Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Large Increase in Leuconostoc citreum KM20 Dextransucrase Activity Achieved by Changing the Strain/Inducer Combination in an E. coli Expression System

  • Ko, Jin-A (Infection Control Material Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Jeong, Hyung-Jae (Infection Control Material Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Ryu, Young-Bae (Infection Control Material Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Park, Su-Jin (Infection Control Material Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Wee, Young-Jung (Department of Food Science and Technology, Yeungnam University) ;
  • Kim, Do-Man (School of Biological Sciences and Technology and the Research Institute for Catalysis, Chonnam National University) ;
  • Kim, Young-Min (Infection Control Material Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Lee, Woo-Song (Infection Control Material Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
  • Received : 2011.11.11
  • Accepted : 2011.12.08
  • Published : 2012.04.28
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Abstract

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction ($OD_{600}$) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of $17^{\circ}C$. These results may effectively apply to the heterologous expression of other large DexT genes.

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References

  1. Ashipala, O. K. and Q. He. 2008. Optimization of fibrinolytic enzyme production by Bacillus subtilis DC-2 in aqueous two-phase system (poly-ethylene glycol 4000 and sodium sulfate). Bioresour. Technol. 99: 4112-4119. https://doi.org/10.1016/j.biortech.2007.09.029
  2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  3. Britton, H. T. S. and R. A. Robinson. 1931. Universal buffer solutions and dissociation constant of veronal. J. Chem. Soc. 1456-1462.
  4. Choi, I. K., S. H. Jung, B. J. Kim, S. Y. Park, J. Kim, and H. U. Han. 2003. Novel Leuconostoc citreum starter culture system for the fermentation of kimchi, a fermented cabbage product. Antonie Van Leeuwenhoek 84: 247-253. https://doi.org/10.1023/A:1026050410724
  5. Henrissat, B. and G. Davies. 1997. Structural and sequence based classification of glycoside hydrolases. Curr. Opin. Struct. Biol. 7: 637-644. https://doi.org/10.1016/S0959-440X(97)80072-3
  6. Jansson. J. C. 1987. On the history of the development of Sephadex. Chromatographia 23: 361-370. https://doi.org/10.1007/BF02316183
  7. Kilikian, B. V., I. D. Suarez, C. W. Liria, and A. K. Gombert. 2000. Process strategies to improve heterologous protein production in Escherichia coli under lactose or IPTG induction. Process Biochem. 35: 1019-1025. https://doi.org/10.1016/S0032-9592(00)00137-0
  8. Kim, J. F., H. Jeong, J. S. Lee, S. H. Choi, M. Ha, C. G. Hur, et al. 2008. Complete genome sequence of Leuconostoc citreum KM20. J. Bacteriol. 190: 3093-3094. https://doi.org/10.1128/JB.01862-07
  9. Kim, Y. M., M. Y. Seo, H. K. Kang, A. Kimura, and D. Kim. 2009. Construction of a fusion enzyme of dextransucrase and dextranase: Application for one-step synthesis of isomalto-oligosaccharides. Enzyme Microbial Technol. 44: 159-164. https://doi.org/10.1016/j.enzmictec.2008.10.007
  10. Malten, M., R. Hollmann, W. D. Deckwer, and D. Jahn. 2005. Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium. Biotechnol. Bioeng. 89: 206-218. https://doi.org/10.1002/bit.20341
  11. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428. https://doi.org/10.1021/ac60147a030
  12. Monchois, V., R. M. Willemot, M. Remaud-Simeon, C. Croux, and P. Monsan. 1996. Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only ${\alpha}$(1-6) and ${\alpha}$(1-3) linkages. Gene 182: 23-32. https://doi.org/10.1016/S0378-1119(96)00443-X
  13. Neubauer, H., A. Bauche, and B. Mollet. 2003. Molecular characterization and expression analysis of the dextransucrase DsrD of Leuconostoc mesenteroides Lcc4 in homologous and heterologous Lactococcus lactis cultures. Microbiology 149: 973-982. https://doi.org/10.1099/mic.0.26029-0
  14. Ryu, H. J., X. G. Jin, J. H. Lee, H. J. Woo, Y. M. Kim, G. H. Kim, et al. 2010. Optimal expression and characterization of fusion enzyme having dextransucrase and dextranase activities. Enzyme Microbial. Technol. 47: 212-215. https://doi.org/10.1016/j.enzmictec.2010.07.006
  15. Seo, E. S., D. Kim, J. F. Robyt, D. F. Day, D. W. Kim, H. J. Park, et al. 2004. Modified oligosaccharides as potential dental plaque control materials. Biotechnol. Prog. 20: 1550-1554. https://doi.org/10.1021/bp049883e
  16. Su, D. and J. F. Robyt. 1993. Control of the synthesis of dextan and acceptor-products by Leuconosotc mesenteroides B-512FM dextransucrase. Carbohydr. Res. 248: 471-476.
  17. van Hijum, S. A., S. Kralj, L. K. Ozimek, L. Dijkhuizen, and I. G. van Geel-Schutten. 2006. Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria. Microbiol. Mol. Biol. Rev. 70: 157-176. https://doi.org/10.1128/MMBR.70.1.157-176.2006
  18. Yang, Y., J. Luo, J. Wang, D. Teng, and Z. Tian. 2008. Expression and characterization of dextransucrase gene dsrX from Leuconostoc mesenteroides in Escherichia coli. J. Biotechnol. 133: 505-512. https://doi.org/10.1016/j.jbiotec.2007.12.002
  19. Zhang, H. B., X. Q. Mao, Y. J. Wang, and X. Q. Hu. 2009. Optimization of cultivation conditions for high-level expression of dextransucrase in Escherichia coli. J. Food Agric. Environ. 7: 75-78.

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