J. Microbiol. Biotechnol.

(2009), 19(2), 000–000

doi: 10.4014/jmb.0901.0045
First published online 12 June 2009

Production of Lactosucrose from Sucrose and Lactose by a Levansucrase

from Zymomonas mobilis
Han, Woo-Cheul1, Sun-Ho Byun1, Mi-Hyun Kim1, Eun Hwa Sohn2, Jung Dae Lim2, Byung Hun Um3,
Chul Ho Kim4, Soon Ah Kang 5, and Ki-Hyo Jang1*

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1
Department of Food and Nutrition, Kangwon National University, Gangwon 245-711, Korea
2
Department of Herbal Medicine Resource, Kangwon National University, Gangwon 245-711, Korea
3
Korean Institute of Science and Technology Gangneung Institute, Gangwon 210-340, Korea
4
Biotechnology Research Division, Jeonbuk Branch Institute Molecular Bioprocess Research Center, Korea Research Institute of
Bioscience and Biotechnology, Daejeon, Korea
5
Department of Fermented Food Science, Seoul University of Venture and Information, Seoul 137-070, Korea
Received: January 22, 2009 / Revised: April 14, 2009 / Accepted: April 19, 2009
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Lactosucrose (4G-β-D-galactosylsucrose) is an oligosaccharide fructose. Lactosucrose can be obtained via a transfructosylation
consisting of galactose, glucose, and fructose. In this study, reaction catalyzed by either a levansucrase (E.C. 2.4.1.10)
we prepared lactosucrose from lactose and sucrose using a or a β-fructofuranosidase (E.C. 3.2.1.26), with lactose and
levansucrase derived from Zymomonas mobilis. Optimum sucrose serving as substrates [2, 6, 15]. Lactosucrose is
conditions for lactosucrose formation were 23oC, pH 7.0, indigestible in the human digestive tract and is therefore
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18.0% (w/v) lactose monohydrate, and 18% (w/v) sucrose low in calories and suitable for use in low-calorie foods. It
as substrates, and 1 unit of enzyme/ml of reaction mixture. is a putative growth stimulator of intestinal bifidobacteria
Under these conditions, the lactosucrose conversion efficiency [1] and therefore could be classified as a prebiotic. Until
was 28.5%. The product was purified and confirmed to be now, most trials in process development for the production
O-β-D-galactopyranosyl-(1 → 4)-O-β-D-glucopyranosyl- of lactosucrose have concentrated on using whole-cell
(1→2)-β-D-fructofuranoside, or lactosucrose. A mixed-enzyme systems [14-16]. However, microbial levansucrases are
system containing a levansucrase and a glucose oxidase usually produced extracellularly or in the outer membrane-
was applied in order to increase the efficiency of lactose bound form [17]. With the exception of a few classes of
and sucrose conversion to lactosucrose, which rose to 43.2% proteins, such as toxins [3] and hemolysins [8], E. coli
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as a result. normally does not secrete foreign proteins extracellularly;

Keywords: Glucose oxidase, lactosucrose, levansucrase, therefore, Yun et al. [23] have suggested that whole-cell
Zymomonas mobilis immobilization of recombinant E. coli containing an
endoinulinase gene is suitable for the production of inulo-
oligosaccharides from inulin. Previously, we isolated the
levansucrase gene from Z. mobilis and successfully expressed
A number of health benefits have been claimed for prebiotics, it in E. coli [9, 19]. In the present work, the optimal
and many products containing them are available worldwide reaction conditions for batchwise production of lactosucrose
[11, 18]. Gibson and Roberfroid [7] defined a prebiotic as using E. coli lysate are described.
a food ingredient that is nondigestible and nonabsorbable
in the upper gastrointestinal tract and that supports the
growth and/or activity of one or more particular bacteria in MATERIALS AND METHODS
the colon, thereby improving the health of the host.
Lactosucrose (4G-β-D-lactosylfructoside, galactosylsucrose) Materials
is a trisaccharide consisting of glucose, galactose, and Fructose, glucose, and sucrose were purchased from Sigma (St.
Louis, MO, U.S.A.) and α-lactose monohydrate was obtained from
Samchun Chemical Inc. (Seoul, Korea). Gluzyme, a mixture of
*Corresponding author
Phone: +82-33-570-6882; Fax: +82-33-570-6889; glucose oxidase and catalase, was obtained from Novo Nordisk
E-mail: [email protected] (Bagsvaerd, Denmark).
2 Han et al.

Preparation of Levansucrase Reactions containing 18.0% (w/v) sucrose and 18.0% (w/v) lactose
Plasmid pELCHis24 carrying the levansucrase gene levU of Z. monohydrate were carried out at pH 7.0 and 23oC as described above.
mobilis, which is expressed when induced by the T7 promoter in E. For the time course, the enzymatic reaction was carried out in a
coli BL21 (DE3, F - ompT r-Bm-B) [20], was used for levansucrase 20-ml mixture containing 18.0% (w/v) sucrose and 18.0% (w/v)
production [19]. E. coli harboring pELCHis24 was grown aerobically lactose monohydrate in 50 mM sodium acetate buffer (pH 7.0) and
at 37oC for 12 h. Cells were harvested and then disrupted by 80 µl (20 U) of enzyme solution at 23oC.
ultrasonication. Cell debris was removed by centrifugation at 10,000 ×g
for 10 min at 4oC. The supernatant was used as the source of levansucrase Mixed Enzymatic Reactor System
without further purification. A Marubishi (Tokyo, Japan) MJ-N14L jar fermenter (7.5 l) was employed
as a mixed enzymatic reactor to produce lactosucrose. The reaction
Measurement of Enzyme Activity was carried out in a 2-l mixture containing 18% sucrose and 18%

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Levansucrase activity was determined by either sucrose hydrolysis lactose monohydrate in 10 mM sodium acetate buffer (pH 6.0), 10 ml
or lactosucrose formation activity. One unit (U) of levansucrase (2,500 U) of levansucrase, and 1.2 g (12,000 GOD U) of gluzyme.
activity was defined as the amount of enzyme releasing one mmole of The reaction temperature was maintained at 30oC and the pH was
glucose per minute at pH 5.0 and 37oC. One unit of glucose oxidase kept at 6.0 through the automatic addition of a CaCO3 slurry. Oxygen
(GOD) activity was defined as the amount of enzyme consuming was continuously supplied at a velocity of 10 l/min and the agitation
one mmole of oxygen per minute at pH 5.1 and 35oC. To calculate speed was 500 rpm.
the conversion efficiency (%), the concentration of lactosucrose was The reproducibility of analyses was verified as follows: five
divided by the sum of the initial concentrations of lactose monohydrate injections were carried out using various concentrations of fructose,
and sucrose, and then expressed as a percentage value.
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Analysis of Products
Glucose, fructose, sucrose, lactose, and lactosucrose were quantitatively
glucose, sucrose, lactose, and lactosucrose; there was a linear relationship
between the peak area detected corresponding to each carbohydrate
and the amount of carbohydrate applied, with a less than 3% variation
in detected amounts between injections. For analysis of carbohydrate
determined by HPLC (Agilent Technologies Inc., Santa Clara, CA,
composition following the mixed enzyme reaction, independent
U.S.A.) with a refractive index and a Kromasil 100-10NH2 column
experiments were performed. Results reported for each type of
(Eka Chemicals AB, Bohus, Sweden) at 50oC. The mobile phase
experiment were consistent between replicates within any one
consisted of 75% acetonitrile:25% water and was used at a flow rate
experiment but some quantitative variation in carbohydrate composition
of 1.0 ml/min. The transfructosylation reaction was performed and
occurred between separate runs. The trends seen for the relative
the product was fractionated by HPLC. The purified sugar was
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proportions of carbohydrates were identical in every analysis; therefore,

concentrated by rotary evaporation and subjected to NMR measurements.
1 representative figures are shown in the results and discussion rather
H NMR and 13C NMR spectra were obtained in pyridine-d5 on a
than an average of percentage composition among experiments, unless
Varian 500 MHz NMR system (Varian, Palo Alto, CA, U.S.A.) equipped
otherwise stated.
with a cold probe, with standard pulse sequences operating at 500
and 125 MHz.

Optimization of Reaction Conditions for Lactosucrose Production RESULTS AND DISCUSSION

The effect of temperature on lactosucrose production was determined
by varying the temperature from 5 to 50oC. The enzymatic reaction Effect of Temperature on Lactosucrose Formation Activity
was carried out in a 2-ml mixture containing 18.0% (w/v) sucrose The optimal temperature for lactosucrose formation was
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and 18.0% (w/v) lactose monohydrate in 50 mM sodium acetate 23oC (Fig. 1), which is low compared with those of other
buffer (pH 6.0) and 8 µl (2 U) of enzyme solution for 30 min and
bacterial levansucrases. For example, this temperature is
6 h. The reaction was terminated by heating the tubes at 95oC for
5 min and the filtrate was analyzed by HPLC.
55oC in the case of levansucrases from Bacillus subtilis,
To determine the optimal pH for lactosucrose production, the reaction Geobacillus stearothermophilus, and Paenibacillus polymyxa
was carried out at varying pH (3.0 to 7.0) in a 2-ml reaction mixture [16] and is 40oC for levansucrases from Pseudomonas
containing 18.0% (w/v) sucrose and 18.0% (w/v) lactose monohydrate syringae and Rahnella aquatili [16]. For further experiments,
at 23oC under the conditions described above. a temperature of 23oC was selected for lactosucrose production
To determine the optimal substrate concentration, lactose monohydrate because levansucrase from Z. mobilis is less thermostable
and sucrose were used at the same concentration (0.9%, 1.8%, 9.0%, than other levansucrases [9, 10].
13.5%, and 18.0% [w/v]) [14]. The enzymatic reaction was carried
out at pH 7.0 and 23oC under the conditions described above. Effect of pH on Lactosucrose Formation Activity
To determine the optimal ratio of lactose monohydrate to sucrose,
Differences in pH gave rise to marked differences in
the enzymatic reaction was performed with varying concentrations
of sucrose (4.5%, 9.0%, 18.0%, and 27.0% [w/v]) whereas the
lactosucrose production. The optimum pH for enzyme
concentration of lactose monohydrate was maintained at 18.0% (w/v). activity was found to be 7.0 (Fig. 2). In contrast, the optimum
Enzymatic reactions were carried out at pH 7.0 and 23oC as described pH for lactosucrose production by levansucrases from B.
above. subtilis, G. stearothermophilus, P. polymyxa, P. syringae,
The effect of enzyme concentration on lactosucrose production R. aquatilis, Sterigmatomyces elviae, and Microbacterium
was determined by varying the concentration from 0.02 to 4 U. laevaniformans is 6.0 [13, 14, 16]. A pH of 7.0 was selected
 PRODUCTION OF LACTOSUCROSE BY ZYMOMONAS MOBILIS 3

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Fig. 1. Effect of temperature on lactosucrose production.
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The enzymatic reaction was carried out with 18% (w/v) sucrose and 18% (w/v) lactose monohydrate in 50 mM sodium acetate buffer (pH 6.0) and 2 U
enzyme for 30 min and 6 h. Symbols: ( △ ), sucrose; ( ▽ ), lactose; ( ◆ ), lactosucrose; ( ■ ), glucose; ( ● ), fructose.

for further experiments since levansucrase is highly unstable of lactose monohydrate was fairly low at 18.0% (w/v) at
at alkaline pH [10]. room temperature. It is interesting to note that in the presence
of a high concentration of sucrose as the sole substrate in
Effect of Substrate Concentration on Lactosucrose the reaction mixture, synthesis of low-molecular-weight
Formation Activity fructooligosaccharide is the dominant reaction catalyzed
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To investigate the effect of substrate concentration, enzyme by the levansucrase rather than synthesis of high-molecular-
reactions were performed using equal concentrations of weight levan [5].
lactose monohydrate and sucrose. Maximum lactosucrose
was produced at the highest concentration (18.0% w/v) Effect of the Ratio of Sucrose to Lactose Monohydrate
(Fig. 3). This concentration was chosen for further experiments on Lactosucrose Formation Activity
rather than designing experiments to cover the full range In order to improve the efficiency of lactosucrose production,
of possible substrate concentrations since the solubility the concentration of sucrose was varied (4.5%, 9.0%, 18.0%,
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Fig. 2. Effect of pH on lactosucrose production.

The enzymatic reaction was carried out at varying pH (3.0-7.0) with 18% (w/v) sucrose and 18% (w/v) lactose monohydrate in 50 mM sodium acetate
buffer and 2 U enzyme at 23oC for 30 min and 6 h. Symbols: ( △ ), sucrose; ( ▽ ), lactose; ( ◆ ), lactosucrose; ( ■ ), glucose; ( ● ), fructose.
4 Han et al.

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Fig. 3. Effect of substrate concentration on lactosucrose production.
The enzymatic reaction was performed with various substrate concentrations in 50 mM sodium acetate buffer (pH 7.0) and 2 U enzyme at 23oC for 30 min
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and 6 h. Equal concentrations of the two substrates were used: 0.9%, 1.8%, 9.0%, 13.5%, 18% (w/v) each of lactose monohydrate and sucrose. Symbols:
( △ ), sucrose; ( ▽ ), lactose; ( ◆ ), lactosucrose; ( ■ ), glucose; ( ● ), fructose.

and 27.0% [w/v]) while the concentration of lactose production of lactosucrose was increased. However, a further
monohydrate was kept at 18.0% (w/v). At the lower ratios increase in enzyme concentration up to 4 units resulted in a
of sucrose to lactose monohydrate, a lower conversion slight decrease in the efficiency of conversion to lactosucrose
efficiency was observed (Fig. 4). The conversion efficiency from the two substrates (Fig. 5). This result is probably due
increased as the ratio of sucrose to lactose monohydrate to the lactosucrose hydrolysis activity of the levansucrase.
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increased. The conversion efficiency was 26.4% when the

ratio of sucrose to lactose monohydrate was 1:1. Time Course of Lactosucrose Production
Levansucrase transfers the fructosyl moiety of sucrose to
Effect of Enzyme Concentration on Lactosucrose lactose and then lactosucrose is formed [12]. However,
Formation Activity levansucrase catalyzes not only the fructose transfer reaction
As the enzyme concentration was increased up to 2 units but also the hydrolysis of lactosucrose to fructose and
(5.4 units enzyme per gram of lactose monohydrate), the lactose [6, 12]. Therefore, the hydrolysis reaction should
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Fig. 4. Effect of the concentration ratio of the two substrates on lactosucrose production.
The enzymatic reaction was performed with a varying ratio of the two substrates in 50 mM sodium acetate buffer (pH 7.0) and 2 U enzyme at 23oC for
30 min and 6 h. Substrate ratios: 18.0%:4.5%, 18.0%:9.0%, 18.0%:18.0%, 18.0%:27.0% (w/v) lactose monohydrate:sucrose. Symbols: ( △ ), sucrose; ( ▽ ),
lactose; ( ◆ ), lactosucrose; ( ■ ), glucose; ( ● ), fructose.
 PRODUCTION OF LACTOSUCROSE BY ZYMOMONAS MOBILIS 5

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Fig. 5. Effect of enzyme concentration on lactosucrose production.
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The enzymatic reaction was performed with 18% (w/v) sucrose and 18% (w/v) lactose monohydrate in 50 mM sodium acetate buffer (pH 7.0) with varying
concentrations of enzyme. Enzyme concentrations: 0.02 units, 0.04 units, 0.2 units, 0.4 units, 2 units, 4 units. Symbols: ( △ ), sucrose; ( ▽ ), lactose; ( ◆ ),
lactosucrose; ( ■ ), glucose; ( ● ), fructose.

be minimized by monitoring the enzymatic reaction time. can be released from lactosucrose by the lactosucrose
Fig. 6 shows the time course of enzyme activity over a hydrolysis activity of the enzyme.
period of 24 h. The production of lactosucrose increased
with increasing cultivation time and consequently reached Measurements of NMR Specta
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a conversion efficiency of 28.5% after 2 h but began to The structure of the sugar was elucidated through analysis
decrease thereafter. HPLC analysis revealed that a prolonged of 1D and 2D NMR data primarily from 1H NMR, 13C
enzymatic reaction of more than 2 h increased the production NMR, 1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC, and
of glucose and lactose, whereas the amount of sucrose and HSQC experiments. The 13C NMR spectrum showed 18
lactosucrose were reduced, a result that has been described carbons including three anomeric sugar carbons (105.80,
by others [6, 12]. Glucose can be produced from sucrose 92.81, 105.80 ppm) and four methylene oxylated carbons
by the sucrase activity of levansucrase, whereas lactose (62.70, 61.39, 61.76, 64.39 ppm), indicating the presence
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Fig. 6. Time course of lactosucrose production.

The enzymatic reaction was performed in a 20-ml reaction mixture containing 18% (w/v) sucrose and 18% (w/v) lactose monohydrate in 50 mM sodium
acetate buffer (pH 7.0) and 20 U enzyme at 23oC. Symbols: ( △ ), sucrose; ( ▽ ), lactose; ( ◆ ), lactosucrose; ( ■ ), glucose; ( ● ), fructose.
6 Han et al.

Table 1. Chemical shifts in H and C NMR spectra of lactosucrose

1 13

produced from lactose and sucrose by Z. mobilis levansucrase.

13 a 1 a
Group Carbon atoms C NMR H NMR
Galactose 1 105.80 5.07
2 72.21 4.46
3 72.68 4.12
4 69.73 4.09
5 76.99 4.11
6 62.70 4.29/4.26
Glucose 1′ 92.81 6.10
2′ 74.92 4.13

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3′ 72.38 4.71
4′ 82.02 4.28
5′ 72.88 4.66
6′ 61.39 4.48/4.44
Fig. 7. Structure of lactosucrose. Fructose 1″ 61.76 4.43/4.36
2″ 105.80 -
3″ 79.57 5.02
of three hexose sugars in the structure. The 1H-1H COSY 4″ 75.40 4.97
and 1H-13C HSQC spectra confirmed that the 3-sugar 5″ 84.50 4.51
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moiety consisted of galacopyranose, glucopyranose, and
fructopyranose. The 1H-13C HMBC spectrum showed a
correlation of glucose H-1' (6.10 ppm) to fructose C-2''
a
6″
NMR data acquired in Pyr-d5.
64.39 4.34/4.29

(105.80 ppm), indicating a glucose (1→2) fructose linkage. levansucrase in a batchwise process owing to the high
The HMBC correlation of galactose H-1 (5.07 ppm) to level of inhibition by released glucose, which competes
glucose C-4' (82.02 ppm) indicated a galactose (1→4) glucose with the glucose moiety of sucrose for the enzyme [17].
linkage. The results of these experiments suggest that the Yun et al. [22] reported an enzymatic method to enhance
sugar product was O-β-D-galactopyranosyl-(1→ 4)-O-α-D- fructooligosaccharide conversion efficiency by removing
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glucopyranosyl-(1→2)-β-D-fructofuranoside, or lactosucrose glucose using glucose oxidase. Using a similar strategy, we

(Fig. 7). The full assignment of 1H and 13C NMR data are applied a mixed enzyme system containing levansucrase
shown in Table 1. and glucose oxidase. As shown in Fig. 8, an increase
in lactosucrose conversion efficiency was achieved by
Production of Lactosucrose by a Mixed Enzyme System converting glucose to gluconic acid with glucose oxidase.
It has been suggested that a conversion approaching 100% The maximal conversion efficiency was 43.2%. The
of the theoretical yield would be difficult to obtain with efficiencies of lactose and sucrose conversion to lactosucrose
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Fig. 8. Time course of lactosucrose production in a batch system with Z. mobilis levansucrase and glucose oxidase.
The reaction was performed in a 7.5-l fermenter with a working volume of 2 l with 2,500 units of levansucrase and 12,000 units of gluzyme. Reaction
conditions: 500 rpm agitation, 10 l/min air flow, 30oC, pH 6.0. Symbols: ( △ ), sucrose; ( ▽ ), lactose; ( ◆ ), lactosucrose; ( ■ ), glucose; ( ● ), fructose.
 PRODUCTION OF LACTOSUCROSE BY ZYMOMONAS MOBILIS 7

Table 2. Lactosucrose yields obtained using enzymes from various microorganisms.

Enzyme source Substrate (g/l) (lactose+sucrose) Conversion efficiency (%) Reference
Arthrobacter mysorens 300+300 5.3 [14]
Klebsiella pneumoniae 300+300 3.2 [14]
Rahnella aquatilis 300+300 13.2 [14]
Sterigmatomyces elviae 250+250 38.4 [14]
Bacillus subtilis 225+225 40.7 [16]
Paenibacillus polymyxa 225+225 37.8 [4]
Zymomonas mobilis 180+180 28.5 This work
Z. mobilis (+glucose oxidase) 180+180 43.2 This work

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reported by others and from this work are summarized in whole cells of Paenibacillus polymyxa harboring levansucrase
Table 2. Lee et al. [14] achieved a 38.4% conversion activity. Biotechnol. Prog. 20: 1876-1879.
efficiency in a batch reaction with an S. elviae strain harboring 5. Crittenden, R. G. and M. J. Playne. 1996. Production, properties,
levansucrase activity. In the same paper, the authors reported and applications of food-grade oligosaccharides. Trends Food
Sci. Technol. 7: 353-361.
a conversion efficiency of 36.0% for 48 days using
6. Fujita, K., K. Hara, H. Hashimoto, and S. Kitahara. 1990.
immobilized S. elviae cells for continuous lactosucrose Transfructosylation catalyzed by β-fructofuranosidase I from
production in a packed-bed reactor. In another report, an Arthrobacter sp. K-1. Agric. Biol. Chem. 54: 2655-2661.
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efficiency of 40.7% was observed using B. subtilis levansucrase
to convert 225 g lactose/l and 225 g sucrose/l [16]. The
7. Gibson, G. R. and M. R. Roberfroid. 1995. Dietary modulation
of the human colonic microbiota: Introducing the concept of
conversion efficiency obtained in this work using a mixed- prebiotics. J. Nutr. 125: 1401-1412.
enzyme system was higher than those obtained using a 8. Goebel, W. and J. Hedgpeth. 1982. Cloning and functional
single enzyme in isolation or in a whole-cell system. characterization of the plasmid-encoded hemolysin determinant
Because we have not optimized the enzymatic conditions of Escherichia coli. J. Bacteriol. 151: 290-298.
for the mixed-enzyme system, further experimentation 9. Jang, K. H., J. W. Seo, K. B. Song, C. H. Kim, and S. K. Rhee.
is warranted. From the present data, it is evident that 1999. Extracellular secretion of levansucrase from Zymomonas
mobilis in Escherichia coli. Bioproc. Eng. 21: 453-458.
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Z. mobilis levansucrase was successfully applied to the

10. Jang, K. H., K. B. Song, C. H. Kim, B. H. Chung, S. A. Kang,
production of lactosucrose from lactose and sucrose U. H. Chun, R. W. Choue, and S. K. Rhee. 2001. Comparison
substrates. However, the results of this work suggest that of characteristics of levan produced by different preparations of
eliminating glucose during the enzymatic reaction is a levansucrase from Zymomonas mobilis. Biotechnol. Lett. 23:
necessity, for which the use of glucose oxidase may be 339-344.
applicable. 11. Jung, S. W., T. K. Kim, K. W. Lee, and Y. H. Lee. 2007.
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Acknowledgment
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12. Kawase, M., A. Pilgrim, T. Araki, and K. Hashimoto. 2001.

Lactosucrose production using a simulated moving bed reactor.
This work was supported by Grant No. RTI05-01-02 from Chem. Eng. Sci. 56: 453-458.
the Regional Technology Innovation Program of the Ministry 13. Kim, M. J., H. E. Park, H. K. Sung, T. H. Park, and J. H. Cha.
of Knowledge Economy (MKE), Korea. 2005. Action mechanism of transfructosylation catalyzed by
Microbacterium laevaniformans levansucrase. J. Microbiol.
Biotechnol. 15: 99-104.
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