation and hydrolytic activity
at 37 °C and 50 °C, respectively [41]. The levansucrase from Pseudomo-
nas syringae pv. phaseolicola exhibited optimal transfructosylation and
hydrolytic activity at 18 °C and 60 °C, respectively [56]. The T/H ratio
barely changed at lower temperatures but reduced when the tempera-
ture was higher than 35 °C (Fig. S1C). Therefore, 35 °C was determined
to be the most appropriate temperature for levan biosynthesis.
As for the effect of temperature on the stability of the recombinant
levansucrase, various temperatures were tested, and the enzyme exhib-
ited excellent thermostability at these temperatures, as shown in
Fig. 5B. The residual activity after preincubation at 55 °C for 12 h was
still 63.8% of the initial total activity. Even at 70 °C, the recombinant
levansucrase retained approximately 7% activity after 9 h. The recombi-
nant enzyme was almost unaffected at lower temperatures (Fig. S2). For
example, after 12 h of preincubation at 35 °C, 85% of the activity was
retained. Previous reports have shown that levansucrase from Bacillus
sp. TH4-2 [57] and Geobacillus stearothermophilus [58] are
thermotolerant. The levansucrase from Bacillus sp. TH4-2 retained 50%
activity after 30 min of incubation at 60 °C [57]. Although the
levansucrase from Geobacillus stearothermophilus retained more than
95% of the initial activity at 4–47 °C after 6 h, the activity dropped dra-
matically at 57 and 67 °C [58]. Apparently, the levansucrase from L.
reuteri LTH5448 possesses the highest thermostability compared to re-
ported levansucrases.

3.5. Optimization of levan production

3.5.1. Optimization of enzyme dosage

Previous studies have indicated that the enzyme dosage in the reac-
tion system plays an important role in levan generation [27,44,54].
Fig. 6A shows the relationship between levan and fructose formation
and enzyme dosage. The amount of levan accumulated increased with
increasing enzyme dosage until the enzyme was at 13 U/g sucrose and
then decreased slightly. However, the production of fructose continued
to increase within the enzyme dosage range tested, which indicated
that sucrose hydrolysis by the recombinant levansucrase was not af- Fig. 6. Optimization of levan production. (A) Effect of enzyme dosage on the carbohydrate
fected by enzyme dosage. yield (fructose and levan). (B) Effect of sucrose concentration on the carbohydrate yield
(fructose and levan). (C) Effect of time on levan yield.

3.5.2. Effect of sucrose concentration

The effect of sucrose concentration on the yield of levan and fructose
was studied using various concentrations of sucrose (Fig. 6B). Sucrose
concentration significantly enhanced levan production at substrate con-
centrations of 10–100%. In contrast, the yield of fructose increased
slowly, and the growth exhibited a tendency to stabilize. This phenom-
enon indicated that the hydrolytic activity of levansucrase catalysis fol-
lows Michaelis-Menten kinetics and can be inhibited by high
concentrations of sucrose [2,42]. However, the enzyme could not be sat-
urated by sucrose and did not exhibit Michaelis-Menten behavior;
therefore, the Hill-type equation was used to describe the total and
transfructosylation activities [2]. When the sucrose concentration was
100 g/L, only 19.8 g of levan was produced with a levan to fructose pro-
portion of 2.02. When the sucrose concentration increased to 1000 g/L,
the proportion was 5.09, which indicates that a high concentration of
sucrose is more beneficial for levan production.
3.5.3. Determination of reaction time
Because the produced levan cannot reach the maximum with the in-
creasing of sucrose concentration, moderate concentration was chosen
to probe the effect of reaction time. The reaction was conducted under
optimized conditions (acetate buffer at pH 6.0, 35 °C, 13 U/g sucrose
of levansucrase and 500 g/L sucrose) for 12 h. The change in levan
yield is shown in Fig. 6C. The levan yield improved rapidly within 2 h
and then increased slowly and exhibited the tendency to stabilize. Tak-
ing the time cost into account, the reaction time was determined to be
12 h, and the concentration of levan in the reaction mixture was 183 g/L.
 D. Ni et al. / International Journal of Biological Macromolecules 113 (2018) 29–37 35

3.6. Structural analysis of the biosynthesized polysaccharide
The structure of the purified polysaccharide was determined by FTIR
and NMR spectroscopy (Fig. 7). Fig. 7A shows the FTIR spectrum
scanned within the wavenumber range of 4000–400 cm−1. The broad
and strong peak at 3600–3200 cm−1 (specifically, at 3414 cm−1) repre-
sents the stretching vibration of O\\H and indicates the existence of in-
termolecular hydrogen bonding [59]. The stretching vibration of C\\H
caused a broad peak to appear at 3000–2800 cm−1 (specifically, at
2933 cm−1) [59,60]. The broad peaks at 1635 and 1019 cm−1 represent
the stretching vibrations of C\\O of the C\\OH group [54,60] and the
C\\O\\C group [61], respectively. The peaks appearing between 1000
and 800 cm−1 were typical for carbohydrates [27,54], and two charac-
teristic absorptions, at 927 and 811 cm−1, were detected, indicating
the existence of the furanoid ring of the sugar units [62,63].
Fig. 7B and C show the 13C and 1H NMR spectra of the purified prod-
uct, respectively. There were six distinct resonance signals, at 104.55,
80.66, 77.25, 75.90, 63.81 and 60.93 ppm, in the 13C spectrum. The six
peaks represent six carbon atoms in different environments of the fruc-
tose unit. The presence of a downfield-shifted signal of C-6 at 63.81 cm−
1
confirmed a β-(2, 6) linkage [63]. The chemical shifts, relative spacing
and shapes of these six signals were similar to those of reported levans
biosynthesized by levansucrases from various microorganisms
(Table 1) such as Bacillus licheniformis ANT 179 [64], Bacillus
methylotrophicus SK 21.002 [54], Brachybacterium phenoliresistens [65],
Paenibacillus bovis sp. nov BD3526 [63] and Leuconostoc mesenteroides
NTM048 [26]. The 1H spectrum (Fig. 7C) was almost consistent with
those of the levans biosynthesized by levansucrases from Bacillus
methylotrophicus SK 21.002 [54] and Brenneria goodwinii [27] in hydro-
gen atom splitting and in the area of peaks. All the structural informa-
tion revealed that the polysaccharide synthesized by L. reuteri
LTH5448 levansucrase was β-(2, 6) levan.
The average molecular weight of the levan produced was measured
by HPSEC-MALLS-RI (Fig. 7D). A signal peak was eluted at
approximately 17 min, indicating that the product was a homogeneous
polysaccharide. The average molecular weight was determined to be

Fig. 7. Structural analysis of biosynthesized polysaccharide. (A) FTIR spectrum of biosynthesized polysaccharide. (B) 13C NMR spectrum of biosynthesized polysaccharide. (C) 1H NMR
spectrum of biosynthesized polysaccharide. (D) HPSEC-MALLS-RI chromatograms of biosynthesized levan.
36 D. Ni et al. / International Journal of Biological Macromolecules 113 (2018) 29–37
Table 1
13
C chemical shifts reported for biosynthesized levan.
Microorganisms C-1 C-2
Bacillus licheniformis ANT 179 62.578 106.801
Bacillus methylotrophicus SK 21.002 61.20 104.66
Brachybacterium phenoliresistens 59.073 103.834
Paenibacillus bovis sp. nov BD3526 62.78 107.08
Leuconostoc mesenteroides NTM048 59.91 104.24
Lactobacillus reuteri LTH5448 60.93 104.55
3.924 × 107 Da after a 12-h reaction, which was higher than that of most
reported levans biosynthesized using microorganism-derived
levansucrases, as mentioned by Liu [27], but lower than that of the
levansucrase from Brenneria goodwinii [27].
4. Conclusion
In this study, a levansucrase from L. reuteri LTH5448 was cloned,
expressed, purified and characterized, and the optimal conditions for
levan biosynthesis were identified. The amino acid sequence shared
high identity with reported inulosucrases, but this enzyme produces
levan-type fructans; this phenomenon warrants further research. The
levan yield reached 183 g/L when the reaction was conducted at
pH 6.0 and 35 °C with an enzyme dosage of 13 U/g sucrose in 500 g/L su-
crose for 12 h. Structural analysis by FTIR and NMR spectroscopy
showed that the fructosyl of the polysaccharide produced was con-
nected via a β-(2, 6) linkage. The average molecular weight was 3.924
× 107 Da, as measured by HPSEC-MALLS-RI. Notably, the recombinant
levansucrase exhibited the highest thermostability among reported
levansucrases. Therefore, the L. reuteri LTH5448 levansucrase could be
a promising candidate for industrial production of levan using an enzy-
matic strategy.
Acknowledgement
This work was supported by the Support Project of Jiangsu Province
(No. BK20130001 and 2015-SWYY-009).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2018.01.187.
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