Purificação e propriedades de uma enzima degradadora de fitato produzida por Enterobacter sakazakii ASUIA279
DOI:
https://doi.org/10.20873/jbb.uft.cemaf.v3n1.faroukKeywords:
Enerobacter sakazakii, phytate-degrading enzyme, phytate, purificationAbstract
An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The enzyme was purified about 66-fold with a recovery of 27%. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Michaelis constant (KM) and turnover number (kcat ) for sodium phytate at pH 5.0 and 50°C were calculated from the Lineweaver-Burk plot to be 760 µM and 4.14s-1, respectively. The enzyme showed narrow substrate specificity and not phytate, but GTP was dephosphorylated with the highest relative rate of hydrolysis. However, according to the kcat/KM values, phytate was concluded to be the in vivo substrate of the enzyme. Optimal activity was determined at pH 4.5 and 45-55°C. The enzyme was strongly inhibited by Fe3+, Cu2+, Zn2+, molybdate, vanadate, fluoride and phosphate (1 mM).
References
Anis Shobirin, M. H.; Farouk, A.; Hamzah, M. S.; Greiner, R. (2007), Phytate-degrading Enzyme Production by Bacteria isolated from Malaysian soil. World Journal of Microbiology and Biotechnology, 23, 1653-1660.
Anis Shobirin, M. H.; Farouk, A.; Greiner, R. (2009), Potential phytate-degrading enzyme producing bacteria isolated from Malaysian maize plantation. African Journal of Biotechnology, 15, 3540-3546.
Bradford., M. (1976), A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 72, 248-254.
Cheryan, M. (1980), Phytic acid interactions in food systems. Critical Review Food Science Nutrition, 13, 297-335.
Choi, Y. M.; Suh, H. J.; Kim, J. M. (2001), Purificationand properties of extracellular phytase from Bacillus sp. KHU-10. Journal Protein Chemistry, 20, 287-292.
Daly, D. (1991), Impact of land spreading manure and other organic wastes on ground water. Geological Survey of Ireland report, Geological Survey of Ireland, Dublin.
Gibson, D. M. and Ullah, A. H. J. (1988), Purification and characterization of phytase from cotyledons of germinating soybean seeds. Archives of Biochemistry and Biophysics, 260, 503-513.
Golovan, S.; Wang, G.; Zhang, J.; Forsberg, C. W. (2000), Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phophatase activities. Canada Journal Microbiology, 46, 59-71.
Greiner, R. (2004), Purification and properties of a phytate-degrading enzyme from Pantoea agglomerans. Protein Journal, 23, 567-576.
Greiner, R.; Haller, E.; Konietzny, U.; Jany, K. D. (1997), Purification and characterization of a phytase from Klebsiella terrigena. Archives of Biochemistry and Biophysics, 341, 201-206.
Greiner, R.; Konietzny, U.; Jany, K. D. (1993), Purification and characterization of two phytases from Escherichia coli. Archives of Biochemistry and Biophysics, 303, 107-113.
Heinonen, J. K. and Lahti, R. J. (1981), A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Analytical Biochemistry, 113, 313-317.
Hübel, F. and Beck, E. (1996), Maize root phytase: Purification, characterization, and localization of enzyme activity and its putative substrate. Plant Physiology, 112, 1429-1436.
Howson, S. J.and Davis, R. P. (1983), Production of phytate hydrolyzing enzymes by some fungi. Enzyme Microbiology Technology, 5, 377-389.
Idriss, E. E.; Makarewicz, O.; Farouk, A.; Rosner, K.; Greiner, R.; Bochow, H.; Richter, T.; Borris, R. (2002), Extracellular phytase activity of Bacillus amyloliquefaciens FZ45 contributes to its plant growth promoting effect. Microbiology, 148, 2097-2109.
Irving, G. C. J. (1980), In Inositol phosphates: Their chemistry, biochemistry and physiology (Cosgrove, D.J., Rd.), pp. 85-98, Elsevier, Amsterdam.
Irving, G. C. J. and Cosgrove, D. J. (1971), Inositol phosphate phosphatases of microbiological origin. Some properties of partially purified bacterial (Pseudomonas sp.) phytase. Australia Journal Biology Science, 24, 547-557.
Jareonkitmongkol, S.; Ohya, M.; Watanabe, R.; Takagi, H.; Nakamori, S. (1997), Partial purification of phytase from a soil isolates bacterium, Klebsiella oxytoca MO-3. Journal Fermentation Bioengineering, 83, 393-394.
Kerovuo, J.; Lauraeus, M.; Nurminen, P.; Kalkinen, N.; Apajalahti, J. (1998), Isolation, characterization, molecular gene cloning and sequencing of a novel phytase from Bacillus subtilis. Applied Environment Microbiology, 64, 2079-2085.
Kim, H. W.; Kim, Y. O.; Lee, J. H., Kim, K. K.; Kim, Y. J. (2003), Isolation and characterization of a phytase with improved properties from Citrobacter braakii. Biotechnology Letters, 25, 1231-1234.
Kim, Y. O.; Kim, H. K.; Bae, K. S.; Yu, J. H.; Oh, T. K. (1998), Cloning of thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli. FEMS Microbiology Letters, 162, 185-191.
Konietzny, U. and Greiner, R. (2004), Bacterial phytase: Potential application, in vivo function and regulation of its synthesis. Brazilian Journal Microbiology, 35, 11-18.
Konietzny, U. and Greiner, R., (2003), Phytic acid (b) Nutritional impact. In: Caballero B, Trugo L, Finglas, P. (eds). Ency food Sci and Nutr, Elsevier, London 4555-4563p.
Lan, G. Q.; Abdullah, N.; Jallaludin, S.; Ho, Y. W. (2002), Culture conditions influencing phytase production of Mitsuokella jalaludinii, a new bacterial species from the rumen of cattle. Journal Applied Microbiology, 93, 668-674.
Maga, J. A. (1982), Phytate: its chemistry, occurrence, food interactions, nutritional significance, and methods of analysis. Journal Agriculture Food Chemistry, 30, 1-9.
Mitchell, D. B.; Vogel, K.; Weimann, B. J.; Pasamontes, L.; Van Loon, A. S. P. G. M. (1997), The phytase subfamily of histidine acid phosphatases: isolation of gene for two novel phytases from the fungi Aspergillus tereus and Myceliophthora thermophila. Microbiology, 143, 245-252.
Nakamura, Y.; Fukuhara, H.; Sano, K. (2000), Secreted phytase activities of yeasts. Bioscience Biotechnology Biochemistry, 64, 841–844.
Nayini, N. R. and Markakis, P. (1986), In Phytic Acid: Chemistry and Applications (Graf, E., Ed.) pp. 101-118, Pilatus Press, Minneapolis, M. N. Nelson, T. S. 1967. The utilization of phytase phosphorus by poultry. Poultry Science, 46, 862- 871.
Ohkawa, T.; Ebisuno, S.; Kitagawa, M.; Morimoto, S.; Miyazaki, Y.; Yasukawa, S. (1984), Rice bran treatment for patients with hypercalciuric stones: Experimental and clinical studies. Journal Urology, 132, 1140-1145.
Potter, S. M. (1995), Overview of proposed mechanisms for the hypocholestrolemic effect of soy. Journal Nutrition, 125, 606S-611S.
Powar, V. K. and Joganathan, V. (1982), Purification and properties of phytate specific phosphatase from Bacillus subtilis. Journal Bacteriology, 51, 1102-1108.
Reddy, N. R.; Pierson, M. D.; Sathe, S. K.; Salunkhe, D. K. (1989), Phytates in cereal and legumes. CRC Press Inc, Boca Raton, Florida.
Sajidan, A.; Farouk, A.; Greiner, R.; Jungblut, P.; Muller, E. C.; Borris, R. (2004), Molecular and physiological characterization of a 3-phytase from the Rhizobacterium Klebsiella pneumoniae ASRI. Applied Microbiology Biotechnology, 65, 110-118.
Sandberg, A. S. and Andlid, T. (2002), Phytogenic and microbial phytases in human nutrition. International Journal Food Science Technology, 37, 823-834.
Sano, K.; Fukuhara, H.; Nakamura, Y. (1999), Phytase of the yeast Arxula adeninivorans. Biotechnology Letters, 21, 33-38.
Seguielha, L.; Moulin, G.; Galzy, P. (1993), Reduction of phytate content in wheat bran and glandless cotton flour by Schwanniomyces castelli. Journal Agriculture Food Chemistry, 41, 2451-2454.
Shimizu, M. (1992), Purification and characterization of phytase from Bacillus subtilis (natto) N-77. Bioscience Biotechnology Biochemistry, 56, 1266-1269.
Sreeramulu, G.; Srinivasa, D. S.; Nand, K.; Joseph, R. (1996), Lactobacillus amylovorus as a phytase producer in submerged culture. Letter Applied Microbiology, 23, 385-388.
Tambe, S. M.; Kaklij, G. S.; Keklar, S. M.; Parek, H. L. J. (1994), Two distinct molecular forms of phytase from Klebsiella aerogenes: Evidence for unusually small active enzyme peptide. Journal Fermentation Bioengineering, 77, 23-27.
Ullah, A. H. J. (1988), Production, rapid purification and catalytic characterization of extracellular phytase from Aspergillus ficuum. Preparation Biochemistry, 18, 443-458.
Van der Kaay, J. and Van Haastert, P. J. M. (1995), Stereospecificity of inositol hexakisphosphate dephosphorylation by Paramecium phytase. Biochemistry Journal, 312, 907-910.
Vucenik, I. and Shamsuddin, A. M. (2003), Cancer inhibition by inositol hexaphosphate (IP6) and inositol: From laboratory to clinic. Journal Nutrition, 133, 3778S- 3784S.
Walz, O. P. and Pallauf, J. (2002), Microbial phytase combined with amino acid supplementation reduces P and N excretion of growing and finishing pigs without loss of performance. International Journal Food Science Technology, 37, 835-848.
Wang, H. L.; Swain, E. W.; Haseltine, C.W. (1980), Phytase of molds used in oriental food fermentation. Journal Food Science, 45, 1264-1266.
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