Production of Fos From Sucrose

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World Journal of Microbiology and Biotechnology 7, 331334

A straln of Aspergiiius niger isolated from

sugarcane fields, produced an extracellular

transfructosylase In the culture medium.

Sucrose and raffinose Induced the produc-

tlon 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 en-

zyme hydrolysed sucro se rapidly and

simultaneously formed fructoollgosacchar-

Ides by transfructosylatlon.

The authors are with the University of

Campinas, College of Food Engineering,

Laboratory of Food Bioche mistry (UNI-

CAMP), Caixa postal 6121, Campinas, SP,

Brazil.

Production of fructooligosaccharides

from sucrose by a transfructosylase from

Aspergillus niger

Y.K. Park and M.M. Almeida

Neosugar (or fructooligo-sugar) is fructooligosaccharides and non-nutritive sweet-

ener, 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.

Previous ly, the mechanism of action of transfructosidase of Aspergikr or_yqye

was studied with regard to its involvement in the formation of fructooligosacchar-

ides 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 fructosy l units to water and leads eventually to the complete

hydrolysis of sucrose. Transfructosidase and invertase activit ies are due to the

same enzyme, P-fructofuranosidase (Bealing & Bacon 1953; Bealing 1953). Fruc-

tooligosaccharides 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

act ivi ty than hydrolysing act ivit y (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 act ivi ty 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 puri fy and characterize the extracellular enzyme from this newly

isolated strain.

Materials and Methods

Reagents

@ 1991 Rapid Comm unications of Oxford Ltd.

All chemical reagents were obtained from either Sigma or Merck. Analyt ical

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

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Y. K. Park and M. M. A lmeida

Table 1. Effect of different carbohydrates on

production of translructosylase activity.

Carbohydrate Enzyme activity

(unitlminlml)

Sucrose 3.0

Raffinose 2.4

Glucose 0.2

Fructose

0.1

Maltose

0.2

Lactose 0.2

Xylose 0.1

Mannose 0.1

Galactose

0.1

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 perfor-

mance liquid chromatography (HPLC).

0 ne unit of the activity of transfructosyla-

tion was defined as the amount of enzyme activity which catalyses the formation

of 1 pmol glucose per min and one unit of hydrolytic act ivi ty 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 refractom-

eter 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 centri fuging. Fina lly, the cells were suspended

in 20 ml of deionized water. Supernatants and cell suspensions were examined for

transfructosylation act ivi ty. One of the strains that produced the highest enzyme

act ivi ty 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.

Production

of

Extra- and

Intracelhlar

Enzymes and Ptirifcation

The supernatant solution (900 ml containing 6600 units of transfructosylase

acti vity ) 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/phos-

phate buf fer, pH 6.0. The mixture of the extracts was centrifuged and the

supernatants (118 ml) were dialysed against the same buffer solution. Total act ivi ty

of intracellular transfructosylase was 2924 units. Proteins in 900 ml supernatants

(extracellular enzyme) were fractionated with ammonium sulphate (80 satura-

tion). The precipitates, obained by centrifugation, were successive ly 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 chromato-

graphy. 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).

332

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Fructooligosaccharider

Table 2. Purlflcation of extracellular enzyme of A. nlger.

Protein Enzyme activity Specific enzyme activity T/H

Wmf)

(U/min/ml) (Ulminlmg protein)

Transfructosylation Hydrolysis Transfructosylation Hydrolysis

Mycelium extracts

(intrac ellular enzyme) 330 25 5 75 14 5

Supernatant solution

(extracellular enzyme) 1800 7 2 4 1 4

DEAE-cellulose column

chromatography 26 6 1 212 34 6

CM-cellulose column

chromatography 10

6

1

550 88 6

T/H-ratio of transfru ctosylase activity to hydrolytic activity.

Production of Fructooligosaccbarides by the Purifed Enzyme

One ml of the purified enzyme (6 units o f 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 transfructo-

sylase and an 88-fold purification of hydrolytic act ivi ty. The ratio of transfructosy-

lase act ivi ty to hydrolytic act ivit y (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 act ivi ty 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 act ivi ty 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 hydrolys is) 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 b y 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 b y the mechanism of transfructosyla-

tion. Sucrose and fructooligosaccharides may act as fructose donors and simultan-

eously, sucrose, fructooligosaccharides and water act as acceptors for fractose.

Therefore, the purified enzyme should classif y as P-fructofuranosidase (EC

3.2.1.26). Previously, Bealing & Bacon (1953) reported that fungal and yeast

Word Journal of Microbiology and Biotechno logy, Vol 7, 1991

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Production of Fos From Sucrose