Cloning of Isoamylase Gene of Pectobacterium carotovorum subsp. carotovorum LY34 and Identification of Essential Residues of Enzyme

Pectobacterium carotovorum subsp. carotovorum LY34에서 Lsoamylase 유전자 클로닝 및 효소 활성의 필수 잔기 확인

  • Cho, Kye-Man (Division of Applied Life Science, Gyeongsang National University) ;
  • Kim, Eun-Ju (Division of Applied Life Science, Gyeongsang National University) ;
  • Math, Renukaradhya K. (Division of Applied Life Science, Gyeongsang National University) ;
  • Asraful Islam, Shah Md. (Division of Applied Life Science, Gyeongsang National University) ;
  • Hong, Sun-Joo (Division of Applied Life Science, Gyeongsang National University) ;
  • Kim, Jong-Ok (Division of Applied Life Science, Gyeongsang National University) ;
  • Shin, Ki-Jae (Division of Applied Life Science, Gyeongsang National University) ;
  • Lee, Young-Han (Division of Plant Environmental Research, Gyeongsangnam-do Agricultural Research and Extension Service) ;
  • Kim, Hoon (Department of Bio-environmental chemistry, Sunchon National University) ;
  • Yun, Han-Dae (Research Institute of Agriculture & Life Science, Gyeongsang, National University)
  • Published : 2007.09.30


The gene encoding for isoamylase of the Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 was cloned and expressed into Escherichia coli $DH5{\alpha}$. Isoamylase catalyzes the hydrolysis of ${\alpha}-1,6-glycosidic$ linkages specifically in amylopectin, glycogen, and derived oligosaccharides, while the enzyme did not hydrolyze ${\alpha}-1,4-glycosidic$ linkages of amylose. The isoamylase gene (glgX) had an open reading frame of 1,977 bp encoding 658 amino acid residues with a calculated molecular weight of 74,188 Da. The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS-PA gel. The mature GlgX had a calculated pI of 4.91. Isoamylase from Pcc LY34 had 70% amino acid identity with isoamylase from Pectobacterium chrysanthemi and contained the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7.0 and $40^{\circ}C$. GlgX was $Ca^{2+}-dependent$. The changes of Asp-335, Glu-370, and Asp-442 into Ala, respectively, using site-directed mutagenesis techniques showed that three residues are essential to isolamyalse (GlgX) activity. The sequences around those residues were highly conserved in isoamylase of different origins and GlgX of the glg operon in glycongen biosynthesis.


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