Optimization of Heteropolysaccharide-7 Production by Beijerinckia Indica

Beijerinckia Indica 배양을 통한 Heteropolysaccharide-7 생산 최적화

  • Wu Jian-Rong (Department of Biotechnology & Bioengineering Pukyong National University) ;
  • Son Jeong Hwa (Department of Biotechnology & Bioengineering Pukyong National University) ;
  • Kim Ki Myong (Department of Biotechnology & Bioengineering Pukyong National University) ;
  • Nam Soo-Wan (Department of Biotechnology, Dong-Eui University) ;
  • Lee Jin-Woo (Division of Biotechnology, Dong-A University) ;
  • Kim Sung-Koo (Department of Biotechnology & Bioengineering Pukyong National University)
  • Published : 2005.06.01

Abstract

Beijerinckia indica was cultured in mineral salts medium (MSM) medium with various carbon and nitrogen sources to improve the production yield of heteropolysaccharide-7 (PS-7). At high C/N ratio, the high concentration of PS-7 was produced until 40 h of the culture, whereas most of the glucose as a carbon source was used for the cell growth at low C/N ratio. However, at the high C/N ratio, PS-7 accumulation stopped at 48 h of the culture due to the increasing viscosity of the culture broth would inhibit the cell growth. Therefore, the optimized value of C/N ratio was 33.3 (20 g/L glucose, 7.5 mM $NH_{4}NO_3$) for the high production of PS-7. In the culture with various carbon sources, B. indica effectively used the hexoses or glucose-generating sugars for PS-7 formation. Especially, sucrose was the best carbon source for the high production of PS-7 (6.96 g/L) with a high viscosity (40772 cp). In the culture of B. indica with MSM medium containing 20 g/L glucose and 7.5 mM $NH_{4}NO_3$ in a 51 fermentor, the highest cell concentration was 2.5 g/L and the highest concentration of PS-7 was 7.5 g/L (35174 cp). The additional nitrogen sources of 7.5 mM $NH_{4}NO_3$, glutamine and glutamate at 12 h of the culture after exhaustion of a nitrogen source regulated the metabolism of carbon sources, therefore the nitrogen sources could control PS-7 synthesis.

Beijerinckia indica를 통한 Heteropolysaccharide-7(PS-7)생산 최적화를 위해 다양한 탄소원과 질소원을 첨가하여 다른 배지 조성들이 PS-7에 미치는 영향을 조사하였다. 높은 C/N ratio에서 PS-7 생산을 배양 40시간까지 효과적으로 증가시킬 수 있었으며 낮은 C/N ratio에선 탄소원으로 첨가한 glucose가 균체의 생산에 사용되었을뿐 PS-7의 생산은 낮았다. 하지만, 높은 C/N ratio에서는 배양액의 점도로 균체의 생산이 정체하는 것으로 보아 본 실험에서는 20g/l glucose와 $NH_{4}NO_3$ 7.5 mM으로 33.3의 C/N ratio가 PS-7 생산에 적절한 비율인 것으로 관찰되었다. 다양한 탄소원을 첨가한 결과, B. indica는 PS-7 생산을 위하여 탄소원으로 hexose 또는 glucose-generating 당을 효율적으로 이용할 수 있었다. Beijerinckia indica를 이용한 PS-7 생산을 위해 첨가한 다양한 탄소원 중 sucrose 농도가 최고의 PS-7(6.96 g/L)을 생산 하였다. 51 배양기를 이용한 배양에서 탄소원 20 g/L glucose, 질소원 7.5 mM $NH_{4}NO_3$이 포함된 MSM 배지내 배양된 B. indica의 세포 농도가 최고 2.5 g/L이였고, 생산된 최고 PS-7농도는 7.5 g/L(35174 cp)으로 나타났다. 질소원이 완전히 소모된 12시간 이후, 7.5 mM의 $NH_{4}NO_3$, glutamine, glutamate을 첨가한 MSM 배지에서의 B. indica 배앙 결과, 질소원이 B. indica 내에서 탄소원 대사를 조절하는 역할을 하는 것을 관찰할 수 있었으며 최종적으로 PS-7의 생산을 조절할 수 있었다.

Keywords

References

  1. Arcondeguy, T., T. Jack, and M. Merrick. 2001. PII signal transduction proteins, pivotal players in microbial nitrogen control. Microbial. Molecul. Biol. Rev. 65: 85-105
  2. Ashtaputre, A. A. and A. K. Shah. 1995. Studies on a viscous, gel-forming exopolysaccharide from sphingomonas paucimobilis GSl. Appl. Environ. Microbial. 61: 1159-1162
  3. Banik, R. M., B. Kanari, and S. N. Upadhyay. 2000. Exopolysaccharide of the gellan family: prospects and potential. World J. Microbial. Biotechnol. 16: 407-414 https://doi.org/10.1023/A:1008951706621
  4. Becker, A, F. Katzen, A. Puhler, and L. Ielpi. 1998. Xanthan gum biosynthesis and application: a biochemical & genetic perspective. Appl. Microbial. Biotechnol. 50: 145-152 https://doi.org/10.1007/s002530051269
  5. Boza, Y, L. P. Neto, F. A. A. Costa, and A. R. P. Scamparini. 2004. Exopolysaccharide production by encapsulated Beijerinckia cultures. Proc. Biochem. 39: 1201-1209
  6. Charbit, A. 1996. Coordination of carbon and nitrogen metabolism. Res. Microbiol. 147: 513-518 https://doi.org/10.1016/0923-2508(96)84005-8
  7. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356 https://doi.org/10.1021/ac60111a017
  8. Falk, C., P. E. Jansson, M. Rinaudo, A. Heyraud, G. Widmalm, and P. Hebbar. 1996. Structural studies of the exocellular polysaccharide from Sphingomonas paucimobilis strain 1-886. Carbohydr. Res. 285: 69-79
  9. Gulin, S., A. Kussak, P. E. Jansson, and G. Widmalm. 2001. Structural studies of S-7, another exocellular polysaccharide containing 2 deoxy-arabino-hexuronic acid. Carbohydr. Res. 311: 285-290
  10. Jin, H., H. SKim, S. K. Kim, M. K. Kim, M. K. Shin, J. H. Kim, and J. W. Lee. 2002. Production of heteropolysaccharide7 by Beijerinckia indica from agro-industrial byproducts. Enzyme Microb. Technol. 30: 822-827 https://doi.org/10.1016/S0141-0229(02)00064-9
  11. Kim, H. S. 1999. Production of Heteropolysaccharide-7 by Beijerinckia Indica with Agro-lndustrial Byproducts as the Substrate. Ms thesis, Dong-A University, Busan, Korea
  12. Kang, S. K. and W. H. McNeely. 1976. Polysaccharide and bacterial fermentation process for its preparation. US Patent 3,960,832
  13. Kim, M. K., I. Y. Lee, J. H. Ko, Y. H. Rhee, and Y. H. Park. 1996. Higher intracellular levels of uridine monophosphate under nitrogen-limited conditions enhance metabolic flux of curdlan synthesis in Agrobacterium species. Biotechnol. Bioeng. 62: 317-323
  14. Klinke, S., M. Dauner, G. Scott, B. Kessler, and B. Withot. 2000. Inactivation of isocitrate lyase leads to increased production of medium-chain-Iength poly(3-hydroxyalkanoates). Appl. Environ. Microbiol. 66: 929-913
  15. Lee, J. W., W. G Yeomans, A. L. Allen, R. A. Gross, and D. L. Kaplan. 1997. Compositional consistency of a heteropolysaccharide-7 produced by Beijerinckia indica. Biotechnol. Lett. 19: 803-807
  16. Oelze, J. 2000. Respiratory protection of nitrogenase in Azotobacter species: is a widely held hypothesis unequivocally supported by experimental evidence? FEMS Microbiol. Rev. 24: 321-333 https://doi.org/10.1111/j.1574-6976.2000.tb00545.x
  17. Peekhaus, N. and T. Conway. 1998. What is for dinner? : Entner-Doudoroff metabolism in Escherichia coli. J. Bacteriol. 180: 3495-3502
  18. Portais, J. C. and A. M. Delort. 2002. Carbohydrate cycling in microorganisms: what can $^{13}C-NMR tell us? FEMS Microbiol. Rev. 26: 375-402
  19. Ramirez-Castillo, M. L. and J. L. Uribelarrea. 2004. Improved process for exopolysaccharide production by Klebsiella pneumoniae sp. pneumoniae by a fed-batch strategy. Biotechnol. Lett. 26: 1301-1306 https://doi.org/10.1023/B:BILE.0000044923.02460.de
  20. Standford, P. A. 1979. Exocellular Microbial polysaccharides, In: Advance in carbohydrate chemistry & biochemistry, Vol. 136. Academic Press, London
  21. Standford, P. A. and J. Baird. 1983. Industrial utilization of polysaccharide, In: polysaccharide II. Academic Press, London
  22. Sutherland, I. W. 1998. Novel and established applications of microbial polysaccharides. TIBTECH 16: 41-46 https://doi.org/10.1016/S0167-7799(97)01139-6
  23. Sutherland, I. W. 2001. Microbial polysaccharides from gram-negative bacteria. Int. Dairy J. 1: 663-674
  24. Thome, L., M. J. Mikolajczak, R. W. Armentrout, and T. J. Pollock. 2000. Increasing the yield and viscosity of exopolysaccharides secreted by Sphingonomas by augmentation of chromosomal genes with multiple copies of cloned biosynthetic genes. J. Ind. Microbiol. Biot. 25: 49-57 https://doi.org/10.1038/sj.jim.7000019
  25. Vartak, N. B., C. C. Lin, J. M. Cleary, M. J. Fagan and M. H. Saier Jr. 1995. Glucose metabolism in Sphingmonas elodea: pathway engineering via construction of a gucose-6-P dehydrogenase insertion mutant. Microbiology 141: 2339-2350 https://doi.org/10.1099/13500872-141-9-2339
  26. Wang, Y. P., A. Kolb, M. Buck, J. Wen, F. O'Gara, and H. Buc. 1998. CRP interacts with promoter-bound ${\sigma}^{54}$ RNA polymerase and blocks transcriptional activation of the dctA promoter. EMBO 17: 786-796 https://doi.org/10.1093/emboj/17.3.786
  27. Wu, J. R., J. H. Son, H. J. Seo, K. H. Kim, Y. K. Nam, J. W. Lee, and S. K. Kim. 2005. Metabolic flux analysis of Beijerinckia indica for PS-7 production. Biotechnol. Bioproc. Eng. 10: 91-98 https://doi.org/10.1007/BF02931189