Synthesis of β-D-fructofuranosyl-(2→1)-2-acetamido-2-deoxy- α-D-glucopyranoside (N-acetylsucrosamine) using β-fructofuranosidase- containing Aspergillus oryzae mycelia as a whole-cell catalyst

Article abstract of DOI:10.1016/j.carres.2012.03.037

Using soft granules consisting of Celite 535 and dried Aspergillus oryzae NBRC100959 mycelia containing β-fructofuranosidase as a whole-cell catalyst, N-acetylsucrosamine [β-d-fructofuranosyl-(2→1)-2-acetamido- 2-deoxy-α-d-glucopyranoside] was produced from sucrose and 2-acetamido-2-deoxy-d-glucose by enzymatic transfructosylation. The isolated yield of N-acetylsucrosamine from the reaction mixture was 22.1% (from sucrose). The result of N-terminal amino acid sequence analysis indicated that the enzyme involved in the synthesis of N-acetylsucrosamine is a product from gene (NCBI accession number; NW-001884675, locus tag; AOR-1-1114084) encoding putative β-fructofuranosidase on chromosome 6 of strain NBRC100959. The N-acetylsucrosamine we produced is highly soluble in water and is more stable in acidic solution than sucrose. The disaccharide was also produced using dried mycelia prepared from another A. oryzae strains.

Full text of DOI:10.1016/j.carres.2012.03.037

Carbohydrate Research 353 (2012) 27–32
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of b-D-fructofuranosyl-(2?1)-2-acetamido-2-deoxy-a-D-glucopyrano-
side (N-acetylsucrosamine) using b-fructofuranosidase-containing
Aspergillus oryzae mycelia as a whole-cell catalyst
Takako Hirano, Toru Wada, Sumire Iwai, Hitoshi Sato, Makoto Noda, Mai Juami, Masatoshi Nakamura,
⇑
Yasuko Kumaki, Wataru Hakamata, Toshiyuki Nishio
Department of Chemistry and Life Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Using soft granules consisting of Celite 535 and dried Aspergillus oryzae NBRC100959 mycelia containing
Received 24 January 2012
Received in revised form 10 March 2012
Accepted 29 March 2012
Available online 5 April 2012
b-fructofuranosidase as a whole-cell catalyst, N-acetylsucrosamine [b-
D
-fructofuranosyl-(2?1)-2-acet-
-glucose
amido-2-deoxy- -glucopyranoside] was produced from sucrose and 2-acetamido-2-deoxy-D
a
-
D
by enzymatic transfructosylation. The isolated yield of N-acetylsucrosamine from the reaction mixture
was 22.1% (from sucrose). The result of N-terminal amino acid sequence analysis indicated that the
enzyme involved in the synthesis of N-acetylsucrosamine is a product from gene (NCBI accession num-
ber; NW_001884675, locus tag; AOR_1_1114084) encoding putative b-fructofuranosidase on chromo-
some 6 of strain NBRC100959. The N-acetylsucrosamine we produced is highly soluble in water and is
more stable in acidic solution than sucrose. The disaccharide was also produced using dried mycelia pre-
pared from another A. oryzae strains.
Keywords:
N-Acetylsucrosamine
Aspergillus oryzae
Whole-cell catalyst
b-Fructofuranosidase
Transfructosylation
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
to develop a new type of GlcNAc-containing functional oligosaccha-
ride, using enzymatic reactions.
Various types of mono-and oligosaccharides that exhibit physio-
logically useful functions have been developed. These carbohydrates
are prepared using chemical or enzymatic degradation of biomass
polysaccharides or through enzymatic conversion of abundant oli-
gosaccharides. Considerable recent research has focused on 2-acet-
Our initial attempts to enzymatically produce oligosaccharide in-
volved attaching fructose to GlcNAc by exploiting the transfructosy-
lation activity of b-fructofuranosidase. Although b-fructofuran
osidase is an enzyme that catalyzes sucrose hydrolysis, certain iso-
forms of the enzyme possess potent transfructosylation activity in
solutions containing a high concentration of sucrose. Due to the en-
zyme’s high transfructosylation activity, a number of researchers
classified it as a b-fructosyltransferase (EC 2.4.1.99) in order to
emphasize the transfructosylation activity. The transfructosylation
reaction catalyzed by b-fructofuranosidase can be used to produce
several beneficial oligosaccharides. For example, fructooligosaccha-
amido-2-deoxy-D-glucose (GlcNAc), a monosaccharide obtained by
the hydrolysis of the biomass polysaccharide chitin, because evi-
dence suggests that intake of this saccharide may prevent skin aging
and ameliorate the effects of arthrosis deformans by increasing the
production of mucopolysaccharides such as hyaluronic acid and
chondroitin.1–3 Reports also suggest that the oligosaccharides that
incorporate GlcNAc may have physiologically beneficial properties.
rides such as 1-kestose [b-
anosyl-(2?1)- -glucopyranoside] and nystose [b-
furanosyl-(2?1)-b- -fructofuranosyl-(2?1)-b- -fructofuranosyl-
(2?1)- -glucopyranoside] can be produced from sucrose by
enzymes found in several Aspergillus strains.7–12 In addition, the tri-
saccharide lactosylfructoside [b- -galactopyranosy-(1?4)-
glucopyranosyl-(1?2)-b- -fructofuranoside] can be produced from
D
-fructofuranosyl-(2?1)-b-
D
-fructofur-
For example, lacto-N-biose I [b-
D
-galactopyranosyl-(1?3)-2-acet-
a-
D
D
-fructo
amido-2-deoxy- -glucopyranose], a disaccharide derived from hu-
D
D
D
man milk oligosaccharides, is a candidate bifidus factor.4 Several
reports confirmed that various disaccharides that incorporate Glc-
NAc inhibit the attachment of a range of pathogens to alveolar epi-
thelial cells.5,6 From these facts, it is expected that various types of
oligosaccharides containingGlcNAcwill showphysiologicallyuseful
functions. However, development of such type of functional oligo-
saccharide has scarcely beenperformed untilnow. Then, we planned
a-D
D
a-D-
D
sucrose and lactose by b-fructofuranosidase isolated from Arthro-
bacter.13–17 These oligosaccharides enhance bowel function by
increasing the number of beneficial enterobacteria, such as a bifido-
bacteia.18–21
Here, we report the synthesis of an oligosaccharide consisting of
fructose and GlcNAc using dried A. oryzae mycelia containing an
⇑
Corresponding author. Tel./fax: +81 466 84 3951.
E-mail address: nishio.toshiyuki@nihon-u.ac.jp (T. Nishio).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.03.037

28
T. Hirano et al. / Carbohydrate Research 353 (2012) 27–32
active form of the transfructosylation-catalyzing b-fructofurano-
sidase as a whole-cell catalyst (Fig. 1). The whole-cell catalysis
method reported here has the following advantages: (1) extraction
of the enzyme from the culture is unnecessary and (2) the enzyme
can be easily removed from the reaction mixture.
filter paper and then dried under decompression at room temper-
ature using an aspirator to produce soft granules consisting of
dried A. oryzae mycelia and Celite 535 (Fig. 2A). The content of
dried mycelia in the granules was about 20% (w/w) with all A. ory-
zae strains used in this study. These values were calculated by sub-
tracting the weight of Celite 535 used from the weight of the
granules. Granules were observed under a TM-1000 tabletop elec-
tron microscope (Hitachi High-Technologies, Tokyo, Japan).
2. Experimental
2.1. Materials
2.3. Oligosaccharide analysis
2-Acetamido-2-deoxy-D-glucose (GlcNAc) was purchased from
Oligosaccharide production was monitored using thin layer
chromatography (TLC) and high performance liquid chromatogra-
phy (HPLC). The TLC plates (Silica Gel 60 N, 230–400 mesh, E.
Merck, Darmstadt, Germany) were developed twice using n-
BuOH/pyridine/H2O (8:3:1) as the mobile phase. Compounds were
visualized by spraying the plates with an aqueous solution con-
taining 2.4% H3(PMo12O40)ꢀnH2O, 5% H2SO4, and 1.5% H3PO4, fol-
lowed by heating. The HPLC analyses were performed using a LC-
10AS pump (Shimadzu, Kyoto, Japan) equipped with a Shodex RI-
71 differential refractometer (Showa Denko, Tokyo, Japan) and a
TCI Fine Chemicals (Tokyo, Japan). Sucrose, glucose, fructose, 1-
kestose, nystose, and diatomaceous earth Celite 535 were obtained
from Wako Pure Chemical Industries (Osaka, Japan). The mold cell
wall dissolution enzyme Yatalase was purchased from Takara Bio
(Shiga, Japan). All A. oryzae strains used in this study were obtained
from the Biotechnology Section of the National Institute of Tech-
nology and Evaluation (Chiba, Japan).
2.2. Preparation of soft granules containing dried
A. oryzae mycelia
COSMOSIL Sugar-D column (size;
u
4.6 ꢁ 250 mm, Nacalai Tesque,
Kyoto, Japan). Oligosaccharides were separated under isocratic
conditions using 77% (v/v) CH3CN in water (flow rate = 0.8 -
mL minꢂ1) as the mobile phase. The amount of oligosaccharide in
the reaction mixture was estimated based on comparison of peak
area with a standard curve. The transfructosylation product was
characterized using 1H and 13C NMR spectrometry as well as mass
spectrometry (MS). The 1H and 13C NMR spectra were recorded in
D2O at 25 °C using a VXR-400 spectrometer (Varian, Palo Alto, CA,
USA). Chemical shifts were expressed as downfield shifts (ppm)
from 3-(trimethylsillyl)propionate-d4. Mass spectra were recorded
using a ZQ4000 LC–MS instrument (Waters, Milford, MA, USA) un-
der positive-ion direct electrospray ionization (ESI) conditions. The
optical rotation was measured in H2O at 20 °C using a P-1030
polarimeter (Jasco, Tokyo, Japan). The stability of the oligosaccha-
ride was measured at several temperatures in the following buf-
fers: 20 mM glycine–HCl (pH 3), 20 mM Na citrate (pH 5), 20 mM
Na phosphate (pH 7), and 20 mM glycine–NaOH (pH 9). Buffer
solutions containing each oligosaccharide (1%, w/v) were main-
tained at various temperatures (25, 50, 75, and 100 °C) for 1 h,
and then the compound in the solution was subjected to TLC and
HPLC analysis.
A 500-lL suspension of A. oryzae spores was mixed with 100 mL
of fresh medium (pH 5.6) containing 2% sucrose, 0.5% peptone, 0.2%
NaNO3, 0.1% K2HPO4, 0.05% MgSO4ꢀ7H2O, 0.05% KCl, 0.001% FeS-
O4ꢀ7H2O, and 4% Celite 535, and mycelia were cultivated by shak-
ing (135 rpm) for 72 h at 28 °C. Wet granules consisting of A. oryzae
mycelia and Celite 535 were collected by suction filtration using
no. 5A filter paper (Kiriyama, Tokyo, Japan), suspended in 50 mL
of cold acetone, and then incubated on ice for 30 min. After the
granules were collected by suction filtration using no. 5A filter pa-
per, they were re-suspended in 50 mL of cold acetone again. Dehy-
drated granules were collected by suction filtration using no. 5A
2.4. Oligosaccharide production and purification
Soft granules containing 700 mg of dried A. oryzae NBRC100959
mycelia were added to a mixture of sucrose (10 g, 29.2 mmol) and
GlcNAc (19.4 g, 87.7 mmol) in 100 mL of 20 mM Na citrate buffer
(pH 5.5) and the mixture was incubated at 30 °C with gentle shak-
ing. It should be noted that this reaction solution is almost satu-
rated with carbohydrates. At specific intervals during the
enzymatic reaction, the progress of oligosaccharide production
was monitored using TLC and HPLC. After 8 h of incubation, the
granules were removed by filtration using no. 5A filter paper. The
resulting supernatant containing the reaction products was loaded
onto a charcoal column (particle size: 63–300 mm, Wako Pure
Chemical Ind.; column size: /5.7 ꢁ 41 cm; solvent: H2O), and the
monosaccharides (GlcNAc, glucose, and fructose) contained in the
reaction mixture were eluted with water. The oligosaccharides that
adhered to the charcoal were eluted with 5% EtOH in water. Frac-
tions containing the products were collected and concentrated by
evaporation, and the target oligosaccharide was further purified
by chromatography using an UltraPack NH-40C column [Yamazen,
Osaka, Japan; size: /5 ꢁ 30 cm; solvent: 80% (v/v) 2-PrOH in
water; flow rate: 8 mL minꢂ1]. After the fractions containing the
Figure 1. Schematic representation of the transfructosylation of sucrose and
GlcNAc.

T. Hirano et al. / Carbohydrate Research 353 (2012) 27–32
29
Figure 2. Soft granules composed of dried A. oryzae NBRC100959 mycelia and Celite 535. (A) Photograph of soft granules. (B) Electron micrograph of a soft granule.
oligosaccharide were collected and concentrated by evaporation,
the sample was lyophilized to produce a white powder as the final
product.
using a TOYOPEARL SuperQ-650 M resin [buffer: 10 mM Na citrate
(pH 6.0); column size: /1.5 ꢁ 20 cm; elution: linear gradient of 0–
0.15 M NaCl]. The enzyme was further purified by affinity chroma-
tography using a Con A sepharose 4B (size: /1 ꢁ 5.5 cm, GE Health-
care, Buckinghamshire, England) column pre-equilibrated with
50 mM McIlvain buffer (pH 6.5) containing 0.5 M NaCl. The en-
zyme was eluted from the column with a linear gradient of 0–
2.5. Assay of b-fructofuranosidase hydrolytic activity
The hydrolytic activity of b-fructofuranosidase was assayed
using sucrose as a substrate, and was monitored by measuring
the amount of reducing sugar released from sucrose. The enzyme
0.6 M methyl
a-D-mannoside in 100 mM McIlvain buffer (pH 6.5)
containing 0.5 M NaCl. The homogeneity of the enzyme prepara-
tion was confirmed by SDS–PAGE. Proteins in the polyacrylamide
gels were stained using Coomassie Brilliant Blue R250 (TCI Fine
Chemicals). The N-terminal amino acid sequence of the purified
enzyme was determined using a Procise 492 cLC protein sequencer
(Life Technologies, Carlsbad, CA, USA).
solution (50 lL) was added to 50 lL of 0.4% sucrose in 20 mM Na
citrate buffer (pH 5.5) and the mixture was incubated at 30 °C.
After incubation, the enzymatic reaction was stopped by heating
the sample at 95 °C for 5 min. The amount of reducing sugar in
the reaction mixture was determined by the Somogyi–Nelson
method using glucose as a standard.22 One unit (U) of enzyme
activity was defined as the amount of enzyme required to hydro-
A 100-
added to a mixture of sucrose (100 mg, 292
(194 mg, 877 mol) in 1 mL of 20 mM Na citrate buffer (pH 5.5),
lL aliquot of the partially purified enzyme solution was
lmol) and GlcNAc
lyze 1
lmol of sucrose per minute under the assay conditions.
l
and the mixture was incubated at 30 °C with gentle shaking. At
specific intervals during the enzymatic reaction, the progress of
oligosaccharide production was monitored using TLC.
2.6. b-Fructofuranosidase purification and oligosaccharide
production
Soft granules containing 5.82 g of dried A. oryzae NBRC100959
mycelia were suspended in 600 mL of 10 mM Na phosphate buffer
(pH 6.0) containing 0.6 M (NH4)2SO4 and 500 mg of Yatalase, and
the suspension was incubated with gentle shaking at 30 °C for
3 h, after which insoluble material was removed from the suspen-
sion by suction filtration using no. 5A filter paper. Proteins in the
resulting supernatant were precipitated by adding (NH4)2SO4
(30–90% saturation) and collected by centrifugation (9100ꢁg,
4 °C, 15 min). The precipitate containing b-fructofuranosidase
was dissolved in 10 mL of 10 mM Na citrate buffer (pH 6.0), dia-
lyzed against the same buffer, and the resulting solution was fil-
2.7. Oligosaccharide production by various strains of A. oryzae
Granules containing 90 mg of dried mycelia of each A. oryzae
strain were added to a mixture of sucrose (1 g, 2.92 mmol) and Glc-
NAc (1.94 g, 8.77 mmol) in 10 mL of 20 mM Na citrate buffer (pH
5.5). The mixture was incubated at 30 °C with gentle shaking for
8 h, after which the reaction solution was centrifuged (3800ꢁg,
4 °C, 5 min) and the transfructosylation product in the resulting
supernatant was quantified using HPLC.
3. Results and discussion
tered using a Dismic-25CS syringe filter unit (pore size: 0.2
Advantec MFS, Tokyo, Japan) to obtain crude enzyme solution.
The crude enzyme solution was loaded onto TOYOPEARL
lm,
3.1. Preparation of soft granules containing dried A. oryzae
mycelia
a
SuperQ-650 M ion exchange resin (Tosoh, Tokyo, Japan) column
(size: /1.5 ꢁ 27 cm) pre-equilibrated with 10 mM Na citrate buffer
(pH 6.0), and the enzyme was eluted with a linear gradient of 0–
0.25 M NaCl in the same buffer. Two b-fructofuranosidase fractions
were collected and concentrated separately using an Amicon Ultra-
15 10 K centrifugal filter device (Millipore, Billerica, MA, USA) to
obtain a solution of partially purified enzyme. The b-fructofurano-
sidase in the first fraction was purified by gel filtration column
chromatography using a Bio-Gel P-100 fine resin (Bio-Rad, Hercu-
les, CA, USA) [size: /2 ꢁ 78 cm; elution: 20 mM Na citrate buffer
(pH 5.5)]. The active fractions were collected and the enzyme
was again subjected to ion exchange column chromatography
b-Fructofuranosidases of several species of Aspergillus, including
A. oryzae, have been extracted from mycelia after cultivation in li-
quid medium.12,23–27 In the present study, we confirmed that b-
fructofuranosidase is released from A. oryzae NBRC100959 mycelia
grown in liquid medium containing sucrose by treating mycelia
with the mold cell wall dissolution enzyme Yatalase. These results
indicate that b-fructofuranosidase is contained within the A. oryzae
mycelia grown in liquid culture. Thereupon, to synthesize the oli-
gosaccharide through a b-fructofuranosidase-catalyzed transfruc-
tosylation reaction, we decided to use A. oryzae mycelia that
incorporate this enzyme as a whole-cell catalyst.

30
T. Hirano et al. / Carbohydrate Research 353 (2012) 27–32
Through preliminary investigations to determine the state of
mixture, and its structure was identified using MS and NMR
spectrometry.
mycelia that would promote the efficient b-fructofuranosidase
reaction, it was found that the soft granules (Fig. 2A) consisting
of dried A. oryzae NBRC100959 mycelia and Celite 535 show high
activity. The soft granules were prepared by performing acetone
treatment followed by drying to mycelia grown in liquid medium
containing sucrose and Celite. Although strain NBRC100959 died
out as a result of the manipulations, the intracellular b-fructofu-
ranosidase remained in an active form. Electron microscopy con-
firmed that a considerable amount of space exists within the soft
granule in which the dried mycelia and the Celite were dispersed
(Fig. 2B). We hypothesize that this structure provides for optimal
contact between the substrate and the enzyme, which is contained
in mycelia, in the reaction solution. Based upon these findings, we
decided to use the soft granules for production of the
oligosaccharide.
The ESIMS spectra of compound 1 corresponded to a [M+H]+ spe-
cies at m/z 384, suggesting that this compound is a disaccharide
consisting of fructose and GlcNAc. The 1H and 13C NMR assignments
of compound 1 are shown inFigure 4A. The position of the glycosidic
linkage between fructose and GlcNAc was determined using 1H-de-
tected multiple bond connectivity (HMBC) NMR experiments. The
anomeric hydrogen of GlcNAc (H-1) exhibited cross peaks with
C-3 and C-5 of GlcNAc and C-2 of fructose (C-20) (Fig. 4B). The
anomeric hydrogen (H-1) of GlcNAc was found to have a coupling
constant (J1,2) of 4.0 Hz. These results enabled us to identify com-
pound 1 as a N-acetylsucrosamine [b-D-fructofuranosyl-(2?1)-2-
acetamido-2-deoxy- -glucopyranoside].
a-D
We had confirmed that N-acetylsucrosamine was produced
most efficiently in the solution containing 10% (w/v) sucrose and
19.4% (w/v) GlcNAc. Of course, the quantity of production of N-ace-
tylsucrosamine may increase if GlcNAc concentration is still high-
er. However, concentration of GlcNAc cannot be raised any more,
because this monosaccharide is mostly saturated in the solution.
The production efficiency of N-acetylsucrosamine was not influ-
enced so much by the shaking speed of the reaction mixture. N-
Acetylsucrosamine was generated as a major transfructosylation
product through the reaction (Fig. 5), and cumulative dosage of this
disaccharide was the highest at the reaction for 8 h. By performing
2 types of column chromatography, 2.47 g (22.1% yield from su-
crose) of the disaccharide was isolated from 100 mL of the reaction
3.2. Oligosaccharide production, identification, and
characterization
Oligosaccharide was synthesized by adding soft granules con-
taining 700 mg of dried A. oryzae NBRC100959 mycelia to 100 mL
of 20 mM Na citrate buffer (pH 5.5) containing a high concentra-
tion of substrate carbohydrates. The reason this buffer solution
was used is that optimum pH of A. oryzae b-fructofuranosidase is
around 5.5. Considering low heat stability of this enzyme, oligosac-
charide synthesis was performed at a comparatively low tempera-
ture (30 °C). A TLC analysis of the reaction mixture in which only
sucrose (10%, w/v) was added as a substrate revealed that fructool-
igosaccharides such as 1-kestose and nystose were generated as
the transfructsylation products (data not shown). This result indi-
cates that the b-fructofuranosidase contained in the dried A. oryzae
mycelia catalyzed transfructosylation reaction. In contrast, no
reaction products were detected in the mixture in which only Glc-
NAc (19.4%, w/v) was added as a substrate (data not shown). On
the other hand, in the reaction mixture containing sucrose (10%,
w/v) and GlcNAc (19.4%, w/v) (sucrose/GlcNAc molar ratio 1:3),
an unknown reaction product was generated in addition to the
fructooligosaccharides (Fig. 3). This unknown product (compound
1, indicated by arrow 1 in Fig. 3) was purified from the reaction
mixture after 8-h shake. The optical rotation (½a D25
ꢃ
) of N-acetylsu-
crosamine obtained in our experiment were 80.7° (c 0.96, H2O)
[lit. ½a 2D5
ꢃ
83.6° (c 0.5, H2O)28]. More than 8 g of N-acetylsucros-
amine was dissolved in 2 mL of water to form colorless high-vis-
cosity syrup, indicating that this disaccharide is highly soluble in
water. Although neither N-acetylsucrosamine nor sucrose decom-
posed upon incubation at 100 °C for 1 h in buffers of pH 5, 7, or 9
(data not shown), both disaccharides were hydrolyzed to monosac-
charides when incubated at 75 °C and 100 °C for 1 h in buffer solu-
tion of pH 3. However, the degree of N-acetylsucrosamine
hydrolysis in buffer of pH3 was lower than that of sucrose, indicat-
ing that N-acetylsucrosamine is more stable under acidic condi-
tions than sucrose. The data about the stability of this
disaccharide are presented in Supplementary Figure S1.
Römer et al. reported the sucrose synthase-mediated synthesis
of N-acetylsucrosamine from sucrose and uridine diphosphate Glc-
NAc (UDP-GlcNAc) [overall molar yield: 59% (from UDP-GlcNAc)],29
while Lichtenthaler et al. synthesized N-acetylsucrosamine from
sucrose using a seven-step chemical synthesis (overall molar yield:
5.76%).28 In our method, N-acetylsucrosamine was synthesized in a
one-step reaction using low-cost carbohydrate substrates and dried
A. oryzae mycelia, which can be prepared easily, as a catalyst. Our
results demonstrate that the method described here is a simpler
and lower cost alternative for the production of N-
acetylsucrosamine.
3.3. Confirmation of N-acetylsucrosamine-producing enzyme
We also investigated the enzyme involved in the synthesis of N-
acetylsucrosamine from sucrose and GlcNAc. The enzyme was ex-
tracted from dried A. oryzae NBRC100959 mycelia by treating the
granules with the mold cell wall dissolution enzyme Yatalase,
and was partially purified using anion exchange chromatography
on a TOYOPEARL SuperQ-650 M column. The b-fructofuranosidase
activity was separated into 2 fractions, Fa and Fb (Fig. 6A). Each
partially purified enzyme fraction was added to a solution contain-
ing high-concentration sucrose and GlcNAc, and the reaction prod-
uct was analyzed using TLC. The enzyme contained in the first
anion exchange fraction (Fa) synthesized N-acetylsucrosamine
Figure 3. TLC of the compounds formed in the transfructosylation reaction.
Carbohydrates in the mixture were identified based on comparison with standards:
sucrose (a), glucose (b), fructose (c), GlcNAc (d), 1-kestose (e), and nystose (f).
Arrow 1 represents the unknown reaction product (compound 1).

T. Hirano et al. / Carbohydrate Research 353 (2012) 27–32
31
Figure 4. Identification of the transfructosylation product. (A) 1H and 13C NMR shifts and the structure of compound 1. 1H NMR data (chemical shift in ppm; multiplicity;
coupling constant in Hz) and 13C NMR chemical shifts (ppm) are designated with arrows and dashed line arrows, respectively. The bent double-headed arrow indicates the
HMBC cross peak. (B) HMBC NMR cross peaks with the GlcNAc anomeric hydrogen of compound 1. Peak H-1 is the anomeric hydrogen of the GlcNAc residue of compound 1.
Peaks C-1, 2, 3, 4, 5, and 6 are ring carbons of the GlcNAc residue of compound 1. Peaks C-10, 20, 30, 40, 50, and 60 are ring carbons of the fructose residue of compound 1. Dashed
lines indicate crossing between the peak of H-1 and those of C-3, C-5, and C-20.
Figure 5. Carbohydrate composition in reaction mixture. The total amount of
carbohydrate in the reaction mixture at each time point was taken as 100%.
(main product) and 1-kestose (minor product) (Fig. 6B1), while the
enzyme contained in the second anion exchange fraction (Fb) cat-
alyzed only the hydrolysis of sucrose (Fig. 6B2).
Sequencing of the A. oryzae NBRC100959 genome (37 Mb) was
accomplished in 2005.30 Then, in order to identify the b-fructofu-
ranosidase involved in the synthesis of N-acetylsucrosamine, we
purified the enzyme contained in Fa and determined its N-terminal
amino acid sequence. Each chromatographic step produced a single
peak showing b-fructofuranosidase activity. The purified protein
gave
a single band on SDS–PAGE analysis (Supplementary
Figure 6. Enzyme purification and N-acetylsucrosamine production by partially
purified b-fructofuranosidases. (A) Chromatogram showing separation of two kinds
of b-fructofuranosidases by ion exchange column chromatography. (B) TLC of the
carbohydrates in the reaction mixture. b-Fructofuranosidase (0.65 U) contained in
Fa and that (1.55 U) contained in Fb were added separately to a substrate solution.
Carbohydrates in the mixture were identified based on comparison with standards:
sucrose (a), glucose (b), fructose (c), GlcNAc (d), N-acetylsucrosamine (e), 1-kestose
(f), and nystose (g).
Fig. S2). We confirmed that this purified protein catalyzed N-ace-
tylsucrosamine production in a reaction mixture containing high-
concentration sucrose and GlcNAc (data not shown). The N-termi-
nal amino acid sequence of this protein was determined as IDYNA.
This sequence exists in N-terminal region of the predicted protein
from gene (NCBI accession number; NW_001884675, locus tag;

32
T. Hirano et al. / Carbohydrate Research 353 (2012) 27–32
AOR_1_1114084 encoding putative b-fructofuranosidase on chro-
mosome 6 of strain NBRC100959. We clarified that N-acetylsucros-
amine is highly water soluble and more stable than sucrose in
acidic solution. Our results also indicate that dried mycelia pre-
pared from a variety of A. oryzae strains produce about the same
quantity of N-acetylsucrosamine.
We plan to develop N-acetylsucrosamine as a functional oligo-
saccharide. Because A. oryzae is widely used in the Japanese food
industry, we believe that from a safety standpoint it would be suit-
able to use this microbe in the production of N-acetylsucrosamine.
In the near future we will begin investigating the possible physio-
logical uses of this disaccharide.
Acknowledgements
This research was supported by a Grant from the Research Pro-
ject of the Life Science Research Center, College of Bioresource Sci-
ences, Nihon University. We thank Dr. Tadatake Oku and Dr.
Masaaki Kurihara for their support.
Supplementary data
Figure 7. N-Acetylsucrosamine production by various strains of A. oryzae. This
experiment was conducted using three lots of granules prepared separately with
each strain. The concentration of N-acetylsucrosamine in each reaction mixture was
determined. Values are represented as average SD of three independent exper-
iments using different granule samples.
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.carres.
2012.03.037.
AOR_1_1114084) encoding putative b-fructofuranosidase on chro-
mosome 6 of strain NBRC100959. A query of the genome database
DOGAN [database of the genomes analyzed at the National Insti-
tute of Technology and Evaluation (NITE), http://www.bio.nite.-
go.jp/dogan/top] indicated that two b-fructofuranosidase genes
are located on chromosome 5 of A. oryzae NBRC100959. The se-
quence IDYNA does not exist in the N-terminal regions of the puta-
tive proteins from these two genes. Based upon these findings, we
concluded that the b-fructofuranosidase involved in the synthesis
of N-acetylsucrosamine in our experiments was the product from
gene AOR_1_1114084 on chromosome 6 of A. oryzae NBRC100959.
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the synthesis of this disaccharide is
a product from gene