Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Gene Cloning of Streptomyces Phospholipase D P821 Suitable for Synthesis of Phosphatidylserine

  • Moon Min-Woo (Laboratory of Microbial Genomes, Korea Research Institute of Bioscience and Biotechnology, Department of Biotechnology, College of Engineering, Yonsei University) ;
  • Lee Jung-Kee (Laboratory of Microbial Genomes, Korea Research Institute of Bioscience and Biotechnology) ;
  • Oh Tae-Kwang (Laboratory of Microbial Genomes, Korea Research Institute of Bioscience and Biotechnology) ;
  • Shin Chul-Soo (Department of Biotechnology, College of Engineering, Yonsei University) ;
  • Kim Hyung-Kwoun (Division of Biotechnology, The Catholic University of Korea)
  • Published : 2006.03.01
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Abstract

A strain, P821, with phospholipase D activity was isolated from soil and identified as a Streptomyces species. The phospholipase D enzyme was purified from a culture broth of the isolated strain using ammonium sulfate precipitation and DEAE-Sepharose, phenyl-Sepharose, and Superose 12 HR column chromatographies. The purified enzyme exhibited an optimum temperature and pH of $55^{\circ}C$ and 6.0, respectively, in the hydrolysis of phosphatidylcholine and remained stable up to $60^{\circ}C$ within a pH range of 3.5-8.0. The enzyme also catalyzed a transphosphatidylation reaction to produce phosphatidylserine with phosphatidylcholine and serine substrates. The optimum conditions for the transphosphatidylation were $30^{\circ}C$ and pH 5.0, indicating quite different optimum conditions for the hydrolysis and transphosphatidylation reactions. The gene encoding the enzyme was cloned by Southern hybridization and colony hybridization using a DNA probe designed from the conserved regions of other known phospholipase D enzymes. The resulting amino acid sequence was most similar to that of the PLD enzyme from Streptomyces halstedii (89.5%). Therefore, the enzyme was confirmed to be a phospholipase D with potential use in the production of phosphatidylserine.

Keywords

References

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