RESEARCH PAPER New Biotechnology  Volume 27, Number 4  September 2010

Expression and activity analysis of

Research Paper

sucrose:sucrose 1-fructosyltransferase
from onion
Yawei Han1, Liping Chen1, Duobin Mao1, Lijun Tang2 and Lihong Guan1
1
Zhengzhou University of Light Industry, Henan Province, 450002, China
2
Shangqiu Medical College, Henan Province, 476100, China

This study was designed to express the onion fructosyltransferase by Escherichia coli DH5a, and obtain
the optimal conditions of FST-1 activity. Thereby, fructosyltransferase gene was obtained by RT-PCR
from onion in this experiment, and named FST-1. The expressed proteins were analyzed by SDS-PAGE.
FST-1 activity was identified by the high performance liquid chromatography (HPLC). The optimal
conditions of FST-1 were analyzed by the dinonylnaphthalene sulfonic acid (DNS) and orthogonal test.
Results revealed that FST-1 was identified to 98% similarity with fructosyltransferase mRNA of onion
(accession number: AJ006066). FST-1 was successfully expressed in E. coli DH5a. HPLC results indicated
that the expressed protein from FST-1 had a good transferring activity for fructose. The optimal
conditions of FST-1 in catalyzing reaction were the pH 5.0, 45 8C and 60% sucrose substrate. The results in
this experiment would lay the foundation for the large-scale of kestose by bio-catalysis method.

Introduction
Fructosyltransferase is a group of enzymes (E.C. 2.4.1.9) mostly
described as acting on sucrose to transfer the resulting fructose to
sucrose. This reaction yields oligosaccharide, that is fructose oli-
gomers mainly composed of 1-kestose, nystose, and 1-fructosyl-
nystose [1]. Oligosaccharide can be used as the alternative
sweetener, it has been commercialized since the beginning in
the 1980s [2]. For example, oligosaccharide gained much interest
as building blocks in pharmaceuticals and functional food. Some
of the desirable characteristics of fructooligosaccharides were low
caloric, safe sugars for diabetics and non-cariogenic [3]. Thereby,
oligosaccharides are important not only because of their sweet-
ness, but also because of their functional properties. From the
point of view of oligosaccharide application in pharmaceuticals
and functional food, the importance of fructosyltransferase should
be realized.
Currently, fructosyltransferase had been isolated and character-
ized from a variety of prokaryotic (Bacillus subtilis, Aspergillus niger,
Scopulariopsis brevicaul, Aerobacter levanicum, Streptococcus mutans)
and eukaryotic organisms (wheatgrass, onion, asparagus, beet,
Corresponding author: Mao, D. (opsar@163.com)
barley) in the previous reports [411]. In 1995, the first gene of
fructan biosynthesis from plant, 6-fructosyltransferase (6-SFT), was
cloned from barley. Since then, several genes encoding enzymes
associated with fructan biosynthesis had been cloned from various
plants: a fructan: fructan 1-fructosyltransferase (1-FFT) from Cichor-
ium intybus [12], Cynara scolymus [13,14], Helianthus tuberosus [15]; 1-
fructosyltransferase (1-SST) and 6G-fructosyltransferase (6G-FFT)
from Allium cepa [16,17]; and 1-SST and/or 6-SFT from the temperate
grasses such as Festuca arundinacea [18], Agropyron cristatum [19], Poa
secunda [20], Triticum aestivum [21], Lolium perenne [22,23] and H.
vulgare [24]. In some studies, the transgenic plants carrying genes of
fructan biosynthesis have shown the increased levels of tolerance to
environmental stresses such as cold, drought and freezing [2528].
Above data indicated that fructosyltransferase were investigated in
detail. Although fructosyltransferase had been obtained from many
species, their industrialized headstream was limited in microorgan-
ism. The vegetal fructosyltransferase was mostly investigated in the
transgenic plant and enzymatic mechanism because of the season,
region and origin of plant, and complexity for extraction of enzyme
from vegetal tissue.
In this paper, fructosyltransferase genes from GenBank were
analyzed. The total length sequence from onion was cloned and

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New Biotechnology  Volume 27, Number 4  September 2010
expressed in Escherichia coli DH5a. The expressed FST-1 had the
biological activity by analysis of high performance liquid chro-
matography (HPLC) and dinonylnaphthalene sulfonic acid (DNS)
methods.
Material and methods
Materials
Onions in this study were from the market of Zhengzhou city. E.
coli DH5a (Promega, USA) was used as the host strain for cloning
and expression of fructosyltransferase. The RevertAidTM First
Strand cDNA synthesis Kit, diethyl pyrocarbonate (DEPC) and
IPTG was from MBI fermentas. The expression vector of pPROEX
Htb was from Addgene Inc. HPLC (Agilent 1200) was from Agilent
Technologies Inc., USA.
Total RNA extraction of onion and RT-PCR
Total RNA extraction of onion and RT-PCR was performed by the
method described [29]. RT-PCR primers were designed according
to the fructosyltransferases sequences in GenBank. The sites of Pst I
and Hind III restriction endonuclease were added to the 50 and 30
ends of primers referred to the clone site of the expression vector,
respectively. 50 -GCCTGCAGATGGAATCC AGAGATATCGAGT-30 ;
50 -GCAAGCTTTTTCAAGGAAGCTGGAAATCCGGGG-30 . The
underlined bases in primers were the sites of restriction enzyme.
Construction of expression vector and protein expression
The PCR product and pPROEX Htb vector were digested doubly by
Pst I and Hind III at 37 8C according to the protocol. The digested
products were ligased by T4 ligase at 16 8C for over night. The
recombined pPROEX Htb was transformed into E. coli DH5a. The
positive clones were identified by PCR and the double digest of Pst I
and Hind III. Gene was expressed referring to the reported method
[30]. Cells were disrupted by sonication. All components of cell
disruption were harvested for identifying the fructosyltransferase
expression by SDS-PAGE.
Activity analysis of FST-1
Preparation of standard sample: Ten milligrams of fructose, glu-
cose, sucrose and 1-kestose were dissolved into 10 mL deionized
water, respectively. The concentration of every sugar was 1.0 mg/
mL.
Catalyzing reaction of FST-1: E. coli DH5a from the induced and
non-induced bacteria containing FST-1 gene was disrupted by
sonication. The supernatant were used as the crude enzyme for
catalyzing the sucrose reaction. 2 mL crude enzymes and 6 g
sucrose was added into 2 mL phosphate and citric acid buffer
(pH 6.2), respectively. The catalyzing reactions were performed
at 42 8C for 24 h. At last, the enzyme was inactivated at 100 8C for
10 min. The reaction mixture was diluted to 3000 times, and then,
5 mL dilution mixture and standard sample were analyzed by
HPLC.
HPLC analysis: HPLC column was D-sugar (Prevail carbohydrate
ES 5 mm, 250 mm  4.6 mm) column (Grace Davison Discovery
Sciences (Deerfield, IL, USA)). The analysis conditions for sample:
75% acetonitrile of mobile phase (acetonitrilewater), 1.0 mL/min
velocity of flow. The detector was the evaporative light detector
(Agilent Technologies Inc., USA). The quantity of analysis sample
was 3 mL.
RESEARCH PAPER
Analysis of the optimal condition for FST-1
The preparation of standard curve for glucose was referred the
protocol of Kit (Megazyme International Ireland Limited), and
DNS reagents were prepared according to the described [31].
Activity analysis of FST-1 at the different factors: Firstly, sucrose
was incubated with the crude FST-1 at the different reaction times
(0, 5, 10, 15, 20, 23, 25, 30, 35, 40, 45, 50, 55 and 70 h), 60%
sucrose, 42 8C and pH 5.0. Secondly, sucrose was catalyzed by the
crude FST-1 at different temperatures (32, 35, 37, 40, 42, 45, 50, 55
and 60 8C), 60% sucrose and pH 5.0 for 1 h. Third, the same
reaction was incubated at different pH values (pH 3.0, 3.5, 4.0,
Research Paper
4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 8.0), 60% sucrose and 42 8C for 1 h. At
last, the same reaction was performed at different substrate con-
centrations (15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60% and 65%), pH 5.0 and 42 8C for 1 h. The crude FST-1 was
inactivated in reaction end at 100 8C for 10 min. For analyzing the
effect of sucrose hydrolysis in the catalyzing reaction, 60% sucrose
solution and phosphate buffer solution (PBS) were used as the
control in measuring the OD value of the catalyzing reaction, the
calibration curves of every factor were constructed according to its
OD values, respectively.
Orthogonal test of three factors for finding the optimal
conditions
On the basis of experiments for single factor, the orthogonal test of
three factors was performed for finding the optimal conditions of
FST-1. The factor-levels were temperature (42, 45 and 50 8C), pH
(5.0, 5.5 and 6.0) and substrate concentration (50%, 60% and 65%)
in this experiment. The scheme of orthogonal test was designed by
the Orthogonality Experiment Assistant II [32], and the detailed
experimental conditions given as supplementary data.
Results
RT-PCR results and analysis of FST-1 sequence
RT-PCR results were identified by the electrophoresis of agarose
gel. About 2000 bp band was obtained and recombined with the
pPROEX Htb vector by T4 ligase. The recombined vectors were
transformed into E. coli DH5a. The positive clones were sequenced.
The sequence (GenBank accession no: GQ214178) was given as
supplementary data, and named as FST-1. 1872 bp FST-1 sequence
included 10 mutation sites. The total length was identified to 99%
homolog with AJ006066 sequences [15]. From these results, fruc-
tosyltransferase gene was obtained successfully in this experiment.
Expression of FST-1
The expression proteins were analyzed by SDS-PAGE (Figure 1). FST-
1 gene should encode 624 amino acids according to its mRNA
length. The molecular weight of FST-1 was about 70 kDa. The similar
results were shown by SDS-PAGE. The lane 3 in Figure 1 was the
results of total proteins from the induced expression. The special
band about 70 kDa was shown clearly. These results were not
identified in the bacteria containing the empty vector and the
non-induced bacteria (lane 1 and lane 2 in Figure 1). The SDS-PAGE
results indicated that FST-1 was successfully expressed in E. coli.
Activity analysis of FST-1
The catalyzing reactions of FST-1 were performed according to the
methods described above in Section Activity analysis of FST-1.
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FIGURE 1
M: protein marker; 1: the total proteins of bacteria containing the empty
vector; 2: the total proteins of the non-induced bacteria containing FST-1
gene; and 3: the total proteins of the induced bacteria containing FST-1 gene.
HPLC results of FST-1 compared with the standard sample were
analyzed in Figure 2.
HPLC results showed that FST-1 not only had the transferase
activity, but also hydrolase activity, because the glucose and
kestose were produced in catalyzing reaction. In contrast to HPLC
results of standard samples and non-induced bacteria, the peak of
glucose and fructose suggested that FST-1 could discompose
sucrose, and then, the peak of kestose in Figure 2C implied that
FIGURE 2
A: HPLC results of standard samples in this experiment; B: HPLC results of the supernatant from the non-induced bacteria catalyzing reaction for the sucrose; and
C: HPLC results of the crude enzyme from the induced bacteria catalyzing reaction for the sucrose.
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New Biotechnology  Volume 27, Number 4  September 2010
FST-1 could transfer fructose. These results (Figure 2B and C)
reliably confirmed that FST-1 gene was successfully expressed,
and the expressed FST-1 had the biological activity.
Optimal conditions for FST-1 activity
The standard curve of glucose is given as supplementary data.
Figure 3A reveals that FST-1 activity was affected strongly in the
temporal factor. The concentration of reducing sugar in reaction
should be upward all through, and then receive a flat from theory
conjecture. However, the undulation of curves was acute. The
results of temporal factor indicate that the concentration of redu-
cing sugar increased in 15 h, and reached the vertex in 55 h.
However, the slope ratio suggests that FST-1 activity was best in
25 h.
The effect of temperature was shown in Figure 3B. From the
concentration of the reducing sugar in different temperatures,
42 8C was the optimal temperature for FST-1 catalyzing sucrose.
FST-1 activity was low at under or above 42 8C. However, the
undulation of activity was smooth relatively.
Figure 3C results indicated that pH 5.5 was the optimal pH value
for FST-1 in the catalyzing reaction. The undulation of the redu-
cing sugar was acute in pH 3.08.0, which indicated that the pH
could acutely affect the hydrolysis of sucrose.
The curve of the reducing sugar was upward along with the
increase of the sucrose concentration as a whole, and reached the
culmination at 60% sucrose concentration. When sucrose con-
centration is above 65%, the reducing sugar decreased. The results
were shown in Figure 3D.
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Research Paper
FIGURE 3
A: Effect of reaction time on FST-1 activity. 1: The results of the PBS solution of 70% sucrose, 2: the results of FST-1. B: Effect for temperature on FST-1 activity.
C: Effect of pH on FST-1 activity. D: Effect of substrate concentration on FST-1 activity.

From analysis of the single factor, the optimal conditions of FST-
1 should be 42 8C, pH 5.5, 60% sucrose concentration and 55 h
reaction times.
Orthogonal test of three factors
After all, above results for the optimal condition were from the
analysis of single factor. The optimal conditions of enzyme ought
to be affected by multi-factors in reaction. Therefore, the optimal
conditions of FST-1 should be obtained from the orthogonal test of
three factors. The results were shown in Table 1.
From the results of range analysis, pH value for effect of FST-1
activity was strong, the sucrose effect was weak. Although three
factors could not receive the remarkable level, Figure 4 results
clearly displayed the effect of three factors for FST-1 activity. The
optimal conditions of FST-1 in reaction should be the pH 5.0, 45 8C
and 60% sucrose substrate.
Discussion
The purpose of this study was to express the activated fructosyl-
transferase from onion genome and find the optimal conditions of

TABLE 1
Analyzing results of the orthogonal test for the three factors
Experimental number Substrate concentration (%) Temperature (8C) pH Results
NO. 1 1 1 1 1 1.613
NO. 2 1 2 2 2 1.600
NO. 3 1 3 3 3 0.340
NO. 4 2 1 2 3 0.821
NO. 5 2 2 3 1 1.124
NO. 6 2 3 1 2 1.589
NO. 7 3 1 3 2 1.310
NO. 8 3 2 1 3 1.645
NO. 9 3 3 2 1 0.491
K1j 1.184 1.248 1.616 1.076
K2j 1.178 1.456 0.971 1.500
K3j 1.149 0.807 0.925 0.935
Range 0.035 0.649 0.691 0.565
Values in table were the mean of three results.

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FIGURE 4
The results of range for substrate, temperature and pH from the orthogonal
test of three factors. 55%, 60% and 65% were the substrate concentrations;
42, 45 and 50 8C were the reaction temperatures; 5.0, 5.5 and 6.0 were pH
values.
FST-1 activity for the industrialized application. Therefore, accord-
ing to the experimental design, the overall work can be divided
into three parts in this paper. The following discussions were for
the three parts.
Clone and expression of FST-1
The sequence and expression of gene indicated that fructosyl-
transferase gene was successfully obtained in this study. The full
length of FST-1 was 1872 bp including 10 mutation sites, which
caused the mutation of 6 amino acids. The sequence analysis of
FST-1 had exhibited that the bases of mutation were the transi-
tion of G and A. The clone and expression of fructosyltransfer-
ase gene was reported in many species, the activity of the
expressed fructosyltransferase was also identified in vitro
[2,12,15,1719,33]. Therefore, expression and identification of
the activated FST-1 was an important step in this experiment.
From SDS-PAGE result, the expressed FST-1 was exhibited
clearly. Especially, 70 kDa protein was shown in the induced
bacteria. The bacteria containing empty vector and the non-
induced bacteria containing FST-1 gene did not exhibit almost
70 kDa protein. These results indicated that FST-1 was success-
fully expressed in E. coli.
Analysis of FST-1 activity
For analyzing the FST-1 activity, fructose, glucose and kestose were
used as the standard samples. According to HPLC results, Figure 2B
had only appeared the sucrose peak, which revealed that FST-1
could not be expressed in no-IPTG treatment. However, the emer-
gence of fructose, glucose and kestose peak in Figure 2C indicated
that the expressed FST-1 could decompose the sucrose, and trans-
fer the fructose, because many experiments proved that sucrose
could be hydrolyzed by fructosyltransferase, and decomposed into
the fructose and glucose. Moreover, fructose could be transferred
to sucrose for producing the kestose [34]. HPLC results suggested
that the FST-1 from onion was successfully expressed in E. coli, the
expressed FST-1 owned the activity of hydrolyzing sucrose and
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New Biotechnology  Volume 27, Number 4  September 2010
transferring fructose. If the FST-1 was purified in this experiment,
HPLC results should be better. The reason for no purification was
serving to the industrial preparation of FST-1.
Pay particular attention, the cell break should be performed in
low temperature conditions, and the interval of sonication was
appropriately prolonged for avoiding the FST-1 inactivation,
because a large amount of energy from the sonication would make
enzyme inactive.
Optimal conditions of FST-1 catalyzing reaction
Any reaction should be an optimal condition. Especially, the
catalyzing reaction of enzyme should require the substrate speci-
ficity, optimal pH, and temperature [33]. Fructosyltransferase is a
kind of glycosidase hydrolyzing carbohydrate, thereby, the opti-
mal conditions of reaction was required also for FST-1. Many
factors could also affect the catalyzing reaction of enzyme, such
as pH, temperature, reaction time, and substrate concentration. In
this study, the reaction conditions of FST-1 were investigated. The
results of single factor revealed that FST-1 activity should be best at
42 8C, pH 5.5 and 60% sucrose concentration and 55 h reaction
times. Whereas, the orthogonal test of three factors indicated that
the pH 5.0, 45 8C and 60% sucrose substrate were the optimal
conditions of FST-1 activity. Although the results of the orthogo-
nal test were different appreciably than those of a single factor, the
results form analysis of the composite factor should be more
representative. Therefore, the optimal conditions of FST-1 activity
should be 45 8C, pH 5.0 and 60% sucrose concentration and 55 h
reaction times.
It was particularly worth noting that results of single factors
were inconsistent with theoretical predictions, such as the tem-
perature activity curve had two peaks at 42 8C and 55 8C, pH
activity curve did not show the representative bell shape. The
reasons might be attributable to the method flaw, because the
DNS method would measure the total amount of reducing sugar.
If this method had been used with glucose oxidase, the results
would be best. Another, the result from the orthogonal test of
three factors was a supplement to DNS method, therefore, DNS
method was only used for the convenient analysis of single
factor.
In conclusion, fructosyltransferase gene was obtained and
expressed successfully from onion in this study. FST-1 activity
was analyzed by HPLC and DNS method, which indicated that FST-
1 had the activity of hydrolase and fructosyltransferase. The
optimal conditions of FST-1 were 45 8C, pH 5.0 and 60% sucrose
concentration and 55 h reaction times. Although the fructosyl-
transferase gene from onion was cloned and expressed in the
reported literature, a comprehensive analysis of fructosyltransfer-
ase activity was investigated for the first time.
Acknowledgements
We are grateful to Huafu Zheng and Xue Wang for helping with
the experiments. This work was supported by National Foundation
of Natural Sciences (No: 20676127) and Key Foundation of
Zhengzhou University of Light Industry (No: xyyjj02).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.nbt.2010.02.004.
New Biotechnology  Volume 27, Number 4  September 2010
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