Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502 Vol. 3(ISC-2013), 147-153 (2014) Res. J. Recent. Sci. International Science Congress Association 147 Study on Fructosyltransferase enzyme from Aspergillus sp. in Fructooligosaccharides production Arthee R and Vijila K Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, INDIAAvailable online at: www.isca.in Received 2nd December 2013, revised 11th February 2014, accepted 26th March 2014 AbstractThe fructosyltransferase intra- and extra- cellular enzyme preparations obtained from Aspergillus sp. was used to produce fructooligosaccharides by enzymatic conversion of sucrose. The crude enzyme preparation of Aspergillus sp. exhibited fructosyltransferase activity of 5.0 U.mg-1 at 60 per cent sucrose concentration. The specific activity was recorded at its highest rate at the substrate concentration of 40 per cent (9.05 U.min-1.mg-1 of protein). The high performance liquid chromatography analysis of the end products of fructosyl enzyme activity on sucrose had shown the formation of, 1-kestose and nystose along with glucose, fructose and unhydrolyzed sucrose. The enzyme activity was stable at a temperature of 52°C and at a pH of 4.5. The activity of enzyme was enhanced by addition of 1 mM FeSO (7.1 U.min-1.mg-1 of protein) and also by addition of Fe2+ and its combination with Ca2+ (9.2 U.min-1.mg-1 of protein). Susceptibility to detergents was observed. The selectivity of conversion of sucrose to fructooligosaccharides obtained by the enzyme was approximately 70 per cent under optimized conditions. The partially purified fructosyltransferase preparation from Aspergillus sp. is found applicable for industrial production of fructooligosaccharides. Keywords: 1-Kestose, Nystose, Fructosyltransferase, HPLC, Parameter optimization. Introduction The design of food products that confer a health benefit is relatively a new trend and recognizes the growing acceptance of the role of diet in disease prevention, treatment and well-being. Consumers believe that foods contribute directly to their health. So an increasingly active population, the pursuit of healthier lifestyles and the desire to live longer has risen to a new category of food products referred to as “Nutraceuticals”. The term “Nutraceuticals” was coined from “nutrition” and “pharmaceutical” in 1989 by Stephen Defelice and was originally defined as a wide range of food and food components with a claimed medical or health benefits. Nutraceuticals can come from plant, marine, animal and microbial sources; specifically the food products include whole foods, food additive, probiotics, vitamins and mineral. A lot of attention is being paid to dietary carbohydrates, especially on the various kinds of oligosaccharides. In the current context of functional foods, it generates a global market in which oligosaccharides could play a major role as functional ingredients compared to dietary fibers, sugar alcohols, peptides, probiotics, polyunsaturated fatty acids and antioxidants. The generic term “oligosaccharides” is customarily used for saccharides having the degree of polymerization of 2-10 and are found either free or in combined forms in all living organisms as polymeric carbohydrates. Structurally, oligosaccharides are composed of 2-10 monosaccharide residues linked by glycosidic bonds that are readily hydrolysed to their constituent monoaccharides either by acids or by specific enzymes. Oligosaccharides are functional food ingredients, they have a great potential to improve the quality of many foods. In this way, a compound named as fructooligosaccharides (FOS) coming under the category of sucrose related oligosaccharides has emerged as one of the important product in the functional food market. They are naturally occurring sugars that has beneficial effects as food ingredient. FOS act as prebiotic which represent selective nutrient for beneficial microorganisms and they have the potential to increase the effectiveness of probiotic products. Furctooligosaccharides consist mainly of 1-kestose, nystose and fructofuranosyl nystose. Fructosyltransferase (FTase) (E.C. 2.4.1.9) catalyze the formation of FOS from sucrose, which have broad applications in the food and pharmaceutical industries. Several microorganisms have been reported to posses FTase activity and produce FOS from sucrose. The characterization of the enzyme was undertaken as a necessary step towards understanding the properties of the FTase enzyme. The activity of the FTase enzyme obtained from the strain Aureobasidium pullulans (CFR 77) was assayed under pre-determined conditions by incubating the FTase enzyme with sucrose (60%w/v). The activity of the enzyme was determined based on the amount of enzyme required to liberate 1 micromole of glucose under the specified conditions . The fructosyltransferase enzyme from Rhodotorulasp. showed a rather sigmoid cooperative type behavior similar to that expressed by the Hill’s model with a Vmax value of 236.1 mol.ml-1 8 A behaviour associated with the ping-pong mechanism for fructosyltransferase produced by Aspergillus sp. N74 leading to the highest sucrose bioconversion at 24 h of Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 147-153 (2014) Res. J. Recent. Sci. International Science Congress Association 148 reaction (84 per cent w/v) was reported. Batch fructooligosaccharides production was done and obtained the yield of 69 per cent (43 per cent kestose and 26 per cent nystose). Fructooligosaccharides production of 51.8 per cent kestose and 74.6 per cent nystose in the batch culture mode and 102.4 g.l-1 for 1-kestose and 59 g.l-1 for nystose by two step batch culture mode using fructosyltransferase from Aureobasidium sp. ATCC 20524 was reported10. The fructooligosaccharides are called as, ‘non digestable food ingredients’ that beneficially affects the host health by selectively stimulating the growth and activity of one or a limited number of bacteria in the colon. The low calorie sweetner like fructooligosaccharides has an important role in control of diabetes. They also prevent the colonization of human gut by pathogenic microorganisms because it encourages the growth of beneficial bacteria. Dietary treatment with FOS significantly potentiated the effects of sub therapeutic doses of cytotoxic drugs commonly utilized in human cancer treatment. On optimizing the conditions for higher enzyme activity, we achieve maximum FOS production and also maximum conversion of sucrose substrate into product. The substrate concentration, at which there is maximum enzyme activity and minimum substrate inhibition, is optimized. Material and Methods All chemicals, reagents and enzyme were of analytical grade. HPLC grade chemicals obtained from Sigma were used for chromatographic studies. Borosilgrade glasswares were used in all the experiments. The Aspergillus sp. obtained from fermented jaggery was developed for fructooligosaccharides production in the previous studies. The crude protein extract of Aspergillus sp. exhibited fructosyltransferase activity, producing fructooligosaccharides from sucrose. The culture was stored and propagated in potato dextrose agar to be used as the inoculum for further experiments. The fungal isolate Aspergillus sp. was developed by transferring 1 ml of spore suspension in to 100 ml Erlenmeyer flask containing 50 ml of pre-inoculum medium (sucrose: 1per cent; yeast extract: 0.2 per cent, pH 5.50). Development of fungal inoculum in fermentation medium: The 36 h old pre-inoculum at 20 per cent (v/v) was transferred to 100 ml of fermentation medium (Sucrose - 20 g.l-1 ,Yeast extract – 5 g.l-1, Sodium nitrate – 1 g.l-1, Potassium hydrogen orthophosphate – 0.5 g.l-1, Dipotassium hydrogen orthophosphate – 0.25 g.l-1, Sodium chloride – 0.25 g.l-1, Magnesium sulphate – 0.05 g.l-1, Ammonium chloride – 0.05 g.l-1, pH – 5.5) prepared in 250 ml Erlenmeyer flasks 11. The culture flasks and uninoculated fermentation medium were incubated at 30 ± 1C in environment incubator shaker (New Brunswick Scientific, USA) at 250 rpm. An uninoculated fermentation medium served as control. Preparation of crude protein extract by ammonium sulphate precipitation: At the end of 48 h of incubation, the culture broths were centrifuged under 6000 g at 4C for 30 min in refrigerated centrifuge (KUBOTA 6800) to separate the biomass from the culture broth. The clear cell free extract thus obtained was used for further assay. The cell free extract and the mycelial / cell extract were pooled and saturated with 30, 40, 50, 60 and 70 per cent ammonium sulphate (AR grade). The precipitate obtained from each fractionation was dissolved in a minimal amount of phosphate buffer (0.2 M, pH 7.0) and used as crude enzyme source for fructosyltransferase assay. Fructosyltransferase enzyme assay: The crude protein extract obtained was used as an enzyme source and fructosyltransferase activity was assayed under the predetermined conditions as suggested11. The substrate sucrose solution was prepared at various concentrations, 20–80 per cent (w/v) (58mM–234mM) in 0.1 M citrate buffer (pH 5.0). The reaction mixture consisted of 250 µl of substrate and 250 µl of enzyme source. The reaction mixture was placed at 55C in shaking water bath (JULABO SW20) at 100 rpm for 1 h. After the period of incubation the reaction mixture was heated at 90C for 15 min to arrest the reaction. The amount of glucose released at the end of the reaction was determined by dinitrosalicylic acid assay12. In case of fructosyltransferase enzyme activity one unit was defined as the amount of enzyme required to liberate 1 µ mole of glucose per minute. Detection of fructooligosaccharides by high performance liquid chromatography: High performance liquid chromatography was used to quantify the fructooligosaccharides synthesized by fructosyltransferase activity in the reaction products at various substrate concentrations13. The fructooligosaccharides present in the samples was analyzed by polar bonded phase high performance liquid chromatography (Shimadzu, Japan). A sample volume of 20 µl was injected into the amino column with a mobile phase of acetonitrile and water in 60:40 as binary gradient at a flow rate of 1.0 ml.min-1. The sample was run for 10 min. The saccharides were identified by comparing retention times with those of known standards viz., glucose, fructose, sucrose, kestose and nystose (HPLC grade, Sigma). Analysis of yield of fructooligosaccharides: The yield of fructooligosaccharides, which was the measure of enzyme activity, was calculated14. The selectivity of conversion from sucrose to fructooligosaccharides (FOS) was calculated from the following formula, 2c (GF) + 3c (GF) S (FOS) = _____________________ 2c (GF) + 3c (GF) + c(F) where c (F) is the molar concentration of fructose. Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 147-153 (2014) Res. J. Recent. Sci. International Science Congress Association 149 Determination of optimal temperature and pH for enzyme activity: This experiment was conducted to find out the temperature and pH for maximum enzyme activity. The fructosyltransferase activity on sucrose was determined by incubating the enzyme (15 U.ml-1) at temperatures of 53C, 54C, 55C, 56C and 57C in medium consisting of sucrose (200 – 800 g.l-1) in 0.1 M citrate buffer (pH 5.0) and enzyme preparation in 0.2 M phosphate buffer (pH 7.0). The fructosyltransferase activity was studied at pH values from 4.0-8.0. The 0.1 M citrate buffer was used to prepare sucrose with pH values of 4.0, 5.0 and 6.0 whereas substrates of pH 7.0 and 8.0 were prepared using 0.2 M phosphate buffer. The enzyme solution was maintained at 55C in each buffer. The fructosyltransferase activity was determined by the fructosyltransferase enzyme assay procedure. Effect of metal ions on fructosyltransferase activity: The metal ions were added as NaCl, CaCl, CuSO and FeSO at concentration of 1 mM to the enzyme and incubated for 60 min. Upon incubation the hydrolytic activity was measured by the standard assay with sucrose at a concentration of 1.76 M. Effect of detergents on fructosyltransferase activity: The detergent susceptibility was assessed by incubating the crude fructosyltransferase enzyme with polyethylene glycol (5 per cent w/v), tween 20 (5 per cent w/v) and tween 80 (5 per cent w/v) in 0.2 M sodium acetate buffer (pH 5.5). Upon incubation an aliquot was withdrawn, chilled on ice and the fructosyltransferase activity from 1.76 M sucrose concentration was measured by the enzyme assay. Statistical analysis: The experimental set up in all the investigations was duplicated and three samples were taken for analysis. Statistical analysis was done in completely randomized block design 15 Results and Discussion Results: The isolate Aspergillus sp. when grown for 48 h in 100 ml of fermentation medium yielded a mycelial biomass of 0.4 g dry weight. In the ammonium sulphate protein extract, the quantity of protein estimated was in the range of 3 to 5.0 mg. The maximum fructosyltransferase activity exhibited in the crude enzyme extract was 9.05 U.mg-1 in the reaction conditions of 250 µl of substrate sucrose at 40 per cent (w/v) concentration in 0.1 M citrate buffer (pH 5.0) and 250 µl of enzyme source (table 1). Table-1 Activity of fructosyltransferase from Aspergillus sp. at various sucrose concentrationsS. No. Sucrose concentration (mM) Total activity (U. min - 1 ) Specific activity (U. min - 1 . mg - 1 of protein) 1 58 (20) 65.90 7.01 2 88 (30) 74.17 7.89 3 116 (40) 85.10 9.05 4 146 (50) 71.95 7.65 5 176 (60) 61.05 6.49 6 204 (70) 54.60 5.81 7 234 (80) 48.50 5.16 Mean 65.64 6.98 CD (0.05) 3.50 0.37 *Values in paranthesis indicate percent substrate (sucrose) concentration, U – amount of enzyme required to liberate 1 mole of glucose Table-2 Fructosyltransferase activity and fructooligosaccharides production at various sucrose concentrations by Aspergillus sp.S. No. Sucrose concentration (mM) Total activity (U. min-1) Specific activity (U. min - 1 . mg-1 of protein) FOS concentration (g.l -1) 1 58 (20)* 60.70 6.89 75 2 88 (30) 72.07 7.45 90 3 116 (40) 84.10 8.90 110 4 146 (50) 70.95 7.15 85 5 176 (60) 60.05 6.19 67 6 204 (70) 53.60 5.61 45 7 234 (80) 47.50 5.06 31 Mean 63.64 6.98 73.43 CD (0.05) 3.50 0.37 2.72 *Values in paranthesis indicate percent substrate (sucrose) concentration, U – amount of enzyme required to liberate 1 mole of glucose Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 147-153 (2014) Res. J. Recent. Sci. International Science Congress Association 150 The total activity and specific activity recorded in different sucrose concentrations were given in table-1. The reaction products of crude fructosyltranferase enzyme from Aspergillus sp.AFJ3 with sucrose concentrations, 58 mM – 234 mM (200 g.l-1 – 800 g.l-1) were analyzed by high performance liquid chromatography for the detection of sugars (figure-1) formed from sucrose due to fructosyltransferase enzyme activity. The sugars formed from sucrose concentrations 58 mM – 234 mM (20-80 per cent) presented in table-2 shows fructooligosaccharides production at all sucrose levels. The rest of carbohydrates in the mixture were fructose, glucose and remaining sucrose. The chromatogram also revealed the presence of sugars with high polymerization. The enzyme activity at various sucrose levels (58 mM – 234 mM) and temperature (53ºC, 54ºC, 55ºC, 56ºC and 57ºC) on frutosyltransferase enzyme activity was determined and the results are presented in figure-2. The fructosyltransferase activity was maximum (8.50 U.min-1.mg-1 of protein) at 55ºC when incubated with 116 mM of sucrose. The activity of fructosyltransferase was minimum (4.40 U.min-1.mg-1 of protein) at 53ºC when incubated with 234 mM of sucrose. Figure-1 Chromatogram of sugars formed by fructosyltransferase activity of Aspergillus sp., from sucrose by high performance liquid chromatography 105354555657Temperature (C)Specific activity (U. min-1.mg-1 of protein) 58mM 88mM 116mM 146mM 176mM 204mM 234mMU – amount of enzyme required to liberate 1 mole of glucose Figure-2 Effect of temperature on fructosyltransferase activity of Aspergillus sp. AFJ3 at various sucrose concentrations Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 147-153 (2014) Res. J. Recent. Sci. International Science Congress Association 151 – amount of enzyme required to liberate 1 mole of glucose Figure-3 Relationship of fructosyltransferase activity and pH at various sucroseconcentrationsThe influence of temperature and pH on the fructosyltranferase activity was studied for substrate sucrose levels at pH of 4.0, 5.0, 6.0, 7.0 and 8.0. The enzyme was stable at temperature of 52°C and in the pH of 4.5. According to the results shown in figure-3, the maximum enzyme activity of 9.30 U.min-1.mg-1 of protein was recorded at pH 4.0 when incubated at sucrose concentration of 116 mM. The crude fructosyltransferase enzyme displayed activity using sucrose as substrate (176 mM) even in the absence of metal ions (table 3). Of those tested, only Fe2+ added in the form of FeSOincreased the fructosyltransferase activity by 13.5 per cent (7.1 U.min-1.mg-1 of protein). Most of the ions tested produced an inhibitory effect. The combination of metal ions Fe2+ and Ca2+added in the form of FeSO and CaCl respectively, had a positive effect on fructosyltransferase activity by 28.6 per cent (9.2 U.min-1.mg-1of protein). The effect of detergents on fructosyltransferase activity is shown in (table 4). None of the detergents tested exhibited positive effect. Discussion: Fructooligosaccharides have enormous functional properties that make them commercially interesting for applications in pharmaceutical and food industries. In the human digestive tract, fructooligosaccharides are almost exclusively fermented by bifidobacteria and lactobacilli which have beneficial health effects. Sucrose can be converted in to fructooligosaccharides by using a range of transfructosylating enzymes originating from plants, bacteria and fungi. Fructooligosaccharides synthesis has been reported for the commercially important fungus Aspergillus sp. as a result of the specific fructosyltransferase enzyme activity 16. The fungi of the genera Aspergillus, Fusarium, Aureobasidium and Penicillium have been described as good producers of fructooligosaccharides with potential for industrial processes 17. Fructooligosaccharides producing fungal strain, Aspergillus sp. N74 was isolated from sugarcane crop18. The use of sugar by-products as substrates for growing fructooligosaccharides producing fungi also have been reported 19. The chromatographic profile of the reaction product from Aspergillus sp. AFJ3 revealed the presence of sucrose, fructose, glucose and fructooligosaccharides namely, kestose and nystose. This shows the presence of transfructosylation activity in enzymatic product. Presence of sugar polymers were also detected in the enzymatic product chromatographic profile. The maximum fructooligosaccharides production was found at 40 per cent initial sucrose concentration. The fructooligosaccharides produced were identified as kestose and nystose. Presence of glucose, fructose and residual sucrose were also found. It is understood that the lower fructooligosaccharides yield above 40 per cent sucrose concentration may be due to poor hydrolysis of substrate at high sucrose concentrations. Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 147-153 (2014) Res. J. Recent. Sci. International Science Congress Association 152 Table-3 Effect of metal ions and EDTA on fructosyltransferase activity S. No. Metal ion/ Chelator Total activity (U. min - 1 ) Specific activity (U. min - 1 . mg - 1 of protein) 1 NaCl 30.15 3.21 3 CaCl 2 54.71 5.82 7 CuSO 4 26.85 2.86 8 FeSO 4 74.20 7.1 12 FeSO 4 + CaCl 2 88.60 9.2 13 Control 61.05 6.49 Mean 41.55 4.39 CD (0.05) 1.56 0.19 – amount of enzyme required to liberate 1 mole of glucose Table-4 Effect of detergents on fructosyltransferase activityS. No. Detergent Total activity (U. min - 1 ) Specific activity (U. min - 1 . mg - 1 of protein) 1 Polyethylene glycol 27.55 1.12 2 Tween 20 25.55 2.10 3 Tween 80 29.40 2.12 7 Control 61.05 6.49 Mean 29.10 3.12 CD (0.05) 1.04 0.15 – amount of enzyme required to liberate 1 mole of glucose The fructosyltransferase activity of crude enzyme from Aspergillus sp. AFJ3 was maximum (8.99 U. min-1.mg-1 of protein) at 55°C and minimum at 53°C. A large number of reports have described the temperature ranges of microbial fructosyltransferase as 50-60°C. The results were found to be similar with that of the enzyme from Aspergillus niger which exhibited an optimal activity at 50-60°C . Temperatures greater than 65°C inactivated the fructosyltransferase enzyme from Aspergillus aculeatus20. The crude fructosyltransferase from Aspergillus sp. AFJ3 exhibited maximum enzyme activity at pH 4.0 irrespective of the substrate sucrose concentration. Production of fructooligosaccharides from sucrose by the enzyme preparation was optimal at pH 5.0 - 6.0. Similarly fructosyltransferase from other fungi are most active in the pH range of 5.0 - 6 .0 7, 21, 22. The activity of fructosyltransferase from Aspergillus aculeatus was elicited 1.4-1.9 fold by ions like Mn2+, K and Co2+ whereas the activity showed inhibition at low concentration of Hg2+ and Zn2+. The addition of ethylene diamine tetraacetic acid (EDTA) did not result in a decrease in activity, indicating that the fructosyltransferase activity was independent of the divalent ions 20. Conclusion In the present study, the fructosyltransferase specific activity exhibited in the crude protein extract of Aspergillus sp. from sucrose was 5.0 U.mg-1 of protein. The chromatographic profile of the cell free extract from Aspergillus sp. revealed the presence of sucrose, fructose, glucose and fructooligosaccharides namely, kestose and nystose. Presence of sucrose, fructose, glucose, kestose and nystose were also detected in the enzymatic product. This shows the presence of transfructosylation activity in both cell free extract and enzymatic product. The study of optimal temperature and pH of fructosyltransferase activity were needed to understand the suitability of the enzyme in industrial applications. The study of how one metal ion could influence the reaction rate is important to understand the structure-function relationship in enzymes. Further elaborate studies are required in optimizing the culture conditions and reaction conditions for maximum production of fructooligosaccharides. Studies are required to find the suitability of the purified enzyme from Aspergillus sp. in food and nutraceutical industries.References 1.Pszczola D.E., The Nutraceutical Initiative: A Proposal for Economic and Regulatory Reform, Food Biotechnol.,4677-79, (1992) 2.Kalra E.K., Nutraceutical – definition and introduction, AAPS Pharm Sci., 5, E25, (2003) 3.Roberfroid M. and Slavin, J., Nondigestible oligosaccharides, Crit. Rev. Food Sci. Nutr.,40, 461–480 (2000) 4.Barreteau H., Delattre C and Michaud P., Production of Oligosaccharides as Promising food additive generation, Food Technol. Biotechnol., 44, 323-333 (2006) 5.Nakakuki T., Oligosaccharides: Production, Properties and Applications, Japanese Technol. rev., 144-157, (1993) Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 3(ISC-2013), 147-153 (2014) Res. J. Recent. Sci. International Science Congress Association 153 6.Nakakuki T., Oligosaccharides: production, properties, and application, Gordon and Breach Science Publishing, 3, 50-117, (1996) 7.Hidaka H., Hirayama M., and Sumi N., A fructooligosaccharide-producing enzyme from Aspergillus niger ATCC 20611, Agric. Biol. Chem., 52, 1181- 1187 (1988)8.Hernalsteens S. and Maugeri F., Purification and characterization of a fructosyltransferase from Rhodotorula sp. Appl., Microbiol. Biotechnol., 79, 589-596 (2008) 9.Sanchez O., Felipe G., Diana G., Edelberto S. and Luis C., Fructooligosaccharides production by Aspergillus sp. N74 in a mechanically agitated airlift reactor, Food and Bioproducts processing, 86, 109-115 (2008) 10.Salinas M.D and Perotti N.I., Production of fructosyltransferase by Aureobasidium sp. ATCC 20524 in batch and two step batch culture, J. Ind. Microbiol. Biotechnol., 36, 39-43 (2009) 11.Lateef A., Oloke J.K and Prapulla S.G., Purification and partial characterization of intracellular fructosyltransferase from a novel strain of Aureobasidium pullulan, Turk. J. Biol., 31, 147-154, (2007)12.Miller G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem., 31, 426-428, (1959) 13.Vigant, M.B.A., Laukevics J., Toma M., Rapoports A. and Zikmanis P., The effect of osmo-induced stress on product formation by Zymomonas mobilis on sucrose, Int. J. Food Microbiol., 55, 147–150 (2000)14.Madlova A., Antosova M., Baráthová, M., Polakovie M., Stefuca V. and Bales V., Screening of Microorganisms for Transfructosylating Activity and Optimization of biotransformation of Sucrose to fructooligosaccharides, Chem. Papers, 53, 366- 369 (1999)15.Anderson V.L and Mc Clean. R.A., Design of experiments: A realistic approach. Mercel Dekker. Inc., New York, 50-55, (1974) 16.Goosen C., Yuan X.L., Van Munster J.M., Ram A.F.J., Van Der Maarel, M.J.E.C. and Dijkhuizen, L, Molecular and biochemical characterization of a novel intracellular invertase from Aspergillusniger with transfructosylating activity, Eukaryotic Cell, 6, 674-681, (2007) 17.Dominguez A., Nobre C., Tores D., Rodrigues L.R., Rocha I., Teixiera J.A., Nelson L. and Ferreeira E.C., Optimization of fermentation conditions for fructooligosaccharides productivity by Aureobasidium pullulans, In: Proceedings of World congress on industrial biotechnology and bioprocessing, Toronto, (2006) 18.Oscar S., Felipe G., Diana G., Edelbert S and Lui C., Fructooligosaccharides production from sucrose by Aspergillus sp. N74 in a hybrid bioreactor, In: Proceedings of European congress of chemical engineering (ECCE), Copenhagen, (2007) 19.Sangeetha P.T., Ramesh M.N. and Prapulla S.G., A process for the production of fructooligosaccharides using jaggery (87/DEL/03) (2003)20.Ghazi I., Fernandez-Arrojo A.L., Arellano H.G., Ferror M., Ballesteros A. and Plou F.J., Purification and kinetic characterization of a fructosyltransferase from Aspergillus aculeatus, J. Biotechnol., 128, 204-211 (2007)21.Yun J.W., Noh J.S., Lee M.G and Song S.K., Production of fructooligosaccharides by the mixed enzyme system of frcutosyltrnasferase and glucose isomerise, J. Korean Inst. Chem. Eng., 31, 846-851 (1993)22.Hang, Y..D., Woodams E.E and Jang K.Y., Enzymatic conversion of sucrose to kestose by fungal extracellular fructosyl transferase, Biotechnol. Lett.,17, 295-298, (1995)