Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Characterization of Leuconostoc mesenteroides B-742CB Dextransucrase Expressed in Escherichia coli

  • Park, Mi-Ran (Department of Biomedical Engineering, Chonnam National University) ;
  • Ryu, Hwa-Ja (Department of Fine Chemical Engineering, Chonnam National University) ;
  • Kim, Do-Man (Division of Chemical Engineering and The Research Institute for Catalysis, Chonnam National University) ;
  • Choe, Jun-Yong (Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University) ;
  • John F. Robyt (Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University)
  • Published : 2001.08.01
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Abstract

Recombinant E. coli DH5$\alpha$ harboring a dextransucrase gene (dsrB742) produced an extracellular dextransucrase in a 2% sucrose medium. The enzyme was purified by DEAE-Sepharose and Phenyl-Sepharose column chromatographies upto a 142.97-fold purification with a 11.11% recovery to near homogeneity. The enzyme had a calculated molecular mass of 168.6 kDa, which was in good agreement with the activity band of 170 kDa on a nondenaturing SDS-PAGE. An expression plasmid was constructed by inserting the dsrB742 into a pRSET expression vector. The activity after expression in E. coli BL21(DE3)pLysS increased about 6.7-fold compared to the extracellular dextransucrase from L. mesenteroides B-742CB. The expressed and purified enzyme from the clone showed similar biochemical properties (acceptor reaction, size of active dextransucrase, optimum pH, and temperature) to B-742CB dextransucrase, however, the ability to synthesize ${\alpha}$-(1$\rightarrow$3) branching decreased in comparison to that of L. mesenteroides B-742CB dextransucrase.

Keywords

References

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