A novel enzyme of Bacillus sp. 217C-11 that produces inulin from sucrose.

Article abstract of DOI:10.1271/bbb.67.1327

We found a bacterium that converts sucrose to a useful material, using about 6,000 samples of bacteria isolated from soil. This bacterium, Bacillus sp. 217C-11, was identified according to Bergey's manual, and produced a highly efficient enzyme that converted sucrose into inulin. So, the enzyme was purified to homogeneity through five chromatographic steps, to identify its enzymatic properties. The molecular mass of the enzyme was estimated to be 45,000, and this enzyme was a monomer protein (by SDS-PAGE). The optimum pH and temperature of this enzyme were 7-8 and 45-50 degrees C, respectively. The enzyme reacted only with sucrose, but did not with other disaccharides, fructooligosaccharides and inulin. This paper will show that our enzyme is a novel one, which is different from the other well-known enzymes concerned in inulin production.

Full text of DOI:10.1271/bbb.67.1327

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A Novel Enzyme of Bacillus sp. 217C-11 That
Produces Inulin from Sucrose
Tadashi WADAa, Masao OHGUCHIa & Yoshio IWAIa
a Fuji Nihon Seito Corporation1-4-10 Seikai, Shimizu City, Shizuoka 424-8737, Japan
Published online: 22 May 2014.
To cite this article: Tadashi WADA, Masao OHGUCHI & Yoshio IWAI (2003) A Novel Enzyme of Bacillus sp. 217C-11 That
Produces Inulin from Sucrose, Bioscience, Biotechnology, and Biochemistry, 67:6, 1327-1334, DOI: 10.1271/bbb.67.1327
To link to this article: http://dx.doi.org/10.1271/bbb.67.1327
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Biosci. Biotechnol. Biochem., 67 (6), 1327–1334, 2003
A Novel Enzyme of Bacillus sp. 217C-11 That Produces Inulin from Sucrose
Tadashi WADA,† Masao OHGUCHI, and Yoshio IWAI
Fuji Nihon Seito Corporation, 1-4-10 Seikai, Shimizu City, Shizuoka 424-8737, Japan
Received December 24, 2002; Accepted February 25, 2003
We found a bacterium that converts sucrose to a
useful material, using about 6,000 samples of bacteria
isolated from soil. This bacterium, Bacillus sp. 217C-11,
was identiˆed according to Bergey's manual, and
produced a highly e‹cient enzyme that converted
sucrose into inulin. So, the enzyme was puriˆed to
homogeneity through ˆve chromatographic steps, to
identify its enzymatic properties. The molecular mass of
the enzyme was estimated to be 45,000, and this enzyme
was a monomer protein (by SDS-PAGE). The optimum
pH and temperature of this enzyme were 7–8 and 45–
Biˆdobacterium is worthy of remark amid the recent
boom in health-consciousness.
Inulin has not been commercialized in Japan,
because commercial cultivation of these plants is
di‹cult. To obtain inulin in Japan, there is no choice
but to import. Such imported inulin is more expen-
sive than some domestic substances having functions
analogous to inulin.
In addition, the yield of plant-derived inulin de-
pends on the harvest conditions, as the raw material
of inulin is extracted from plant bodies. Also, the
inulin content is rapidly reduced by autolysis, unless
the inulin extraction is done just after the harvest.
These facts must be fully considered in the industrial
production of inulin.
Furthermore, the puriˆcation of plant-derived
inulin is extremely di‹cult because of diŠerent fruc-
tose-chain lengths. Currently available plant-derived
inulin is commercialized by spray-drying of a roughly
fractionated crude solution containing inulin of
diŠerent chain lengths.
509C, respectively. The enzyme reacted only with
sucrose, but did not with other disaccharides, fruc-
tooligosaccharides and inulin. This paper will show that
our enzyme is a novel one, which is diŠerent from the
other well-known enzymes concerned in inulin produc-
tion.
Key words: inulin; sucrose; fructosyltransferase;
inulin-producing enzyme (IPE); Bacillus
sp. 217-11
Therefore, even though the purity of inulin may be
high, a lack of uniformity in the chain lengths is
unavoidable.
Inulin is a kind of polysaccharide, and is widely
distributed throughout the plant kingdom. Among
inulin-containing plants, the Asteraceae had been
mainly used as raw materials for inulin production in
Europe. For example, inulin exists as a reserve sub-
stance in the tubers or tuberous roots of the Aster-
aceae plant, such as dahlia, Jerusalem artichoke, and
The above-mentioned higher plants from which
inulin can be extracted naturally contain some
enzymes for producing inulin. Luscher et al.,3) and
Edelman and JeŠord4) reported that inulin is pro-
duced from sucrose using two cooperative enzymes
which were extracted from tubers of Helianthus
tuberosus. Two enzymes were (1) sucrose:sucrose
1-fructosyltransferase (1-SST; EC2.4.1.99), which
performs the transfer of fructosyl between sucroses,
and (2) fructan:fructan 1-fructosyltransferase
(1-FFT; EC2.4.1.100), which transfers fructose
moieties between fructans having a degree of poly-
merization of 3 or more. But it is industrially imprac-
tical to use the above process requiring a large
amount of enzymes from plant bodies, because it is
time- and labor-consuming.
Inula japonica
chicory.1) In contrast with starch, inulin dissolves in
warm water, and is a linear polymer having -(2 1)
linkages by which -fructoses were polymerized.
, i.e. British inula, and in the root of
b
ª
D
Plant-derived inulins are a group of compounds
diŠerent in their molecular weights, and the average
degree of fructose polymerization ranges from 32 to
34. Praznik and Beck2) reported that the mean
molecular weight is 2,282 to 17,000. Thus, the degree
of fructose polymerization varies depending on plant
species, but the molecular weights of inulin are limit-
ed within a certain range.
In addition to the plant-derived inulin, a method
for producing analogues of inulin by the action of
microbial enzymes has been reported. KopeloŠ et
Inulin is a dietary ˆber that is di‹cult to digest.
Further, the promoting eŠect on the growth of
†
To whom correspondence should be addressed. Fax:
81-543-35-5046; E-mail:tadasi.wada fnsugar.co.jp
＋ ＠
Abbreviations: IPE, inulin-producing enzyme; 1-SST, sucrose: sucrose 1-fructosyltransferase; 1-FFT, fructan: fructan 1-fructosyltrans-
ferase; DP, degree of polymerization

1328
T. WADA et al
.
al.5) reported in 1920 that the conidiospores of Asper-
gillus sydowi have invertase activity and can produce
a levan-type fructan from sucrose. After a fairly long
time, Loewenberg and Reese6) revealed in 1957 that
the polysaccharide shows an inulin-typed structure
tiˆcation and chemical compositions of the reaction
products derived from sucrose.
Materials and Methods
having
b
-(2
ª
1) linkages of fructose.
Chemicals. Inulin was purchased from Orafti,
Tienen, Belgium. Raftiline ST is unfractionated,
spray-dried inulin isolated from the roots of Cichori-
um intybus L. This unfractionated inulin is poly-
disperse, and the average DP was 10. Raftiline HP is
spray-dried inulin, in which low DP of inulin is
removed by fractionation. The DP of Raftiline HP
was 23. Levan obtained from Serratia levanicum was
purchased from Wako Pure Chemicals. DEAE-
Toyopearl 650M, Toyopearl HW55F, and phenyl-
Toyopearl 650M were purchased from Tosoh Co.
Other chemicals were purchased from Wako Pure
Chemicals.
7)
Later, Kawai et al
.
reported in 1973 that both
polyfructan and oligofructan are produced by
conidiospores of A. sydowi, and that, like higher
plant-derived inulin, the polyfructan consists of a
linear chain which has b-(2ª1) linkages lacking glu-
cose at its end, and then that its molecular weight
≒
(about 20,000,000; DP 123,000) is much larger than
that of higher plant-derived inulin.
8)
In 1986, Nakakuki et al
.
proposed a method for
producing oligofructan and polyfructan by treating
sucrose with cells of A. sydowi. The produced fruc-
tan was a linear polyfructan having fructose linked
by
b-(2ª1) linkages with glucose at its end. This
oligofructan showed the DP of 5 (828 in M.W.) or
less, while the macromolecular fructan showed
Screening method. The soil sample (1 gram) was
suspended in sterilized water and spread on agar
5
7
×
×
molecular weights ranging from 1.8 10 to 1.4 10 .
Recently, Harada et al
plates containing 2
peptone, 0.5
0.1 K2HPO4, and 0.05
incubated for several days at 30
were picked up, and were suspended in the same
liquid medium. Twenty (w w) sucrose solution
(ˆnal concentration) was added to the suspension,
and it was incubated for 15 h at 30 C. Then, the
z
sucrose as a carbon source, 1
yeast extracts, 0.3 malt extract,
MgSO4 (pH 7.2), and
C. Growing colonies
z
9)
.
also proposed a method
z
z
for producing polyfructan from sucrose using the
conidiospores of A. sydowi, and reported that the
molecular weight of the polyfructan is about
z
z
9
≒
10,000,000 (DP 61,700).
z
W
10)
On the other hand, Hidaka et al
method for producing a linear fructan of
.
proposed a
-(2 1)
b
ª
9
linkages from sucrose by using fructosyltransferases
produced by Aspergillus or Fusarium. However, the
fructan was an oligosaccharide in which 1 to 4 molec-
ules of fructose are bound to sucrose. So, the fructan
was deˆned as a substance diŠerent from inulin in
molecular size.
resulting products were detected with thin-layer chro-
matography (TLC).
Microorganisms and their cultivation. One of the
soil microorganisms screened was Bacillus sp.
217C-11. The strain was cultivated at 30
with agitation at 150 rpm in culture medium (pH 8.0)
containing 0.5 sucrose, 1 peptone, 0.5 yeast
extracts, and 0.2 K2HPO4. After cultivation, the
9
C for 15 h
Furthermore, Rosell and Birkhed 11) reported that
Streptococcus mutans, which was considered as the
pathogen of dental caries, induces an enzyme capable
of producing an analogue of inulin. However, the
inulin analogue is diŠerent from inulin, since it was a
giant molecule having the molecular weight of
z
z
z
z
bacterial cells were removed by centrifugation at
15,000 rpm for 15 min.
20,000,000 and it had
b
-(2
ª
6) linkages in a linear
Thin-layer chromatography (TLC). TLC of sugars
chain of -(2 1) linkages.
b
ª
were done on a silica-gel 60 plate (Merck) developed
The above-described substances shall be referred
to as inulin-type polyfructan, in order to distinguish
them from higher-plant-derived inulin. Their prop-
erties are largely diŠerent from those of higher-plant
inulin; for example, their molecular sizes are larger,
and they have diŠerent structures. Concerning the
inulin production by enzymes of microorganisms, no
industrial technology has been established yet.
We continued the screening of microorganisms
which change sucrose into valuable materials, and
discovered a microorganism that can produce a high-
ly e‹cient enzyme that converts sucrose into inulin.
In this paper, we shall report the identiˆcation of the
isolated microorganism, the puriˆcation of an in-
duced enzyme, its enzymatic properties, and the iden-
with a solvent of acetone-water (9:1, v v), and sugar
W
spots were detected with 10
on a hot plate.
z
sulfuric acid by heating
Quantitative analysis of sugars. High pressure liq-
uid chromatography (HPLC) was done for quantita-
tive analysis of sugars with the following equipment
and detection conditions: column, Shinwa Chemical
×
Industries ULTRON PS-80N (300 8.0 mm); mobile
phase; distilled water; ‰ow rate, 0.5 ml min; temper-
W
ature, 509C; pump, Hitachi L-6000; and detector,
Hitachi L-7490 RI detector.
Measurement of the average DP of inulin.
Enzyme hydrolysis. The average DP of inulin was

A Novel Inulin-producing Enzyme of Bacillus sp.
1329
deˆned as the fructose glucose ratio, after complete
500 m
were pooled, concentrated by addition of ammonium
sulfate to 70 saturation, and centrifuged. The
precipitate was dissolved in a minimum volume of
M
KCl in the same buŠer. The active fractions
W
hydrolysis with Fructozyme inulinase (Novo Nor-
disk) for 15 h at 37
9
C at pH 7.0.12,13) The amount of
z
sugars was measured by HPLC.
HPLC. The average DP of inulin was measured by
HPLC with a Tosoh TSK-Gel G3000PWXL (300
20 m phosphate buŠer pH7.0. The enzyme solution
M
×
was dialyzed against the same buŠer.
Toyopearl HW55S gel ˆltration chromatography.
The enzyme obtained after DEAE Toyopearl 650M
treatment was put on a Toyopearl HW55S gel ˆltra-
7.8 mm) gel permeation chromatography column
under the following conditions: mobile phase, dis-
tilled water; ‰ow rate, 0.5 ml min; temperature,
W
50
9
C; pump, Hitachi L-6000; and detector, Hitachi
tion column (
phosphate, pH 7.0, containing 0.15
active fractions were pooled and dialyzed against
1.7 ammonium sulfate in 20 m phosphate,
pH 7.0.
Phenyl Toyopearl 650M column chromatography
The dialyzed enzyme solution was put on a Phenyl
q
1.8
×
137 cm) equilibrated with 20 m
M
＝
L-7490 RI. Raftiline ST (DP 10) and Raftiline HP
(DP 23) were used as standard materials.
M
KCl. The
＝
M
M
Measurement of the polydispersity of inulin. The
polydispersity in the chain length of inulin was
evaluated qualitatively. The Dionex series DX-500
apparatus was equipped with an eluent degassing
module, Dionex ED40, pulsed electrochemical detec-
tor, GP50 gradient pump and AS50 autosampler.
.
×
Toyopearl 650M column (
with 1.7 ammonium sulfate in 20 m
pH 7.0. The column was washed with this buŠer, and
the enzyme was eluted with a linear gradient of 1.7
ammonium sulfate in the same buŠer. The
active fractions were pooled and dialyzed.
q
1.2 6 cm) equilibrated
M
M
phosphate,
The injected samples (50
CarboPac PA-1 column (4 250 mm). The bound
material was eluted by a NaOH-NaOAc gradient.
m
l, 3
z
solution) passed a
M
×
¿0 M
1H-and 13C-NMR spectra. A sample was dissolved
in D2O, and the NMR spectrum was recorded at
500 MHz using a JOEL lambda-500FT-NMR spec-
trometer (Nippon Bunkoh).
Measurement of molecular weight of the enzyme.
Gel ˆltration method. The molecular weight of the
enzyme was estimated by Fast protein liquid chro-
matography (FPLC) with a Sephacryl S300 gel ˆltra-
tion column (Pharmacia). Molecular weights of the
marker proteins used were thioglobulin (670,000),
gamma-globulin (158,000), ovalbumin (44,000), and
myoglobin (17,000).
Enzyme assay. The activity of the inulin-producing
enzyme was assayed by measuring the amount of
glucose released from sucrose. The reaction mixture
(0.4 ml) containing 10 m
M
phosphate buŠer (pH 7.0),
sucrose, and enzyme solution was incubated at
C for 30 min. The reaction was stopped by adding
HCl. The glucose released was measured by the
SDS-polyacrylamide gel electrophoresis (SDS-
20
37
z
z
PAGE). SDS-PAGE was done on a 2–15 poly-
9
acrylamide separating gel (Multy Gel 2 15, Daiichi
W
1
N
Pure Chemicals Co., Ltd.) under reducing condi-
tions. Protein was stained by Coomassie brilliant
blue (CBB R-250). Molecular weights of calibration
glucose oxidase-peroxidase method. One unit of
enzyme activity was deˆned as the amount of enzyme
that liberates glucose 1
tions.
m
mol min under these condi-
proteins used were myosin (200,000), b-galactosidase
W
(116,248), bovine serum albumin (66,267), aldolase
(42400), carbonic anhydrase (30,000), and myoglobin
(17,000).
Puriˆcation of inulin-producing enzyme (IPE).
The puriˆcation of IPE was done by the following
ˆve steps.
Ultraˆltration. The crude enzyme was concentrat-
ed 5 times by the ultraˆlter (Nihon pole) for cutting
oŠ the molecular weight below 30,000.
Protein assay. The protein concentrations were
estimated with the Bio-Rad protein assay reagent
(Bio-Rad Laboratories, Hercules, Calif.) for the
Bradford dye-binding method, with bovine serum
albumin as the protein standard.14)
Ammonium sulfate fraction. Powdered ammoni-
um sulfate was added to the crude enzyme solution to
obtain 70
z
saturation. After this was left standing
Analysis of N-terminal amino acid sequence of
inulin-producing enzyme (IPE). After SDS-PAGE of
the puriˆed enzyme, the protein on the gel was trans-
for 2 h, the precipitation was collected by centrifuga-
tion and was dissolved in a minimum volume of
20 m
M
phosphate buŠer, pH 7.0. Then, the enzyme
ferred to PVDF membrane at 200 m
M
for 1 h. After
solution was dialyzed against the same buŠer.
the protein was stained as described above, the mem-
brane corresponding to the protein band of IPE was
cut oŠ and analyzed with a HP G1005A Protein
Sequencing system (Hewllett-Packard Co.).
DEAE Toyopearl 650M column chromatography
.
The dialyzed solution was put on a DEAE Toyopearl
×
(q1.8 14 cm)
650M anion exchange column
equilibrated with 20 m phosphate buŠer, pH 7.0.
The enzyme was eluted with a linear gradient of 0 to
M

1330
T. WADA et al.
Fig. 1. TLC Analysis on Reaction Products from Sucrose.
The reaction mixture was composed of 20
Producing Enzyme). The reaction was done at 37
z
(w w) sucrose, 5 m
M
phosphate buŠer (pH 7.0), and crude enzyme of IPE (Inulin-
W
9
C for 16 h. The reaction products were analyzed by TLC with acetone distilled water
W
(9:1, v v) as a solvent.
Lane R, reaction mixture; Lane 1, R
W
＋
inulinase; Lane 2, R
＋
dextranase; Lane 3, levan
＋
inulinase; Lane L, levan; Lane I, inulin;
Standard materials were fructooligosaccharide (FO), fructose (F), glucose (G), sucrose (GF), 1-kestose (GF2), nystose (GF3), and fruc-
tosylnystose (GF4).
and the reaction products were analyzed by TLC. As
shown in Fig. 1, one of the products caused a strong
spot. This product was not digested by dextranase
(Sankyo Co., Japan), but was digested completely by
inulinase. This inulinase could not digest levan, a
kind of fructan, so it seems that this reaction product
is inulin.
Results and Discussion
Screening
During the screening, we found three types of
bacterial strains producing inulin from sucrose. The
ˆrst strain produced together the well-known fruc-
tooligosaccharides (1-kestose, nystose, and fructosyl-
nystose) and inulin from sucrose. The second one
showed very low invertase activity, and produced
glucose, fructose, and also inulin from sucrose. The
third one produced a relatively large amount of
inulin from sucrose together with a slight amount of
fructose. We took notice of the last strain, by which
highly e‹cient inulin production will be obtained,
and detailed investigation was done on the puriˆca-
tion of related enzyme, reaction products, and so on.
Taxonomical study of the isolate was done by
NCIMB Japan Co., Ltd (Shizuoka). Taxonomic
characteristics of the isolate, 217C-11, were Gram-
The lower molecular weight fructans indicated
higher
Rf-values in developing solvent systems of
TLC analysis. Since reaction products were devel-
oped on lower positions than those of fructosyl-
nystose, (pentasaccharide), it seems that their degrees
of fructose polymerization are above hexasaccharide.
Time-course of inulin production by inulin-
producing enzyme (IPE)
The time-course of changes of inulin production
by IPE of Bacillus sp. 217C-11 were investigated. In
the ˆnal concentration of 40
z
sucrose solution, the
reaction was done at 37 C (Fig. 2). Inulin and
9
×
variable, spore forming, motile, and rod-shaped (1
m). The organism grew in both aerobic and
glucose were gradually increased with the decrease in
sucrose. When the reaction reached an the equilibri-
um, the composition of inulin and glucose had
2
¿3 m
anaerobic conditions. It was positive for both cata-
lase and oxidase production, and negative for urease
production and nitrate reduction. Thus, this strain
was identiˆed as a Bacillus sp.
z
reached to 45 , respectively, and the release of fruc-
tose was hardly recognized. This will make the inulin
puriˆcation easy.
Thus, the conversion rate of inulin from sucrose is
TLC analysis on reaction products of sucrose
The enzyme was put in to a 20
45z. When the concentration of the remaining
z
sucrose solution
z
sucrose solution reached to about 7 , the enzyme

A Novel Inulin-producing Enzyme of Bacillus sp.
1331
reaction balanced. In the case of higher concentra-
tions than about 7 , the reaction proceeded to the
direction of inulin production. And, in lower than
about 7 solution, inulin degradation occurred. So,
product did not agree with that of levan, but did so
with the standard inulin. These results and an addi-
tional analysis by mass spectroscopy indicated that
the reaction product is inulin.
z
z
this enzyme reaction seems to be a equilibrium
reaction.
DP of the reaction products
The result on the average DP of inulin was meas-
ured by the above-described method (Fig. 4). The DP
Identiˆcation of reaction product
After removal of low molecular weight sugars
from reaction products by ˆltering through a reverse-
osmosis membrane, the residue was reˆned three
times with ethanol precipitation. The sample pre-
pared to be uniform on HPLC was analyzed with
NMR (Fig. 3). The same analyses were done for stan-
dard inulin and levan. The chart of the reaction
obtained by this enzyme was 18. The glucose fruc-
W
tose ratio of the sample digested completely with
inulinase was 1:17. It was estimated that the DP is 18
(Table 1). Also, the polydispersity in chain length
was investigated, and it was found that the DP of
sucrose-origin inulin is in the range from 10 to 25, in-
dicating a peak of about 16–17 (Fig. 5). On the other
hand, the DP of plant-origin inulin was dispersed in a
wider range from 10 to 60. Thus, each molecular
weight of sucrose-origin inulin produced by this en-
zyme was relatively low, and these DPs concentrated
in a narrower range from 10 to 30, compared with
those of plant-origin inulin. Fortunately, we succeed-
ed in producing a group of inulin molecules having
approximately similar chain-length, using this en-
zyme. The inulin obtained by IPE was quite diŠerent
from naturally occurring plant inulin. The sig-
niˆcance of this diŠerence is under consideration. At
present, we are interested in several speciˆc functions
of the inulin produced by IPE. The details will be
described in our next paper.
Fig. 2. Inulin Formation from Sucrose by IPE (inulin-producing
enzyme).
M
Puriˆcation of inulin-producing enzyme (IPE)
Table 2 shows a summary of the puriˆcation of
IPE derived from Bacillus sp. 217C-11. The enzyme
The reaction mixture containing 40
phosphate buŠer (pH 7.0), and 1.5U of IPE was incubated at
37 C for 10 h. Samples were withdrawn at various intervals, and
sugar composition in the reaction mixture was measured by
z (w w) sucrose, 10 m
W
9
was puriˆed about 7-fold with 17
z yield from the
#



HPLC. ( ), sucrose; ( ), inulin; ( ), glucose; ( ), fructose;
culture. The speciˆc activity of the puriˆed enzyme
$
(
), 1-kestose.
Fig. 3. NMR Spectrum of Reaction Product and Authentic Fructan.
1
(A) 13C-NMR Spectrum, (B) H-NMR Spectrum.

1332
T. WADA et al
.
preparation obtained by a phenyl-Toyopearl 650M
of the enzyme was low. It seems that this slightly low
recovery was caused by an unexpected large adsorp-
tion of the enzyme protein to gel materials.
column chromatography was 53.3 U mg protein.
W
The puriˆed enzyme preparation indicated a single
band by SDS-PAGE. IPE puriˆcation was relatively
easy.
Some properties of IPE
On the gel ˆltration chromatography, the recovery
The eŠects of pH and temperature on the IPE
activity and stability are shown in Table 3. The opti-
mum pH was 7 to 8, and the enzyme was stable from
pH 6 to 9, and was unstable below pH 5. The opti-
mum temperature was 45
stable up to 40 C. The enzyme activity was complete-
ly inhibited by Cu2 and Hg2 (5m
9
C, and the enzyme was
9
＋
＋
M
), but was not
inhibited by EDTA. So, heavy metal ions were not
necessary to IPE activity. The molecular weight of
the enzyme was estimated to be 45,000 by SDS-
PAGE (Fig. 6), and 44,000 by gel ˆltration on a
Sephacryl S-300 column. These results indicate that
this enzyme is a monomer protein.
Substrate speciˆcity
In order to investigate the substrate speciˆcity of
IPE, various disaccharides were examined as sub-
strates. IPE acted speciˆcally on sucrose to produce
inulin with highly polymerized fructoses (DP
À6),
but was inactive on the other disaccharides such as
maltose, lactose, trehalose, and cellobiose.
Furthermore, members of low DP fructooligosac-
charide, that is, 1-kestose, 1-kestose and nystose, 1-
kestose and fructosylnystose, did not act as sub-
strates of high DP inulin production (Table 3). Also,
in the enzyme reaction given sucrose as fructosyl
Fig. 4. Measurement of Average Degree of Polymerization of
Reaction Product.

Symbols: ( ), authentic inulin (Raftiline HP and Raftiline

ST); ( ), reaction product.
Fig. 5. HPAEC Analysis of Inulin on a CarboPack PA-1 Column.
EŒuent, 150 mmol l NaOH; Flow rate, 1 ml min; Injection, 25 l; Detector, pulsed amperometric detector.
m
W
W

A Novel Inulin-producing Enzyme of Bacillus sp.
Table 1. Sugar Composition of Inulin Treated with Inulinase
1333
Molar ratio to glucose
Glucose Fructose
Raftiline ST
Product
1
1
9
17
Glucose and fructose were measured by HPLC.
*
Table 2. Summary of Puriˆcation Procedures of IPE from
Bacillus sp. 217C-11
Total
Total
Speciˆc
Yield
Step
activity protein activity
(
z)
(U)
(mg)
(U mg)
W
Culture ˆltrate
Ultra ˆltration (30kDa)
3720
3534
3225
2780
990
466
349
375
97
26
12
8.0
10.1
9.2
28.7
38.1
53.3
100
95
87
75
27
17
70
z (NH4)2SO4 ppt.
DEAE-Toyopearl 650M
Toyopearl HW55S
Phenyl-Toyopearl 650M
Fig. 6. Homogeneity of Puriˆed IPE (inulin-producing enzyme)
and the Molecular Weight Measurement by SDS-PAGE.
Lane 1, puriˆed IPE; lane 2, standard protein mixture con-
640
*
IPE , Inulin- producing Enzyme.
taining myosin (Mw 200,000), b-galactosidase (Mw 116,248),
bovine serum albumin (Mw 66,267), aldolase (Mw 42,400),
carbonic anhydrase (Mw 30,000), and myoglobin (Mw 17,000).
*
Table 3. Properties of IPE from Bacillus sp. 217C-11
Optimum pH
pH stability
Optimum temp.
Theromostability
Molecular weight
Gel ˆltration
SDS-PAGE
7–8
6–9
459C
Acknowledgment
up to 409C
We thank Dr. Kawagishi of the Faculty of Agricul-
ture, Shizuoka University, Japan, for the NMR
assay.
44 kDa
45 kDa
＋
Inhibitor
Cu2＋, Hg2 (5 m
M)
Substrates GF
ª
ª
×
ª
×
ª
×
high DP inulin
high DP inulin
high DP inulin
high DP inulin
GF2
GF2
GF2
＋
＋
＋
GF2
GF3
GF4
References
1) Van Loo, J., Coussement, P., De Leenheer, L.,
Hoebregs, H., and Smits, G., On the presence of
inulin and oligofructose as natural ingredients in the
western diet. Crit. Rev. Food Sci. Nutr., 35, 525–552
(1995).
2) Praznik, W., and Beck, R. H. F., Application of gel
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matography, 348, 187–197 (1985).
3) Luscher, M., Erdin, C., Sprenger, N., Hochstrasser,
U., Boller, T., and Wiemken, A., Inulin synthesis by
a combination of puriˆed fructosyltransferases from
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39–42 (1996).
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(1968).
*
IPE , Inulin-producing Enzyme.
GF, sucrose; GF2, 1-kestose; GF3, nystose; GF4, fructosylnystose.
donor together with low DP fructooligosaccharides
as the fructosyl acceptor, the production of high DP
inulin was not recognized.
N-terminal amino acid sequence
The N-terminal amino acid sequence up to 30
amino acid residues of IPE was as follows: EEIN-
SDYTSIWSRQQAEKVTP TDKTTAPKI. A com-
parison was made with the corresponding regions of
other proteins using two data bases (FASTA and
BLASTP), but no similarity to this sequence was
found. A levansucrase derived from Clostridium
acetobutylicum had a homologous arrangement
5) KopeloŠ, N., KopeloŠ, L., and Welcome, C. J.,
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(59
z). This enzyme can produce levan ( b-(2ª6)
fructan) from sucrose. This is a very interesting fact.
In conclusion, our results suggest that our enzyme,
i.e., the inulin-producing enzyme (IPE) of Bacillus
sp. 217C-11, is a novel one unlike others found so
far. The mechanism of inulin synthesis by this
enzyme is being investigated.
formation and in‰uence of reaction. J. Biol. Chem.
,
43, 171 (1920).
6) Loewenberg, J. R., and Reese, E. T., Observations
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Microbiol., 3, 643–650 (1957).

1334
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14) Bradford, M., A rapid and sensitive method for the
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