A recombinant levansucrase from Bacillus licheniformis 8-37-0-1 catalyzes versatile transfructosylation reactions

Article abstract of DOI:10.1016/j.procbio.2014.05.012

This work disclosed the broad transglycosylation capability of the levansucrase from Bacillus licheniformis 8-37-0-1 for the first time. The levansucrase was firstly purified from the strain 8-37-0-1 and found to be a monomer of ～51 kDa with KETQDYKKSY as the N-terminus. Then, the gene encoding the enzyme was cloned and it contained an ORF of 1449 nucleotides, encoding a 482 amino-acid protein with a predicted 29 amino-acid signal peptide. The deduced mature protein without the signal showed the same N-terminus to the purified enzyme. The mature enzyme was subsequently expressed in Escherichia coli. The recombinant enzyme showed similar biochemical properties to the native one. It produced maximal yield of 7.1 mg/mL levan (M_r 9.6 × 10⁶) from 0.8 M sucrose (pH 6.5) at 40 °C for 24 h in vitro. When using sucrose as the donor, the enzyme displayed a wide range of acceptor specificity and was able to transfer fructosyl to a series of sugar acceptors including hexose, pentose, β- or α-disaccharides, along with the difficult branched alcohols that have not been investigated before. Chemical structures of the resultant products were analyzed by MS and NMR spectra.

Full text of DOI:10.1016/j.procbio.2014.05.012

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Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
A recombinant levansucrase from Bacillus licheniformis 8-37-0-1
catalyzes versatile transfructosylation reactions
Lili Lu, Feng Fu, Renfei Zhao, Lan Jin, Chunjuan He, Li Xu, Min Xiao∗
State Key Lab of Microbial Technology and National Glycoengineering Research Center, Shandong University, Jinan 250100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
This work disclosed the broad transglycosylation capability of the levansucrase from Bacillus licheniformis
8-37-0-1 for the first time. The levansucrase was firstly purified from the strain 8-37-0-1 and found to
be a monomer of ∼51 kDa with KETQDYKKSY as the N-terminus. Then, the gene encoding the enzyme
was cloned and it contained an ORF of 1449 nucleotides, encoding a 482 amino-acid protein with a
predicted 29 amino-acid signal peptide. The deduced mature protein without the signal showed the same
N-terminus to the purified enzyme. The mature enzyme was subsequently expressed in Escherichia coli.
The recombinant enzyme showed similar biochemical properties to the native one. It produced maximal
yield of 7.1 mg/mL levan (Mr 9.6 × 106) from 0.8 M sucrose (pH 6.5) at 40 ◦C for 24 h in vitro. When using
sucrose as the donor, the enzyme displayed a wide range of acceptor specificity and was able to transfer
fructosyl to a series of sugar acceptors including hexose, pentose, ␤- or ␣-disaccharides, along with the
difficult branched alcohols that have not been investigated before. Chemical structures of the resultant
products were analyzed by MS and NMR spectra.
Received 28 February 2014
Received in revised form 30 April 2014
Accepted 19 May 2014
Available online xxx
Keywords:
Bacillus licheniformis 8-37-0-1
Levansucrase
Transfructosylation
Levan
Oligosaccharide
Fructoside
© 2014 Published by Elsevier Ltd.
1. Introduction
In addition to levan, fructooligosaccharides (FOS) are also pro-
duced by some levansucrases via oligomerization from sucrose.
Levansucrases (EC 2.4.1.10) catalyze fructosyl transfer following
the mechanism of non-Leloir glycosyltransferases with sucrose as
the main non-activated donor substrate. Such non-Leloir enzymes
have great biotechnological potential, since they do not require
expensively activated donors such as the nucleotide phospho-
sugars that are necessary for Leloir enzymes. The Gibbs binding
energy released from hydrolysis of sucrose is available for saccha-
ride formation [1].
Levansucrases are widely distributed in nature, especially abun-
dant in gram-negative and gram-positive bacteria. They are of
particular interest for the formation of levan, a ␤-(2 → 6)-linked
fructose polymer with small amount of ␤-(2 → 1)-linked branch
chains [2–4]. The degree of polymerization and type of branch-
ing are various depending on enzyme sources. These polymers
possess wonderful physiological and biochemical characteristics
and thus have broad applications. They can be widely used in food
and non-food industries as viscosifier, stabilizer, emulsifier, gelling,
or water-binding agent. Also, they have potential pharmaceutical
applications owing to diverse bioactivities, such as antiviral, anti-
tumor and immunostimulating activities [5–8].
Such kind of oligomers are described as healthful prebiotics, which
exhibit low caloric values, decrease levels of lipids and cholesterol,
help gut absorption of ions, and stimulate the bifidobacteria growth
in the human colon [9]. Using sucrose as glycosyl donor, levan-
sucrases can also catalyze fructosyl transfer to a wide range of
acceptors including alcohols and mono- or oligosaccharides. The
different sugar residues (i.e. galactose and xylose) that cap fruc-
tooligosaccharides may alter prebiotic and biochemical properties
[10]. Some of the hetero-oligosaccharides have been found appli-
cations as sweeteners and as prebiotics [11]. Despite the promising
prospect of developing fructosyl compounds with novel extended
functions, the reported levansucrases are mostly focused on the
formation of levan and FOS, while only a few of them have been
fully characterized with respect to their acceptor specificity for
transglycosylation [12–16].
In the previous work, a strain of Bacillus licheniformis 8-37-0-
1 with high level of levan production (41.7 g/L) was isolated from
the soil [17]. The levan product was subsequently purified from
the fermentation broth and identified to be a novel polysaccha-
ride, which contained a ␤-(2 → 6)-linked backbone with a single
␤-D-fructose at the C-1 position every seven residues, on aver-
age, along the main chain. Preliminary in vitro tests revealed it
could significantly stimulate the proliferation of spleen lymphocyte
[18].
∗
Corresponding author. Tel.: +86 531 88365128; fax: +86 531 88365128.
E-mail address: minxiao@sdu.edu.cn (M. Xiao).
http://dx.doi.org/10.1016/j.procbio.2014.05.012
1359-5113/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Lu L, et al. A recombinant levansucrase from Bacillus licheniformis 8-37-0-1 catalyzes versatile
transfructosylation reactions. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.05.012

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In this work, the levansucrase was purified from B. licheniformis
8-37-0-1, and the gene of the enzyme was cloned and over-
expressed in Escherichia coli. The resulting recombinant enzyme
was found to possess similar biochemical properties to the native
one and thus was used in the synthesis of levan in vitro. Also,
the enzyme was found to catalyze oligomerization reactions. It
could efficiently transfer fructosyl to a lot of saccharides as well
as novel branched alcohol acceptors. This is the first report of
the powerful transglycosylation ability of the levansucrase from B.
licheniformis 8-37-0-1. The excellent characteristics endowed the
enzyme with a high capacity for obtaining novel fructosyl com-
pounds and made it an alternative to the current synthetic enzyme
sources.
of 0.3 mL/min at 4 ◦C. The elution patterns were compared with
those of the standard proteins, including ovalbumin (44 kDa),
conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa), and
thyroglobulin (669 kDa).
2.5. Gene cloning and heterogenous expression
Primers for the gene cloning were designed based on the
sequence of the levansucrase from B. licheniformis DSM 13 (Gen-
Bank No. AE017333). The forward and reverse primers designed
for the entire gene were 5ꢀ-CAGGTCGACATGAACACATCAAAAAC-
ATTGC-3ꢀ and 5ꢀ-CGACTCGAGTTTGTTTACCGTTAGTTGTCC-3ꢀ (Sal I
and Xho I restriction sites are underlined), respectively. As for the
gene without the signal-encoding sequence, the forward primer
was 5ꢀ-CAGCATATGAAAGAAACGCAGGATTACAAG-3ꢀ (Nde I site is
underlined). The PCR reactions were performed in the presence
of TaKaRa LA Taq polymerase, following the procedures includ-
ing 5 min at 94 ◦C, 30 cycles of 30 s at 94 ◦C, 30 s at 55 ◦C, 2 min
at 72 ◦C, and a final 5 min at 72 ◦C. PCR products were purified and
sequenced.
For enzyme expression, the purified PCR products were subse-
quently digested by restriction enzymes, ligated onto the pET-21b
(+) vector, and transformed into E. coli BL21 (DE3). The correct
transformant was grown in LB medium at 37 ◦C, and the enzyme
was induced by adding isopropyl-1-thio-␤-d-galactoside (IPTG)
when the cell density reached 0.6–1.0 at 600 nm. After continuous
cultivation for three to 5 h, cells were harvested and disrupted by
ultrasonic treatment. The lysate was centrifuged and the enzyme
was purified from the suspension by Ni2+ chelation chromatogra-
phy (Qiagen, Germany).
2. Materials and methods
2.1. Bacterial strains, plasmids and culture conditions
B. licheniformis 8-37-0-1 was cultured at 37 ◦C for 24 h in the
medium (pH 7.0) containing 30 g/L sucrose, 1.0 g/L beef extract,
0.5 g/L (NH4)2SO4, 2.5 g/L K2HPO4·3H2O, 2.5 g/L KH2PO4, 1.0 g/L
NaCl, 0.2 g/L MgSO4·7H2O, and 0.001 g/L FeSO4·7H2O. E. coli strains
DH5␣ and BL21 (DE3) were grown at 37 ◦C in LB medium. The pET-
21b (+) plasmid (Novagen, US) was used for the construction of
the expression vector with the His tag. The medium for the E. coli
cells containing the pET-21b (+) plasmid was supplemented with
ampicillin (100 ␮g/mL).
2.2. Enzyme and protein assays
The activity of levansucrase was measured by addition of
enzyme solution (10 ␮L) to 40 ␮L of sucrose (finally at 0.1 M) in
50 mM phosphate buffer (pH 6.0). The reaction was performed at
37 ◦C for 60 min and then stopped by heating at 100 ◦C for 10 min.
The resulting mixture was centrifuged and the glucose content in
the mixture was analyzed using the glucose oxidase kit (Biosino
Biotechnology and Science Inc., China). One unit of enzyme activ-
ity (U) is defined as the amount of enzyme that produces 1 ␮mol
glucose per minute under the assay conditions. The concentrations
of protein were measured according to Bradford method using BSA
as the standard [19]. All enzyme and protein determinations were
performed in triplicate.
2.6. Characterization of native and recombinant levansucrase
Kinetic constants of native and recombinant enzymes were
estimated by Lineweaver–Burk double reciprocal plots. Various
concentrations of sucrose (0.1–1 M) were used to determine kinetic
constants. Reactions were performed under assay conditions as
described above. The effect of pH on the activity of the enzyme
was determined by incubating the enzyme with 0.1 M sucrose
in broad-range buffers containing 6.008 g/L citric acid, 3.893 g/L
KH2PO4, 1.769 g/L boric acid and 5.266 g/L barbitone and using
NaOH to adjust the pH from 4.0 to 9.0. The effect of tempera-
ture was assayed at 20 ◦C to 65 ◦C. All assays were performed in
triplicate.
2.3. Enzyme purification
All the procedures described below were performed at 4 ◦C. The
culture of B. licheniformis 8-37-0-1 was centrifuged at 12,000 rpm
for 5 min. The resultant supernatant was precipitated with ammo-
nium sulfate at 80% saturation, followed by desalting. The sample
was subsequently applied to a 1.1 × 20-cm DEAE Sepharose Fast
Flow column (GE Healthcare, US) which had been pre-equilibrated
with 50 mM phosphate buffer (pH 7.5). Then it was eluted by
NaCl solution (pH 7.5) at concentration gradients from 0 to 0.4 M.
The collected enzyme fragments were concentrated through cen-
trifugal filters (Millipore, Germany) with a 30-kDa molecular mass
cutoff.
2.7. Levan synthesis and analysis
Levan synthesis was carried out with the recombinant mature
levansucrase in the presence of sucrose as substrate. The effects of
sucrose concentrations were determined by incubating enzymes
(50 U/mL) with sucrose solutions ranging from 0.1 M to 1 M in the
pH 8.0 phosphate buffer at 37 ◦C for 24 h. The influence of the reac-
tion time was assayed in 0.8 M sucrose at 37 ◦C. Aliquots were
serially removed at 12 h intervals within 60 h period. The effects of
temperature were assayed at 25, 30, 35, 40, 45, and 50 ◦C for 24 h.
The impacts of pH on levan synthesis were performed in broad-
range buffers with pH values ranging from 4.0 to 9.0 at 40 ◦C for
24 h. All the reactions were stopped by boiling at 100 ◦C for 10 min.
Then the mixtures were centrifuged at 12,000 rpm for 5 min. Levan
in the suspension was precipitated by 75% ethanol and quanti-
fied by the phenol-sulfuric acid method [17]. In the method, the
polysaccharide was hydrolyzed by the concentrated sulfuric acid
and released the monosaccharides that were quickly dehydrated,
reacted with phenol and converted into colorimetric compounds.
The absorbance of the compounds at certain wave length showed
linear relationship with the sugar content in some range. A
2.4. Molecular mass determination
The molecular mass of levansucrase was determined by SDS
polyacrylamide gel electrophoresis (SDS-PAGE) as well as gel fil-
tration chromatography. Proteins in the polyacrylamide gel were
visualized by Coomassie brilliant blue R-250 staining. Gel filtration
chromatography was performed via a Superdex 200 (10 × 300 mm)
column (GE Healthcare, US) pre-equilibrated with 150 mM NaCl
in 50 mM phosphate buffer. Samples were eluted at a flow rate
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standard curve was firstly established between the concentration
of fructose and the absorbance at OD490 after incubation with sul-
furic acid and phenol. Then, the content of levan could be measured
referring to the curve.
2.9. Isolation of oligosaccharides and fructoside products
The oligosaccharide or glycoside products were concentrated
by vacuum freeze dehydration. The resulting samples were loaded
The homogeneity and molecular weight of the levan produced
under the optimal condition were determined on a Waters High-
Performance Size-Exclusion Chromatography (HPSEC) apparatus
equipped with Two TSK columns, G3000 PWXL and G4000 PWXL
in series, coupled to a refractive index detector (RID), and a Wyatt
Technology Dawn-EOS Multi-Angle Laser Light Scattering detector
(MALLS). The carrier solution was 0.2 M NaNO3, and the samples
were dissolved in 0.2 M NaNO3 with stirring. The injection volume
was 200 ␮L and the flow rate was 0.6 mL/min. The normalization
of RID was conducted with bovine albumin monomer. The spe-
cific RI increment (dn/dc) at 658 nm and 25 ◦C was determined
using an interferometric refractometer (Optilab/DSP, Wyatt Tech-
nology, USA). The dn/dc value was averaged to 0.135 mL/g and was
assumed to be constant during the sample elution. ASTRA soft-
ware (http://www.wyatt.com/) was utilized for data acquisition
and analysis [18].
on preparative TLC 1-mm plates (Silica gel 60 F254, Merck,
Germany) using the developing solvent of n-butanol: ethanol:
water (5:3:2, v/v/v) or subjected to a Bio-Gel P2 (Bio-Rad, US) col-
umn (1.5 × 100 cm) eluted by the deionized water for purification.
The eluates were subjected to sugar determination by TLC, and the
correct fractions were collected and lyophilized to dry powder.
2.10. TLC and HPLC analysis
TLC was performed with Silica gel 60 F254 plates (Merck,
Germany). The developing solvent was a mixture of n-butanol:
ethanol: water (5:3:2, v/v/v). Sugars on the TLC plate were detected
by spraying with a solution of 0.5% (w/v) 3,5-dihydroxytoluene dis-
solved in 20% (v/v) sulphuric acid and subsequent heating at 120 ◦C
for 5 min. HPLC was performed by Agilent 1200 series equipped
with Agilent Zorbax carbohydrate analysis column (4.6 × 250 mm)
and Agilent G1362A refractive index detector. Samples were eluted
with acetonitrile: water (4:1, v/v) at a flow rate of 1.0 mL/min, with
the column temperature maintained at 30 ◦C.
2.8. Oligosaccharide and fructoside synthesis
2.11. MS and NMR analysis
Using the sucrose as glycosyl donor, a series of mono-, di-, tri-
saccharides as well as sugar and aliphatic alcohols were tested as
acceptors for transglycosylations. The reactions were performed
at 40 ◦C for 24 h by incubation of the recombinant enzyme with
0.5 M sucrose and 0.5 M acceptors at pH 6.5, in the case of sugar or
sugar alcohols as acceptors which included d-xylose, l-arabinose, l-
rhamnose, d-mannose, d-galactose, melibiose, cellobiose, maltose,
lactose, trehalose, sucralose, raffinose, dulcitol, sorbitol, mannitol
and inositol. As for alkyl alcohols such as isopropanol, 1-pentanol,
1-hexanol, 1-octanol and 1-dodecanol, the reactions were oper-
ated under the same conditions except for the 10% (v/v) acceptor
concentration. All the reactions were terminated by heating at
100 ◦C for 10 min. Sugar analysis was performed by TLC and
HPLC as described below. The glycoside yields were calculated
from the products using 50 U/mL enzymes based on the acceptor
conversion.
Mass spectra were recorded on a Shimadzu LCMS-IT-TOF
instrument (Kyoto, Japan) equipped with an ESI source in pos-
itive/negative ion mode at a resolution of 10,000 full width at
half-maximum. 1H and 13C NMR spectra were recorded at 26 ◦C
with a Bruker DRX Advance-600 spectrometer (Bruker Biospin AG,
Fallanden, Switzerland) at 600 MHz for 1H and 150 MHz for 13C.
Chemical shifts were given in ppm downfield from internal TMS of
D2O. Chemical shifts and coupling constants were calculated from
a first-order analysis of the spectra. Assignments were fully sup-
ported by homo- and hetero- nuclear correlated 2D techniques,
including COSY (correlation spectroscopy), HSQC (hetero-nuclear
single quantum coherence) and HMBC (hetero-nuclear multiple
band correlation) experiments following standard Bruker pulse
programs.
Fig. 1. SDS-PAGE analysis of native (a) and recombinant (b and c) levansucrase from B. licheniformis 8-37-0-1. M, marker proteins; lane 1, crude native enzyme from sulfate
precipitation, lane 2, purified native enzyme; lane 3 and 4, crude enzyme from sacB-containing cells of E. coli with and without IPTG, respectively; lane 5, the purified
recombinant SacB; lane 6, recombinant proteins from sacB1-containing cells of E. coli, which were purified through affinity and anion-exchange chromatography; lane 7,
crude enzyme from sacB1-containing cells of E. coli in the presence of IPTG.
Please cite this article in press as: Lu L, et al. A recombinant levansucrase from Bacillus licheniformis 8-37-0-1 catalyzes versatile
transfructosylation reactions. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.05.012

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2.12. Nucleotide sequence accession number
The GenBank accession number for the levansucrase gene of B.
licheniformis 8-37-0-1 is KF647836.
3. Results and discussion
3.1. Purification, gene cloning and expression of levansucrase
The native levansucrase was purified from B. licheniformis 8-
37-0-1 through ammonium sulfate precipitation, anion-exchange
chromatography and ultrafiltration. The enzyme reached elec-
trophoretic homogeneity with a molecular mass (Mr) of ∼51 kDa
as detected by SDS-PAGE (Fig. 1). The native form of the enzyme
turned out to be a monomer since its natural molecular mass was
less than 51 kDa as determined by gel filtration chromatography.
It was in the range of the molecular masses (45–65 kDa) of mostly
reported microbial levansucrases [15,20–26].
The purified enzyme in the SDS-polyacrylamide gel was subse-
quently electroblotted onto a polyvinylidene difluoride membrane.
The protein band in it was cut out and sequenced by the method
of Edman degradation. The N-terminal amino acid sequence was
determined as KETQDYKKSY (Fig. S1).
Meanwhile, the gene of the levansucrase was obtained by PCR. It
contained an open reading frame (ORF) of 1449 nucleotides, encod-
ing a 482 amino-acid protein with a predicted Mr of ∼53.7 kDa.
Further analysis of the deduced protein by the software SignalP 4.0
revealed it had a putative signal peptide in the N-terminal region,
with a predicted cleavage site between the 29th and 30th amino
acids. The initial ten amino acids of the putatively mature pro-
tein which lacked the signal peptide were identical to those of the
purified enzyme. Additionally, the theoretical Mr of the putatively
mature protein (50.8 kDa) was consistent with the Mr of the puri-
fied enzyme (∼51 kDa). These confirmed the actual existence of the
signal peptide. The presence of the signal peptide seemed common
in the extracellular levansucrase. The SacB enzymes from B. subtilis
and B. megaterium were also found to bear a 29 amino-acid signal
peptide in the reported literatures [21,27].
The nucleotide sequences of the entire and signal-deleted genes
(designated as sacB1 and sacB) of the levansucrase were ligated onto
pET-21b (+) and transformed into E. coli, respectively. As shown in
Fig. 1, either sacB1 or sacB genes from B. licheniformis 8-37-0-1 was
successfully expressed in E. coli cells. A protein band of ∼52 kDa
appeared in both kind of cells, while an additional protein band of
∼57 kDa occurred only in the sacB1-containing cells and it could
not be separated from the smaller one through affinity and anion-
exchange chromatography. Thus, both of the protein bands were
subjected to peptide mass fingerprinting. Two sequenced peptides
(VMKPLIASNTVTDEIER and WYLFTDSR) were found to fully match
the deduced levansucrase sequence from B. licheniformis 8-37-0-1
(Fig. S2). It proved the two proteins were actually the precursor and
mature forms of a same enzyme. Similar phenomenon was previ-
ously observed when expressing B. subtilis levansucrase in E. coli,
where appeared a mature enzyme of ∼50 kDa along with its puta-
tive precursor of approximately 53 kDa [27]. As the sacB-containing
cells only formed the mature protein that was comparable to the
native purified protein, they were used for the production and
purification of the recombinant SacB for the following analysis.
Fig. 2. The effect of pH (a) and temperature (b) on the activity of native (ꢁ) and
recombinant (᭹) SacB.
the range of those of other microbial levansucrases (6.6–160.0 mM)
[21,25,26,28–32].
The effects of pH on the activity of native and recombinant SacB
were in similar patterns. Both enzymes were highly active at pH
6.0 and dropped by more than 50% activity at pH <4.0 or pH >8.5
(Fig. 2a). A lot of levansucrases were found to be of high activ-
ity at pH values near neutrality. For example, the enzyme from
B. megaterium DSM319 presents an optimum range of pH 6.0–7.0.
The hydrolysis activity significantly fell at pH values below 5.6 and
above 7.6 [21]. Another example was that the highest enzyme activ-
ity from L. mesenteroides ATCC 8293 was found in the 6.5–7 pH
range but with a rapid decrease in activity at pH >8 [33].
The optimal temperature for the activity of the recombinant
SacB was 45 ◦C, slightly lower than that of the native enzyme
(45–50 ◦C) (Fig. 2b). Both enzymes lost 80% activities at temper-
atures below 20 ◦C and were nearly inactive at above 60 ◦C. A
number of reported levansucrases exhibited maximal activity at
temperatures between 35 and 45 ◦C, including the enzymes from
L. mesenteroides ATCC 8293 (35 ◦C) [32], B. circulans (45 ◦C) [11], B.
megaterium DSM319 (45 ◦C) [21], and Z. mobilis UQM 2716 (45 ◦C)
[29].
3.2. Characterization of the native and recombinant levansucrase
The Michaelis–Menten constants for native and recombinant
SacB were consistent. The Km values of them for sucrose were
140.81 and 152.79 mM, respectively, while the Vmax values were
equal to be about 0.03 mM/min. The Km values of SacB were in
According to the above results of biochemical assays, only a
few insignificant differences were found between the recombinant
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by enzymes from B. subtilis (3 × 106) [21] and L. sanfranciscensis
TMW 1.392 (5 × 106) [31] but significantly higher than that pro-
duced from native cells of B. licheniformis 8-37-0-1 (2.8 × 104) [18].
It was reported that the levans with high molecular masses were
more effective for a direct effect on tumor cells, which is related
to a modification in the cell membrane, including changes in cell
permeability [35].
and native SacB from B. licheniformis 8-37-0-1. The recombinant
enzyme had a negligible increase of Km value as compared with the
native one, which might be due to the introduced vector sequence
that could slightly affect the affinity of the enzyme toward sub-
strate. In addition, the native enzyme was a little more resistant
for pH below 5.0 and beyond 8.0 as well as for temperatures
above 45 ◦C. That might be related to the presence of native levan-
enzyme complexes of which the substrate protection made the
enzyme stronger [29]. Since the properties of the recombinant
enzyme were generally in agreement with the native one, it was
used as the enzyme source for the following transglycosylation
reactions.
3.4. Oligosaccharide and fructoside synthesis by the recombinant
levansucrase
In the presence of sucrose as glycosyl donor, the recombinant
SacB from B. licheniformis 8-37-0-1 exhibited a wide range of accep-
tor specificity. It was able to catalyze fructosyl transfer to various
sugars such as galactose, cellobiose, xylose, maltose, lactose, arabi-
nose, and trehalose (Fig. S3), along with challengeable branched or
long-chain alcohols like isopropanol and 1-pentanol. As a whole,
the recombinant SacB exhibited better transglycosylation activ-
ity toward sugar acceptors than branched or long-chain alcohols
from which only trace glycoside products were available (data not
shown). As for sugar acceptors, the enzyme catalyzed the glyco-
syl transfer to maltose, arabinose, cellobiose, and xylose with high
product yields of 37.5%, 36.8%, 34.7% and 31.5%. It showed mod-
erate activity toward lactose with 25.8% glycoside yield and lower
activity for galactose and trehalose with 10.1% and 8.5% glycoside
yields.
The newly-produced glycosides were purified and subjected
to MS analysis for the confirmation of fructosylations (Fig. S4).
All of the derivatives from maltose, cellobiose, lactose and tre-
halose showed peaks of [M+Na]+ ions in the MS spectra at m/z
527.15, consistent with fructosyl disaccharides (Mr 504). The galac-
tose derivative displayed the peak of [M+Na]+ ion at m/z 365.10,
in agreement with fructosyl galactose (Mr 342). The signals of
[M+H2O]+ ions at m/z 330.33 and 330.13 revealed the presence
of fructosyl arabinose and xylose (Mr 312). The alkyl fructosides
from isopropanol and 1-pentanol showed characteristic peaks of
[M+Na]+ ions 245.09 and 273.12, corresponding to isopropyl and
pentyl fructosides (Mr 222 and 250), respectively.
3.3. Synthesis of levan by the recombinant levansucrase
Levan synthesis was performed by incubation of the recom-
binant SacB with sucrose. Several efforts were directed toward
evaluating the influence of various factors on the transglycosyla-
tion yield. These factors included substrate concentrations, reaction
time, temperature and pH.
The effects of sucrose concentrations on levan synthesis were
shown in Fig. 3a. The yield of levan was improved when the sucrose
concentrations were increased from 0 to 0.8 M. The transferase
activity of levansucrase was usually dependant on the sucrose
concentration. An instance was that the enzyme from B. amyloliq-
uefaciens showed enhanced transferase activity with the increased
sucrose concentrations from 0.01 to 1.0 M [15]. Another instance
was that the enzyme from L. mesenteroides ATCC 8293 displayed
highly transferase activity at sucrose concentrations higher than
146 mM and retaining the activity up to a sucrose concentration as
high as 875 mM [33].
Reaction time ranging from 0 to 60 h was tested (Fig. 3b). The
levan yield was rapidly increased within the initial 24 h. When
continuously extending the incubation time, the yields kept sta-
ble. Reaction time was seldom investigated as a factor involved in
the levan formation. In this work, the initial incubation time of the
enzyme with sucrose had a large influence on the amount of fruc-
tose transferred to levan. The levan yield at 12 h only achieved a
half of the yield at 24 h.
The reaction temperature also exhibited significant impact on
the levan production by the recombinant SacB (Fig. 3c). The yield
of levan reached a maximum of 5.1 mg/mL at 40 ◦C and decreased
at either lower or higher temperatures. It dropped to be around
2 mg/mL at 25 ◦C or 50 ◦C. The optimal temperature for levan syn-
thesis (40 ◦C) was slightly lower than that for hydrolysis (45 ◦C).
Similar phenomenon was observed in the enzyme from B. circu-
lans, where the optimal temperature for transferase activity was
40 ◦C while for hydrolysis the highest initial rate extended to 45 ◦C
[11]. As for the levansucrase from Z. mobilis, however, the situation
was quite different. The sucrose hydrolysis was maximal at 45 ◦C
whereas the levan-forming activity displayed the optimal activity
at a low temperature of about 15 ◦C and it dropped to zero at 35 ◦C
[29].
The pH affected the levan formation significantly (Fig. 3d). The
yield reached the maximum of 7.1 mg/mL at pH 6.5, whereas it
fell to around 4.0 mg/mL at pH 5.0 and 8.5. A number of levansu-
crases exhibited excellent activity for levan formation in the range
of pH 5.5–8.0, such as the enzymes from L. sanfranciscensis TMW
1.392 (pH 5.5) [31], L. mesenteroides B-512 FMC (pH 6.2) [25], P.
syringae NCPPB 1321 (pH 6.2) [28], Geobacillus stearothermophilus
(pH 6.0–6.5) [16] and B. amyloliquefaciens (pH 8.0) [34].
Furthermore, transglycosylation products from maltose, cel-
lobiose, lactose and xylose were analyzed by NMR for the structure
information (Fig. S5). They were identified to be ␣-D-glucopyra-
nosyl-(1 → 4)-␣-D-glucopyranosyl-(1 → 2)-␤-D-fructofuranoside,
␤-D-glucopyranosyl-(1 → 4)-␣-D-glucopyranosyl-(1 → 2)-␤-D-
fructofuranoside,
␤-D-galactopyranosyl-(1 → 4)-␣-D-glucopyr-
anosyl-(1 → 2)-␤-D-fructofuranoside, and ␣-D-xylopyranosyl-
(1 → 2)-␤-D-fructofuranoside. Their structures were shown in
Fig. 4 and the relevant data were listed in Table 1. The fructose
was added to all acceptors in ␤-(2 → 1) glycosidic bonds. The same
phenomenon was also found in other levansucrase-catalyzed
glycosylations toward sugar acceptors, such as the enzymes from
B. amyloliquefaciens and B. subtilis NCIMB 11871 [12,15].
Levansucrases have already been known for their transferase
activity when acceptors other than sucrose are added to the reac-
tion medium. Nonetheless, the specificity of the enzymes toward
acceptors differs considerably [11]. According to the product
yields based on acceptor conversions, the enzyme from B. amy-
loliquefaciens showed higher activity toward disaccharides than
monosaccharides [15]. In contrast, the enzyme from B. subtilis
NCIMB 11871 fructosylated galactose and xylose more efficiently
than disaccharides [12]. In this work, however, the acceptor
specificity did not follow an obviously uniform principle. The com-
mon acceptors such as maltose, cellobiose, xylose, lactose and
galactose were sequentially less efficient acceptors. Interestingly,
the enzyme from B. licheniformis 8-37-0-1 showed high activity
toward an unusual pentose, L-arabinose, which is a low-calorie
Thus, the optimal conditions for the levan synthesis were an
initial sucrose concentration of 0.8 M at pH 6.5 and 24 h incuba-
tion at 40 ◦C. The levan produced under the conditions was purified
and its molecular mass was determined to be 9.6 × 106 by multi-
angle laser light scatting, which was comparable to those formed
Please cite this article in press as: Lu L, et al. A recombinant levansucrase from Bacillus licheniformis 8-37-0-1 catalyzes versatile
transfructosylation reactions. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.05.012

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Fig. 3. The effect of substrate concentrations (a), reaction time (b), temperature (c) and pH (d) on the levan synthesis by the recombinant SacB.
Table 1
1H and 13C NMR data assignment for hetero-oligosaccharides produced by the recombinant SacB.
C-atom
Mal-Fru
Cel-Fru
Lac-Fru
Xyl-Fru
ıC
ıH
ıC
ıH
ıC
ıH
ıC
ıH
C-1
C-2
C-3
C-4
C-5
C-6
99.6
71.4
72.7
69.1
72.4
59.8
91.8
70.6
72.8
76.4
70.7
60.2
61
5.25
3.43
3.53
3.27
3.58
3.68
5.24
3.43
3.87
3.54
3.81
3.70–3.61
3.51
–
102.4
73
4.38
3.17
3.36
3.27
3.34
3.77–3.59
5.25
3.45
3.72
3.54
3.82
3.76–3.71
3.52
–
4.06
3.88
3.74
3.66
102.6
70.8
77.7
68.3
72.2
62.2
91.6
70.6
71
75.1
70.9
59.2
61.1
103.4
76.1
73.8
81.2
60.9
4.32
3.4
92.2
70.8
72.6
69
61.5
–
60.5
103.7
75.8
73.4
81.2
61.6
–
5.21
3.4
3.55
3.48
3.56
–
3.52
–
4.09
3.97
3.75
3.67–3.56
–
–
–
–
–
–
75.2
69.3
75.7
60.4
91.7
70.6
70.9
78.1
70.9
59.2
61.1
103.5
76
3.56
3.78
3.52
3.66
5.25
3.46
3.74
3.59
3.83
3.75–3.71
3.52
–
C-1ꢀ
C-2ꢀ
C-3ꢀ
C-4ꢀ
C-5ꢀ
C-6ꢀ
C-1ꢀꢀ
C-2ꢀꢀ
C-3ꢀꢀ
C-4ꢀꢀ
C-5ꢀꢀ
C-6ꢀꢀ
103.4
76.2
73.8
81.1
62.1
–
–
–
–
4.05
3.87
3.73
3.65
4.06
3.89
3.74
3.64–3.61
73.8
81.2
62.2
–
sweetener with promising application to reduce weight and con-
trol diabetes. The compound could selectively inhibit intestinal
sucrase in an uncompetitive manner and suppress the increase of
blood glucose [36]. Fructosylation of arabinose might expand its
function to be as prebiotics besides as sweeter and anti-diabetic
agent.
It was also interesting to note that the enzyme from B. licheni-
formis 8-37-0-1 was able to modify unusual alcohols such as
Please cite this article in press as: Lu L, et al. A recombinant levansucrase from Bacillus licheniformis 8-37-0-1 catalyzes versatile
transfructosylation reactions. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.05.012

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Fig. 4. Structures of hetero-oligosaccharides formed by the recombinant SacB. Mal-Fru, Cel-Fru, Lac-Fru and Xyl-Fru represent the products from maltose, cellobiose, lactose
and xylose, respectively.
isopropanol and 1-pentanol, forming novel alkyl glycosides. Such
kind of glycosides are widely used as biosurfactants in manufactur-
ing of cosmetics and household chemicals as well as building blocks
in pharmaceutical industry and molecular biology. In the case
of levansucrase-mediated reactions, the known aliphatic-alcohol
acceptors were limited to primary alcohols with less than four
carbon atoms. The enzyme from B. circulans was reported to glyco-
sylate methanol and butanol [11,37]. In this work, the recombinant
SacB from B. licheniformis 8-37-0-1 exhibited transferase activity
toward secondary alcohol like isopropanol, which was difficult for
glycosylation due to steric hindrance even by chemical method.
Additionally, it also glycosylated 1-pentanol with one carbon longer
chain than the reported butanol. When continuously extending the
chains to hexyl, octyl, and dodecyl alcohols, the enzyme showed
no transferase activity. This might be related to the increasing
hydrophobicity of those acceptors in one aspect, which made them
unavailable to the hydrophilic enzyme.
In conclusion, the levansucrase derived from B. licheniformis 8-
37-0-1 seemed to be a powerful catalyst for fructosylation and
other synthetic applications. It showed excellent polymerization
activity toward sucrose and displayed a wide range of acceptor
specificity for oligomerization. The enzyme may be useful not only
for the efficient synthesis of levan which shows significant biolog-
ical, pharmaceutical, hygienical or industrial values, but also for
the expansion of the repertoire of fructose-containing chemicals
that could either possess inherent values by themselves or act as
intermediates for further modification as valuable products.
Development Program of China (No. 2012AA021504), National
Natural Science Foundation of China (No. 31070064), and Inde-
pendent Innovation Foundation of Shandong University (No.
2012TS014).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.procbio.2014.05.012.
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