생명과학회지 (Journal of Life Science)

한국생명과학회 (Korean Society of Life Science)
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Pectobacterium carotovorum subsp. carotovorum LY34에서 Lsoamylase 유전자 클로닝 및 효소 활성의 필수 잔기 확인

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

  • 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)
  • 발행 : 2007.09.30
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초록

연부균인 Pectobacterium carotovorum subsp. carotovorum LY34로부터 이소아밀라제 유전자 (glgX)를 클로닝한 후 대장균 숙주에서 발현시켰다. 이 효소는 ${\alpha}-1$,6-글루코시드 결합을 가수분해하였으나 ${\alpha}-1$,4-글루코시드 결합은 가수분해 하지 못하였다. 유전자는 658개의 아미노산을 암호화하는 1,977개의 DNA 염기서열로 이루어져 있었고 이 유전자에 의해 암호화되는 아미노산 서열을 다른 아밀라제 효소들과 비교한 결과 이소아밀라제 유전자와 유사하였으며 4개의 보존 지역을 확인하였다. SDS-PAGE에 의해 확인된 단백질의 크기는 약 74 kDa 이었다. 효소 활성은 pH 7.0, $40^{\circ}C$에서 가장 높은 활성을 나타났으며 $Ca^{2+}$ 첨가로 활성이 증가되었다. 이 효소의 보존되어 있는 아미노산 중에 글루탐산 370번, 아스파르트산 335번 및 442번 잔기를 알라닌으로 치환시킨 결과 활성이 약해졌다. 이 결과로부터 이들 잔기들이 효소활성에 중요한 역할을 하는 것으로 추정된다.

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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