Asian-Australasian Journal of Animal Sciences

  • 제29권1호
  • /
  • Pages.126-133
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  • 2016
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  • 1011-2367(pISSN)
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  • 1976-5517(eISSN)
아세아태평양축산학회 (Asian Australasian Association of Animal Production Societies)
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Cloning and Characterization of an Endoglucanase Gene from Actinomyces sp. Korean Native Goat 40

  • Kim, Sung Chan (Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University) ;
  • Kang, Seung Ha (CSIRO Animal, Food and Health Science, Queensland Bioscience Precinct) ;
  • Choi, Eun Young (Department of Biological Science, Silla University) ;
  • Hong, Yeon Hee (Department of Animal Resources Technology, Gyeongnam National University of Science and Technology) ;
  • Bok, Jin Duck (Institute of Green-Bio Science and Technology, Seoul National University) ;
  • Kim, Jae Yeong (Department of Animal Resources Technology, Gyeongnam National University of Science and Technology) ;
  • Lee, Sang Suk (Department of Animal Science and Technology, Sunchon National University) ;
  • Choi, Yun Jaie (Institute of Green-Bio Science and Technology, Seoul National University) ;
  • Choi, In Soon (Department of Biological Science, Silla University) ;
  • Cho, Kwang Keun (Department of Animal Resources Technology, Gyeongnam National University of Science and Technology)
  • 투고 : 2015.07.26
  • 심사 : 2015.11.14
  • 발행 : 2016.01.01
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초록

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-${\beta}$-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) $DH5{\alpha}$. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli $DH5{\alpha}$ harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was $55^{\circ}C$, but it retained over 90% of maximum activity in a broad temperature range ($40^{\circ}C$ to $60^{\circ}C$). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, $K_m$ and $V_{max}$ of rEG1 were 0.39% CMC and 143 U/mg, respectively.

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