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Agarivorans sp. JA-1 유래 신규 GH-16 β-agarase의 클로닝, 발현 및 특성

Cloning, Expression, and Characterization of a Novel GH-16 β-Agarase from Agarivorans sp. JA-1

  • 전명제 (신라대학교 의생명과학대학 제약공학과) ;
  • 김아람 (신라대학교 의생명과학대학 제약공학과) ;
  • 이동근 (신라대학교 의생명과학대학 제약공학과) ;
  • 이상현 (신라대학교 의생명과학대학 제약공학과)
  • Jeon, Myong Je (Department of Pharmaceutical Engineering, College of Medical Life Sciences, Silla University) ;
  • Kim, A-Ram (Department of Pharmaceutical Engineering, College of Medical Life Sciences, Silla University) ;
  • Lee, Dong-Geun (Department of Pharmaceutical Engineering, College of Medical Life Sciences, Silla University) ;
  • Lee, Sang-Hyeon (Department of Pharmaceutical Engineering, College of Medical Life Sciences, Silla University)
  • 투고 : 2012.10.23
  • 심사 : 2012.11.14
  • 발행 : 2012.11.30

초록

이전 연구에서 저자들이 Glycoside hydrolase family 50 (GH-50)과 GH-118 ${\beta}$-agarase들의 발현과 특성을 보고한 Agarivorans sp. JA-1 균주로부터 신규의 GH-16 ${\beta}$-agarase를 보고하고자 한다. 본 유전자는 1,362 염기쌍으로 구성되어 있으며, 453 아미노산 잔기로 구성된 49,830 Da의 단백질을 암호화한다. 본 효소는 Pseudoalteromonas sp. CY24 유래의 GH-16 ${\beta}$-agarase와 98%의 염기서열 상동성과 99%의 아미노산서열 상동성을 나타냈다. 신호서열을 제외한 429 아미노산으로 구성된 성숙단백질에 해당하는 유전자를 E. coli BL21 (DE3) 세포에서 재조합 발현시킨 후, 친화성 크로마토그래피로 효소를 정제하였다. 정제된 효소는 $40^{\circ}C$와 pH 5.0에서 67.6 U/mg의 최적 활성을 보였다. 아가로스를 기질로 한 효소분해산물의 박막크로마토그래피 분석결과, neoagarohexaose와 neoagarotetraose가 주산물로 생산되는 것을 알 수 있었다. 본 효소는 기능성 한천올리고당의 산업적 생산에 활용 가능할 것으로 기대된다.

Authors report the glycoside hydrolase (GH) family 16 ${\beta}$-agarase from the strain of Agarivorans sp. JA-1, which authors previously stated as recombinant expression and characterization of GH-50 and GH-118 ${\beta}$-agarase. It comprised an open reading frame of 1,362 base pairs, which encodes a protein of 49,830 daltons consisting of 453 amino acid residues. Valuation of the total sequence showed that the enzyme has 98% nucleotide and 99% amino acid sequence similarities to those of GH-16 ${\beta}$-agarase from Pseudoalteromonas sp. CY24. The gene corresponding to a mature protein of 429 amino acids was recombinantly expressed in Escherichia coli, and the enzyme was purified to homogeneity by affinity chromatography. It showed maximal activity at $40^{\circ}C$ and pH 5.0, representing 67.6 units/mg. Thin layer chromatography revealed that mainly neoagarohexaose and neoagarotetraose were produced from agarose. The enzyme would be valuable for the industrial production of functional neoagarooligosaccharides.

키워드

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