Asian-Australasian Journal of Animal Sciences

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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Molecular cloning, purification, expression, and characterization of β-1, 4-endoglucanase gene (Cel5A) from Eubacterium cellulosolvens sp. isolated from Holstein steers' rumen

  • Park, Tansol (Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University) ;
  • Seo, Seongwon (Department of Animal Biosystem Sciences, Chungnam National University) ;
  • Shin, Teaksoon (Life and Industry Convergence Research Institute, Department of Animal Science, Pusan National University) ;
  • Cho, Byung-Wook (Life and Industry Convergence Research Institute, Department of Animal Science, Pusan National University) ;
  • Cho, Seongkeun (Life and Industry Convergence Research Institute, Department of Animal Science, Pusan National University) ;
  • Kim, Byeongwoo (Life and Industry Convergence Research Institute, Department of Animal Science, Pusan National University) ;
  • Lee, Seyoung (Division of Animal Husbandry, Yonam College) ;
  • Ha, Jong K. (Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University) ;
  • Seo, Jakyeom (Life and Industry Convergence Research Institute, Department of Animal Science, Pusan National University)
  • Received : 2017.07.26
  • Accepted : 2017.09.04
  • Published : 2018.04.01
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Abstract

Objective: This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated microorganism. Methods: To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens) Ce2 (Accession number: AB163733). The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21. Results: The maximum activity of recombinant Cel5A (rCel5A) was observed at $50^{\circ}C$ and pH 4.0. The enzyme was constant at the temperature range of $20^{\circ}C$ to $40^{\circ}C$ but also, at the pH range of 3 to 9. The metal ions including $Ca^{2+}$, $K^+$, $Ni^{2+}$,$Mg^{2+}$, and $Fe^{2+}$ increased the endoglucanase activity but the addition of $Mn^{2+}$, $Cu^{2+}$, and $Zn^{2+}$ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and $45.66{\mu}mol/min/mg$. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was $96.69(s^{-1})$ and 6.88 (mL/mg/s), respectively. Conclusion: Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.

Keywords

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