Agbaje LATEEF, Julius Kola OLOKE, Siddalingaiya G. PRAPULLA

Purification and partial characterization of intracellular fructosyltranferase from a novel strain of Aureobasidium pullalans

Dokuz saatlik reaksiyon süresinde %59 fructosiltransferaz üretebilen yeni bir Aureobasidium pullulans (CFR77) suşunun hücre içi FOS’u wet-milling yöntemi ile elde edilmiş, saflaştırılmış ve karakterize edilmiştir. Saflaştırılan FTaz 147 ve 170 KD ağırlığında iki bant vermiştir, aktivitesi yaklaşık pH 5 ve 55 °C’de optimum olarak bulunmuştur. Sonuçta saflaştırılan materyalin özel aktivitesi 42’dir ve 79,44 saşaştırma faktörü, %43 kazanca sahiptir. Enzim stabildir ve optimum reaksiyon koşullarında 12 saatten sonra orijinal aktivitesinin %8’inden fazlasını geri kazanmaktadır. FOS’un %59’u 9 saatlik bir reaksiyon süresinde ham hücre içi FTaz kullanılarak üretilmiştir. Bu süre literatürde belirtilen 12-15 saatlik sürenin belirgin bir şekilde azalmasıdır. Saflaştırılan FTaz 3 saatlik reaksiyon süresinde %59 FOS kazanımını sağlamaktadır.

Aureobasidium pullulans suşundan hücreiçi fruktoziltransferaz enziminin saflaştırılması ve kısmi karekterizasyonu

The intracellular fructosyltransferase (FTase) of a novel strain of Aureobasidium pullulans (CFR 77) capable of producing 59% of fructooligosaccharides (FOS) within 9 h of reaction time was obtained by wet-milling, and then purified and characterized. The purified FTase revealed 2 bands of 147 and 170 KD; its activity was optimum at an approximate pH of 5.0 and temperature of 55 °C. The specific activity of the final purified material was 42, representing a purification factor of 79.44 and yield of 43%. The enzyme is very stable, retaining more than 80% of its original activity at the optimum reaction conditions after 12 h. Using the crude intracellular FTase, 59% of FOS was produced within 9 h of reaction time, which is a considerable reduction in the reaction time of 12-25 h that has been reported in the literature. The purified FTase yielded 59% of FOS within 3 h of reaction time.

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1. Prapulla SG, Subhaprada V, Karanth NG. Microbial Production of Oligosaccharides: a review In: Neidleman, S.L., Laskin, A.I, Bennet, J.W., and Gadd, G. eds. Advances in Applied Microbiology. New York, Academic Press. 2000: pp. 299-337.
2. Sangeetha PT, Ramesh MN, Prapulla SG. Microbial production of fructooligosaccharides. Asian J Microbiol Biotechnol Environ Sci 5: 313-318, 2003.
3. Yun JW, Jung KH, Jeon YJ et al. Continuous production of fructooligosaccharides by immobilized cells of Aureobasidium pullulans. J Microbiol Biotechnol 2: 98-101, 1992.
4. Yun JW. Fructooligosaccharides - Occurrence, preparation, and applications. Enzyme Microb Technol 19: 107-117, 1996.
5. Yun JW, Kim D, Moon H et al. Simultaneous formation of fructosyl- and glycosyltransferase in Aureobasidium pullulans. J Microbiol Biotechnol 7: 204-208, 1997.
6. Shin HT, Park KM, Kang KH et al. Novel method for cell immobilization and its application for production of oligosaccharides from sucrose. Letters Applied Microbiol 38: 176- 179, 2004.
7. Vandáková M, Platková Z, Antosová M et al. Optimization of cultivation conditions for production of fructosyltransferase by Aureobasidium pullulans. Chemical Papers 58: 15-22, 2004.
8. Hayashi S. Nonokuchi M, Takasaki Y et al. Purification and properties of ß-fructofuranosidase from Aureobasidium sp ATCC 20524. J Industrial Microbiol 7: 251-256, 1991.
9. Chang CT, Lin YY, Tang MS et al. Purification and properties of ß-fructofuranosidase from Aspergillus oryzae ATCC 76080. Biochem Mol Biol Int 32: 269-277, 1994.
10. Chen JS, Saxton J, Hemming FW et al. Purification and partial characterization of the high and low molecular weight (S- and Fform) of invertase secreted by Aspergillus nidulans. Biochemistry Biophysics Acta 1296: 207-218, 1996.
11. Patil VB, Patil NB. Purification and immobilization of fructosyltransferase for production of fructooligosaccharides from sucrose. Indian J Exp Biol 37: 830-834, 1999.
12. L’Hocine L, Wang Z, Jiang B et al. Purification and partial characterization of fructosyltransferase and invertase from Aspergillus niger AS0023. J Biotechnol 81: 73-84, 2000.
13. Hidaka H, Eida T, Adachi T et al. Industrial production of fructooligosaccharides and its application for human and animals. Nippon Nogeik Kaishi 61: 915-923, 1987.
14. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680-685, 1970.
15. Wray W, Boulikas T, Wray VP et al. Silver staining of proteins in polyacrylamide gels. Anal Biochem 118: 197-203, 1981.
16. Hirayama M, Sumi N, Hidaka H. Purification and properties of a fructooligosaccharides-producing ß-fructofuranosidase from Aspergillus niger ATCC 20611. Agric Biol Chem 53: 667-673,1989.
17. Hidaka H, Hirayama M, Sumi N. A fructooligosaccharidesproducing enzyme from Aspergillus niger ATCC 20611. Agric Biol Chem 52: 1181-1187, 1988.
18. Fujita K, Hara K, Hashimoto H et al. Transfructosylation catalyzed by ß-fructofuranosidase I from Arthrobacter sp. K-1. Agric Biol Chem 54: 2655-2661, 1990.
19. Chen CT, Liu CH. Production of ß-fructofuranosidase by Aspergillus japonicus. Enzyme Microb Technol 18: 153-160,1996.
20. Henry RJ, Darbyshire B. Sucrose fructosyltransferase and fructan: Fructan fructosyltransferase from Allium cepa. Phytochem 19: 1017-1020, 1980.
21. Shiomi N, Izawa M. Purification and characterization of sucrose:sucrose fructosyltransferase from the roots of asparagus (Asparagus officinalis L). Agric Biol Chem 44: 603-614, 1980.
22. Yun JW, Song SK. The production of high content fructooligosaccharides from sucrose by the mixed-enzyme systems of fructosyltransferase and glucose oxidase. Biotechnology Letters 15: 573-576, 1993.
23. Shin HT, Baig SY, Lee SW et al. Production of fructooligosaccharides from molasses by Aureobasidium pullulans cells. Bioresource Technology 93: 59-62, 2004.
24. Sangeetha PT, Ramesh MN, Prapulla SG. Production of fructooligosaccharides by fructosyltransferase from Aspergillus oryzae CFR 202 and Aureobasidium pullulans CFR 77. Process Biochem 39: 753-758, 2004.