Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
권호 표지

Isolation of Phytase Producing Pseudomonas fragi and Optimization of its Phytase Production

Acid Phytase를 생산하는 Pseudomonas fragi의 분리와 phytase의 생산조건

  • Kim, Young-Jin (Department of Civil Engineering and Applied Mechanics, McGill University) ;
  • Jang, Eun-Seok (Department of Food Science and Technology, and Food Science Research Institute, Kongju National University) ;
  • In, Man-Jin (Department of Human Nutrition and Food Science, Chungwoon University) ;
  • Oh, Nam-Soon (Department of Food Science and Technology, and Food Science Research Institute, Kongju National University)
  • 김영진 (맥길대학교 도시공학 및 응용공학과) ;
  • 장은석 (공주대학교 식품공학과 및 식품과학연구소) ;
  • 인만진 (청운대학교 식품영양학과) ;
  • 오남순 (공주대학교 식품공학과 및 식품과학연구소)
  • Published : 2003.11.30
Download PDF
⟨ Previous Next ⟩

Abstract

A bacterial strain producing a high level of an extracellular phytase was isolated from livestock waste water, identified as a strain of Pseudomonas fragi and designated as Pseudomonas fragi Y9451. Under the phytase production medium, the activity of phytase reached the highest level after 120 hours of incubation. On the effect of carbon sources on the phytase production, the most favorable carbon source for phytase production was fructose. As for the effect of nitrogen sources, high levels of phytase activity were detected in the medium containing nutrient broth as the nitrogen source. Free $PO_4^{3-}$ inhibited phytase production with increasing concentration of $KE_2PO_4$ and phytate in the media. The addition of $CaCl_2$ and $MgSO_4$ also resulted in the inhibition of phytase production. To investigate the effect of aeration on the phytase production, different volumes of culture broth in Erlenmeyer flasks were incubated in rotary shaker at the speed of 200 rpm. As a result, a high level of phytase activity was detected at small volume of culture broth as compared to larger volume because of its more aerobic condition.

축산폐수가 방류되는 단위동물 밀집사육장 인근의 하천수로부터 phytase를 생산하는 세균들 중 활성이 가장 우수한 균주를 분리하여, Pseudomonas fragi Y9451로 동정, 명명하였다. 탄소원으로 fructose를 첨가할 경우에 phytase의 생산능이 가장 우수하였고, 배지에 7% 첨가할 경우에 72시간 배양 후 효소활성이 1,078 mU/ml로 최대의 phytase 생산능을 보였다. 또한, 질소원으로는 nutrient broth(NB)가 적합하였으며, NB를 3%의 농도로 첨가할 경우에 96시간 배양 후 효소활성이 1,425 mU/ml까지 향상되었다. 인산원인 $KH_2PO_4$, phytate는 첨가 농도가 높을수록 phytase 생산은 억제되었으며, 무기염으로 $MgSO_4$와 $CaCl_2$를 첨가하는 경우는 첨가농도와 상관없이 phytase의 생산을 현저히 억제하였다. 산업생산을 목적으로 하는 scale-up에 있어서의 통기교반 효과는 phytase의 생산량에 큰 영향을 미치는 것으로 나타났다.

Keywords

References

  1. Sandberg, A.-S. (1994) Antinutrient effects of phytate. Ern$\"{a}$hrung/Nutrition 18, 429-432
  2. Erdman, J.W. apd Poneros-Schneier, A. (1989) Phytic acid interactions with divalent cations in foods and in the gastrointestinal track. Adv. Exp. Med. Biol. 249, 161-171 https://doi.org/10.1007/978-1-4684-9111-1_11
  3. Chang, C. W. (1967) Study of phytase and fluoride effects in germinating corn seeds. Cereal Chem. 44, 129-132
  4. Schwarz, G. and Hoppe, P. P. (1992) Phytase enzyme to curb pollution from pigs and poultry. Feed magazine 22-26
  5. Ahn, B. and Yang, C. B. (1985) Effects of soaking, germination, incubation and autoclaving on phytic acid in seeds. Kor. J. Food Sci. Technol. 17, 516-521
  6. Jung, J. H., Kang, S. G., Kim, Y. S. and Chung, H. J. (1990) Degradation of phytic acid in Chungkookjang fermented with phytase producing bacteria. Kor. J. Appl. Microbiol. Biotechnol. 18, 423-428
  7. Anno, T., Nakanishi, K. and Matsuno R. (1985) Enzymatic elimination of phytate in soybean milk. Nippon Shokuhin Koeyo Gakkaishi 32, 174-180 https://doi.org/10.3136/nskkk1962.32.3_174
  8. Zyta, K. (1992) Mould phytases and their application in the food industry. World J. Microbiol. Biotechnol. 8, 467-472 https://doi.org/10.1007/BF01201941
  9. Howson, S. J. and Davis, R. P. (1983) Production of phytate hydrolyzing enzyme by some fungi. Enzyme Microb. Tech. 5, 377-382 https://doi.org/10.1016/0141-0229(83)90012-1
  10. Ullah, A. H. J. and Cummins, B. J. (1988) Aspergillus ficuum extracellular phytase: immobilization on glutaraldehyde activated silicate. Ann. NY. Acad. Sci. 542, 102-106 https://doi.org/10.1111/j.1749-6632.1988.tb25812.x
  11. Han, Y. W., Gallagher, D. J. and Wilfred, A. G. (1987) Phytase production by Aspergillus ficuum on semisolid substrate. J. Ind. Microbiol. 2, 195-200 https://doi.org/10.1007/BF01569540
  12. Ebune, A., Asheh, A. S. and Duvnjak, Z. (1995) Effects of phosphate, surfactants and glucose on phytase production and hydrolysis of phytic acid in canola meal by Aspergillus ficuum during solid-state fermentation. Bioresource Technol. 54, 241-24 https://doi.org/10.1016/0960-8524(95)00133-6
  13. Lambrechts, C., Boze, H., Moulin, G. and Galzy, P. (1992) Utilization of phytate by some yeasts. Biotechnol. Lett. 14, 61-66 https://doi.org/10.1007/BF01030915
  14. Sano, K., Fukuhara, H. and Nakamura, Y. (1999) Phytase of the yeast Arxula adeninivorans. Biotechnol. Lett. 21, 33-38 https://doi.org/10.1023/A:1005438121763
  15. Shah, V. and Parekh, L. J. (1990) Phytase from Klebsiella sp. No. PG-2: purification and properties, Indian J. Biochem. Biophys. 27, 98-102
  16. Shimizu, M. (1992) Purification and characterization of phytase from Bacillus subtilis (natto) N-77. Biosci. Biotechnol. Biochem. 56, 1266-1269 https://doi.org/10.1271/bbb.56.1266
  17. Kim, Y-O., Kim, H-K., Bae, K-S., Yu, J-H. and Oh, T. K. (1998) Purification and properties of a thermostable phytase from Bacillus sp. DSll. Enzyme Microb. Tech. 22, 2-7 https://doi.org/10.1016/S0141-0229(97)00096-3
  18. Choi, Y, M., Noh, D. O., Cho, S. H., Lee, H. K., Suh, H-J. and Chung, S-H. (1999) Isolation of a phytase producing Bacillus sp. KHU-10 and its phytase production. J. Microbiol. Biotechnol. 9. 223-226
  19. Yoon, S. J., Choi, Y. J., Min, H. K., Cho, K. K., Kim, .J. W., Lee, S. C. and Jung, Y. H. (1996) Isolation and identification of phytase-producing bacterium, Enterobacter sp. 4, and enzymatic properties of phytase enzyme. Enzyme Microb. Tech. 18, 449-454 https://doi.org/10.1016/0141-0229(95)00131-X
  20. Irving, G. C. J. and Cosgrove, D. J. (1997) Inositol phosphate phosphatase of microbiological origin, some properties of a partially purified bacterial (Pseudomonas sp.) Phytase. Aust. J. Biol. Sci. 24, 547-557
  21. Richardson, A.E. and Hadobas, P. A. (1997) Soil isolates of Pseudomonas sp. that utilize inositol phosphate. Can. J. Microbiol. 43, 509-516 https://doi.org/10.1139/m97-073
  22. Kim, Y. H., Gwon, M-N., Yang, S-Y., Park, T-K., Kim, C-G., Kim, C-W. and Song, M-D. (2002) Isolation of phytas producing Pseudomonas sp. and optimization of its phytase production. J. Microbiol. Biotechnol. 12, 279-285
  23. Jareonkitmongkol, S., Ohya, M., Watanabe, R., Takagi, H. and Nakamori, S. (1997) Partial purification of phytase from a soil isolate bacterium, Klebsiella oxytoca M0-3. J. Ferment. Bioeng. 83, 393-394 https://doi.org/10.1016/S0922-338X(97)80149-3
  24. Greiner, R., Haller, E., Konietzny, U. and Jany, K. D. (1997) Purification and characterization of a phytase from Klebsiella terrigena. Arch. Biochem Biophys. 341, 201-206 https://doi.org/10.1006/abbi.1997.9942
  25. Greiner, R., Konietzny, U. and Jany, K. D. (1993) Purification and characterization of two phytases from Escherichia coli. Arch. Biochem. Biophys. 303, 107-113 https://doi.org/10.1006/abbi.1993.1261
  26. Tomschy, A., Brugger, R., Lehmann, M., Svendsen, A., Vogel, K., Kostrewa, D., Lassen, S. R, Burger, D., Kronenberger, A., van Loon, A., R., G., M., Pasamontes, L. and Wyss, M. (2002) Engineering of phytase for improved activity at low pH. Appl. Environ. Microbiol. 68, 1907-1913 https://doi.org/10.1128/AEM.68.4.1907-1913.2002
  27. Jang, E. S. (2003) Production and characterization of phytase from Psedomonas fragi Y9451. MS Thesis, Kongju National University, Kongju, Korea
  28. Sumino, Y, Sonoi, K. and Doi, M. (1993) Scale-up of purine nucleoside fermentation from a shaking flask to a stirred-tank fermentor. Appl. Microbiol. Biotechnol. 38, 581-585