Table 1. Comparison of precipitation methods for A. niger ATCC 20611 inulinase.

1. Introduction Abstract Table 1. Comparison of precipitation methods for A. niger ATCC 20611 inulinase. Inulinase (2,1-b-D-fructanfructanohydrolase; EC 3.2.1.7) hydrolyses inulin liberating fructose (F) and inulooligosaccharides (IOS). Inulinase has been in the production of fructose syrup and inulooligosaccharides. (Grootwassink and Fleming 1979; Yun, et al. 1997.) Crude enzyme from Aspergillusniger showed the mixed activities of inulinase (I) and invertase (S) with different optimal pH (4.5 and 6.0) and temperature (40 and 45 oC), respectively. To separate inulinase from invertase, salting-out with (NH4)2SO4 and precipitation with absolute ethanol and acetone, were studied for use in the production of inulooligosaccharides. The maximum inulinase activity (1.88 U/ml) and yield (9.06%) were obtained by precipitation with at ethanol at -10oC. All I/S activity ratios were decreased after precipitation. This indicated that these precipitation methods were not suitable for recovery and separating inulinase from invertase. Crude enzyme activitiesa Precipitated enzyme activities Recovery yield (U/ml) (U/ml) (%) Methods Experimental Procedure Inulinase Invertase I/S ratiob Inulinase Invertase I/S ratiob Inulinase Invertase Crude enzymes were produced from Aspergillus niger ATCC 20611 using sucrose:inulin (12.5:4 g/l) as substrates. Precipitations were carried out with (NH4)2SO4 (4 oC), absolute ethanol (4 oC and –10 oC) and acetone (-10 oC) for 24 h. Inulinase and invertase activities were assayed by measuring the concentrations of reducing sugars released from inulin and sucrose, respectively. (modified from Nukamura et al., 1994). 75% (NH4)2SO4 (4 oC) 1.19 0.46 2.57 0.19 3.88 0.05 1.55 83.78 (4 oC) 1.71 0.66 2.60 0.43 1.44 0.30 2.50 22.00 60%Absolute ethanol (-10 oC) 2.07 1.09 1.91 1.88 4.69 0.40 9.06 43.20 1.4 (a) (b) 3.5 3.5 1.4 1.2 1.2 3.0 3.0 1.0 1.0 2.5 2.5 60%Acetone (-10 oC) 2.05 1.09 1.89 1.26 2.71 0.47 6.15 24.89 .8 .8 2.0 2.0 Inulinase activity (U/ml) Invertase activity (U/ml) Inulinase activity (U/ml) invertase activity (U/ml) .6 .6 1.5 1.5 .4 .4 1.0 1.0 .2 .2 .5 .5 0.0 0.0 0.0 0.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 25 30 35 40 45 50 55 60 65 70 Temperature (oC) pH A study on separating inulinase from invertase with precipitation method Thikumporn Tantivimongkola* and Sarote Sirisansaneeyakula+ a*,a+Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand Results and Discussion The crude enzyme solution showed activities of inulinase and invertase with different optimum conditions of pH (4.5 and 6.0) (fig. 1a) and temperature (40 and 45 oC) (fig. 1b), respectively. According to the precipitation method studied, inulinase activity (U/ml) and enzyme yield (%) of 0.19, 1.55; 0.43, 2.50; 1.88, 9.06 and 1.26, 6.15 were obtained with using ammonium sulfate (4 oC), absolute ethanol (4 oC and –10 oC) and acetone (-10oC), respectively (Table 1). Fig. 1 (a) pH and (b) temperature affecting activity of inulinase ( ) and invertase ( ). From Table 1, the precipitation with absolute ethanol at –10 oC gave the maximum inulinase activity (1.88 U/ml) and recovery yield (9.06 %), but I/S activity ratio was decreased from 1.91 to 0.40. At present, a semipurified inulinase with partial activity of invertase was prepared by precipitation with absolute ethanol at –10 oC. • a) activities of crude enzyme obtained from A. niger ATCC 20611 culture. • b) I/S = inulinase activity/invertase activity. References Nakamura, T., Nagatomo, Y., Hamada, S., Nishino, Y. and Othta, K. 1994. Occurrence of two form of extracellular endoinulinase from Aspergillus niger mutant 817. J. Ferment. Bioeng. 78(2.): 134-139. Yun, J. W., Kim, D. H., Uhm, T. B. and Song, S. K. 1997. Production of high content inulooligosaccharides from inulin by a purified endoinulinase. Biotechnol. Lett. 19(9): 935-938. Grootwassink, J. W. D. and Fleming, S. 1979. Preparation of high fructose syrup from the tubers of the Jerusalem artichoke (Helianthus tuberosusl). CRC Crit. Rev. Food Sci. Nur. 12: 45-53. Conclusion This data indicated that these precipitation methods were not suitable for recovery and separating inulinase from invertase. For producing IOS, an optimal condition for inulinase (pH 4.5, 40oC) was controlled to minimize an interfered invertase activity. Acknowledgement The authors would like to thank Asst. Prof. Dr. W. Kanlayakrit for his suggestion on enzyme recovery and purification. * Fax :662-579-4096; Email: sarote.si@ku.ac.th