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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Purification and Characterization Sucrose phosohorylase in Leuconostoc mesenteroides NRRL B-1149

Leuconostoc mesenteroides NRRL B-1149의 Sucrose phosohorylase의 분리와 특성 연구

  • Lee Jin Ha (The Engineering Research Institute, Chonnam National University) ;
  • Park Jun Seong (Department of Materials and Biochemical Engineering, Chonnam National University) ;
  • Park Hyen Joung (Department of Materials and Biochemical Engineering, Chonnam National University) ;
  • Cho Jae Young (Department of Materials and Biochemical Engineering, Chonnam National University) ;
  • Choi Jeong Sik (Biology Research Center for Industrial Accelerator, Dongshin University) ;
  • Kim Do Man (School of Biological Sciences and Technology and Research Institute for Catalysis, Chonnam National University)
  • 이진하 (전남대학교 공업기술연구소) ;
  • 박준성 (전남대학교 물질생물화학공학과) ;
  • 박현정 (전남대학교 물질생물화학공학과) ;
  • 조재영 (전남대학교 물질생물화학공학과) ;
  • 최정식 (동신대학교 산업용 가속기 이용 생물 연구센터) ;
  • 김도만 (전남대학교 생명과학기술학부, 촉매연구소)
  • Published : 2004.10.01
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Abstract

Leuconostoc mesenteroides NRRL B-1149 produces various glucoseyltransferases for the synthesis of dextran, levan and glucose-1-phosphate using sucrose as a substrate. A sucrose phosphorylase (1149SPase) was purified from L. mesenteroides NRRL B-1149 culture by using hollow fiber filtration (30 kDa cut off), Toyopearl DEAE 650 M column chromatography and following two times of DEAE-Sepharose column chromatographies. The specific activity of the purified 1149SPase was 25.7 (U/mg) with $16\%$ yield. The 1149SPase showed a molecular size of 56 kDa on denatured $10\%$ SDS-PAGE. The N-terminal amino acid sequence of the enzyme was MEIQNKAM. The optimum pH and temperature of this enzyme were 6.2~6.5 and 37^{circ}C, respectively. It had an apparent K_{m} of 6.0 mM and K_{cat} of 1.62/s for sucrose. 1149SPase crystal was formed by hanging drop diffusion technique using 20 mM calcium chloride dihydrate, 100 mM sodium acetate trihydrate pH 4.6 and $30\%$ 2-methyl-2,4-pentanediol as vaporizing and reservation solution. The 1149SPase catalyzes transferring of glucose from isomaltose or sucrose to salicin and salicyl alcohol by disproportionation reaction or acceptor reaction and synthesized two acceptor products, respectively.

Keywords

References

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