World

Journal

of Microbiology

and

Biotechnology

7, 331334

Production of fructooligosaccharides
from sucrose by a transfructosylase
Aspergillus
niger

Y.K.

A straln of Aspergiiius
niger isolated
from
sugarcane
fields, produced
an extracellular
transfructosylase
In the culture
medium.
Sucrose
and raffinose
Induced the productlon fo the enzyme,
which was purified
by
133fold.
The optlmum
pH for activity
and
stablllty
were 5.5 and 6.5, respectively.
Its
optimum
temperature
was 55C. The enzyme
hydrolysed
sucrose
rapidly
and
simultaneously
formed
fructoollgosaccharIdes by transfructosylatlon.
The authors
are with the University
of
Campinas,
College
of Food Engineering,
Laboratory
of Food
Biochemistry
(UNICAMP),
Caixa postal 6121, Campinas,
SP,
Brazil.

Park

Communications

of Oxford

Ltd.

Almeida

Neosugar (or fructooligo-sugar)

is fructooligosaccharides
and non-nutritive
sweetener, which is composed of sucrose attached in a &2-1)linkage
to 2, 3 or 4
fructose units. Resulting structures are designated as I-kestose (GF,), nystose
(GF,) and If-fructofuranosyl
nystose (GF,) (Dziezak
1986). These sugars are
produced by action of fungal enzyme on sucrose.
Previously, the mechanism of action of transfructosidase
of Aspergikr
or_yqye
was studied with regard to its involvement
in the formation of fructooligosaccharides from sucrose (Pazur 1952). Hydrolysis
of sucrose by extracts of Penicillium
spinulosm is initially rapid and is accompanied by the formation of non-reducing
fructooligosaccharides.
The hydrolysis of these oligosaccharides
proceeds by the
transference of fructosyl units to water and leads eventually to the complete
hydrolysis of sucrose. Transfructosidase
and invertase activities are due to the
same enzyme, P-fructofuranosidase
(Bealing & Bacon 1953; Bealing 1953). Fructooligosaccharides
were also produced
by a mycelium
extract from Fusarium
oxysporum and Aureobasidim
pnbklan (Gupta & Bhatia 1980; Jung et al. 1989).
Formation
of fructooligosaccharides
was also investigated
using cell suspension
of various fungi: Aspergillu
niger ATCC 20611 produced higher transfructosidase
activity than hydrolysing
activity (Hidaka et al. 1988) and the intracellular
enzyme
was subsequently purified and characterized
(Hirayama et al. 1989).
We have examined a number of fungi from sugarcane fields for production
of
transfructosidase
activity and found that one strain of A. niger produced
the
highest enzyme activity in the culture medium. The purpose of this present
research was to purify and characterize the extracellular
enzyme from this newly
isolated strain.

Materials

@ 1991 Rapid

and M.M.

from

and Methods

Reagents
All chemical reagents were obtained from either Sigma or Merck. Analytical
standards of 1-kestose (GF,), nystose (GF,) and If-fructofuranosyl
nystose (GF,)
were obtained from Daiichi Chemical Co (Tokyo, Japan).

World

Journal

of Microbiology

and

Biotechnology,

Vol 7, 1991

331

Y. K.

Park

and

M. M. A lmeida
Etqyme A ssays
The supernatant
of the culture was used as a source of enzyme but had to be
diluted with water. (0.5 ml of Enzyme solution was mixed with 4.5 ml of 4%
sucrose (w/v) in 0.05 M citrate/phosphate
buffer pH 6, and incubated at 55C for
30 min. Glucose, fructose, and I-kestose were then determined
by high perfor0
ne
unit
of
the
activity
of
transfructosylamance liquid chromatography
(HPLC).
tion was defined as the amount of enzyme activity which catalyses the formation
of 1 pmol glucose per min and one unit of hydrolytic activity was defined as the
amount of enzyme which catalyses the formation of 1 pmol fructose per min under
these conditions.
Ana&% of Stigars
Qualitative
analysis of sugars was performed
by paper chromatography
using
Whatman No. 1 paper developed with pyridine/butanol/water
(4:6:3). Sugars
were detected by spraying silver nitrate (Trevelyan
et al. 1950). Quantitative
analysis of sugars was performed by HPLC equipped with a differential refractometer detector using a Cl8 column. The developing solution was acetonitrile/water
(75:25 v/v) with a flow rate of 1 ml/min. The samples were filtered through a
membrane filter before injection.
Screening of Micro-organisms
Nine hundred strains of fungus were isolated from sugarcane fields and waste
water from sugar plants using potato dextrose agar (PDA). Each strain was
cultivated in 20 ml of medium in 125 ml Erlenmeyer
flasks at 30C for 3 days
with shaking at 250 rev/min. The liquid culture medium contained 5 g sucrose,
1 g yeast extract, 1 g peptone, and 0.3 g NaCl per 100 ml water. After incubation,
the culture medium was centrifuged
and the cells were washed 3 times by
suspension in deionized water and centrifuging.
Finally, the cells were suspended
in 20 ml of deionized water. Supernatants and cell suspensions were examined for
transfructosylation
activity. One of the strains that produced the highest enzyme
activity in the culture medium was identified as A. niger. This strain was used in
the following
studies.
Effect of Carbohydrates on Production of Eqyme
One ml of a spore suspension (lO/ml) of A. niger was placed in 100 ml of culture
medium in which sucrose was replaced by equal amounts of other sugars (Table
1) and incubated as described above. After incubation,
the enzyme activities were
determined.

Table 1. Effect of different

carbohydrates
production
of translructosylase
activity.
Carbohydrate

Enzyme
activity
(unitlminlml)

Sucrose
Raffinose

3.0
2.4
0.2
0.1
0.2

Glucose
Fructose
Maltose
Lactose
Xylose
Mannose

0.2
0.1
0.1
0.1

Galactose

332

on

World

Journal

of Microbiology

and

Production of Extra- and Intracelhlar

Enzymes and Ptirifcation
The supernatant
solution (900 ml containing
6600 units of transfructosylase
activity) was prepared as mentioned above and used as extracellular
enzyme. The
intracellular
enzyme from 150 g (wet) of mycelia was prepared by packing the
washed cell mass with nylon cloth, pressing to extract more water and crushing
with glass powder. The crushed mycelia were extracted with 0.05 M citrate/phosphate buffer, pH 6.0. The mixture of the extracts was centrifuged
and the
supernatants (118 ml) were dialysed against the same buffer solution. Total activity
of intracellular
transfructosylase
was 2924 units. Proteins in 900 ml supernatants
(extracellular
enzyme) were fractionated with ammonium
sulphate (80% saturation). The precipitates,
obained by centrifugation,
were successively dialysed
against deionized water and 0.05 M citrate/phosphate
buffer, pH 5.0. The dialysates
were further purified by DEAE-cellulose
and CM-cellulose
column chromatography. The columns were equilibrated
with 0.05 M citrate/phosphate
buffer,
pH 5.0, and elution was carried out with a concentration
gradient of NaCl (0.1
to 0.9 N).

Biotechnology,

Vol 7, 1991

Fructooligosaccharider
Table

2. Purlflcation

of extracellular

enzyme

of A. nlger.

Protein

Enzyme
activity
(U/min/ml)

Wmf)

Transfructosylation
Mycelium
extracts
(intracellular
enzyme)
Supernatant
solution
(extracellular
enzyme)
DEAE-cellulose
column
chromatography
CM-cellulose
column
chromatography
T/H-ratio

of transfructosylase

Specific enzyme
activity
(Ulminlmg
protein)
Hydrolysis

Transfructosylation

T/H

Hydrolysis

330

25

75

14

1800

26

212

34

10

550

88

activity

to hydrolytic

activity.

Production of Fructooligosaccbarides by the Purifed Enzyme

One ml of the purified enzyme (6 units of transfructosylase)
was added to 9 ml
of sucrose substrate to give final concentrations
of 10, 30 and 60% in 0.05 M
citrate/phosphate
buffer, pH 6, and incubated at 55C for 80 h. The time course
of enzymatic reaction was followed by HPLC analysis.

Results

and Discussion

The results (Table 1) show that the newly isolated strain produced a large quantity
of extracellular
transfructosylase
as compared to the intracellular
enzyme when
either sucrose or raffinose was added to the culture medium as a carbon source.
This indicates that transfructosylase
is an inducible enzyme.
Purification of the enzyme (Table 2) led to a 138-fold purification of transfructosylase and an 88-fold purification
of hydrolytic activity. The ratio of transfructosylase activity to hydrolytic activity (T/H) was 5 for crude mycelia extract, whereas
the ratio in the supernatant solution from the culture medium was 4. These data
indicate a lower fructose transfer activity in the supernatant. However, the purified
extracellular
enzyme had the highest T/H ratio of 6. These results suggest that
an interfering
factor for enzymatic transfructosylation
was removed during the
enzymatic purification.
The purified enzyme had an optimum
pH of 5.5 to 6.5 and was also stable
between 5.5 and 6.5. The optimum temperature
for activity was 55C.
Fructooligosaccharides
were produced by the purified enzyme (Figure 1) and
identified as I-kestose (GF,), nystose (GF,) and l-fructofuranosyl
nystose (GF,),
as well as sucrose, glucose and fructose. Time course of enzymatic reaction
demonstrated
an initial rapid hydrolysis of sucrose (77 to 86% hydrolysis) in the
reaction mixture, accompanied
by a simultaneous
formation
of mainly GF,.
During hydrolysis of sucrose, fructosyl units were transferred to sucrose to form
trisaccharides
and glucose was liberated
simultaneously.
After the maximum
concentration
of GF2 was reached, the trisaccharides slowly decreased by transfer
of fructose units on to them. With higher concentrations
of sucrose (30 and 60%),
about 0.5 to 0.7% of GF, was formed by enzymatic fructose transfer.
These results also demonstrate that more free fructose was liberated in reaction
mixtures containing low sucrose concentrations.
Thus, hydrolysis of sucrose and
formation of fructooligosaccharides
occurred by the mechanism of transfructosylation. Sucrose and fructooligosaccharides
may act as fructose donors and simultaneously, sucrose, fructooligosaccharides
and water act as acceptors for fractose.
Therefore,
the purified
enzyme should classify as P-fructofuranosidase
(EC
3.2.1.26). Previously,
Bealing & Bacon (1953) reported that fungal and yeast

World

Journal

of Microbiology

and

Biotechnology,

Vol 7, 1991

333

Park: and M.M.

Almeida

10% SUCROSE

30% SUCROSE

60%

ENZYMATIC
Figure 1. Production
of fructooligosaccharides
1-kestose;
GF,-nystose;
GF4-l-fructofuranosyl

from sucrose
nystose.

SUCROSE

REACTION TIME. h

by purified

enzyme

from

A.

niger.

G-glucose;

F-fructose;

S--sucrose;

GF,-

invertases are /?-fructofuranosidase

but differ in their specificity toward fructose
acceptors. Hidaka et al. (1.988) reported that yeast invertase has more hydrolytic
activity whereas fungal enzyme has more transfructosylase
activity.

References
BEALING,
F. J. 1953 Mould glucosaccharase: a fructose. Biochemical Journal 55, 9S-101.
BEALING,
F.J. & BACON, J.S.D. 1953 The action of mould enzymes on sucrose. Biochemical
Journal 53, 277-285.
DZIEZAK,
J.D. 1986 Special report: Sweeteners and product development. Food Technology
40,111-128.

GUPTA, A.K. & BHATIA, T.S. 1980 Glucofructosan

Pbytocbemistty

biosynthesis in Ftisarium oxysporum.

19, 2557-2563.

HIDAI<A, H., HIRAYAMA, M. & SUMI, N. 1988 A fructooligosaccharide-producing

enzyme
from Aspergiihs
niger ATCC 20611. Agricultural
and Biological Chemistrj, 52, 1181-1187.
HIRAYAMA, M., SUMI, N. & HIDAKA, H. 1989 Purification and properties of a fructooligosaccharide-producing
j?-fructofuranosidase from A spergilhs niger ATCC 20611. Agricultural

and Biological

Chemistry

53, 667-673.

JUNG, K.H., YLJN, J.W., KANG, K.R., LIM, J.Y. & LEE, J.H. 1989 Mathematical model
for enzymatic production of fructooligosaccharides from sucrose. Envme and Microbial
Technology

11, 491-494.

PAZUR, J.H. 1952 Transfructosidation

of Biological

Chemistry

reactions of an enzyme of Aspergihs

oyxae.

JournaL

199, 217-225.

TREVELYAN, W.E., PROCTER, D.P. & HARRISON, J.G. 1950 Detection of sugars on paper
chromatograms. Nature 166, 444445.

(Received

334

World

Journal

of Microbiology

and

Biotechnology,

13 September

Vol 7, 1991

1990;

revised

20 November

1990;

accepted

3 December

1990)