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Asian-Australas J Anim Sci > Accepted Articles
DOI: https://doi.org/10.5713/ajas.17.0552    [Accepted] Published online September 18, 2017.
Molecular cloning, purification, expression, and characterization of β-1, 4-endoglucanase gene (Cel5A) from Eubacterium cellulosolvens sp. isolated from Holstein steer’s rumen
Tansol Park1,2, Seongwon Seo3, Teaksoon Shin4, Byung-Wook Cho4, Seongkeun Cho4, Byeongwoo Kim4, Jong K. Ha1, Jakyeom Seo4,* 
1Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University, Seoul 151-951, Korea
2Department of Animal Sciences, The Ohio State University, Columbus, Ohio, USA, Columbus, United States
3Department of Animal Biosystem Sciences, Chungnam National University, Daejeon 305-764, Korea
4Life and Industry Convergence Research Institute, Department of Animal Science, Pusan National University, Miryang, Korea
Correspondence:  Jakyeom Seo, Tel: +82-55-350-5513, Fax: +82-55-350-5519, Email: jseo81@pusan.ac.kr
Received: 26 July 2017   • Revised: 25 August 2017   • Accepted: 4 September 2017
Abstract
Objective: This study was conducted to isolate the cellulolytic microorganism from a rumen of Holstein steer and characterize endoglucanase gene (Cel5A) from the isolated microorganism.

Methods

To isolate anaerobic microbes having endoglucanase, the rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 99% similarity with Eubacterium cellulosolvens Ce2 (Accession number: AY178842.1). The Cel5A from isolated E. cellulosolvens sp. was cloned using the published genome sequence and expressed through the E. coli BL21.

Results

The maximum activity of recombinant Cel5A (rCel5A) was observed at 50°C and pH 4.0. The enzyme was constant at the temperature range of 20 to 40°C but also, at the pH range of 3 to 9. The metal ions including K+, and Fe2+ significantly increased the endoglucanase activity but the addition of Mn2+, Cu2+, and Zn2+ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and 45.66 µmol/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 beneficial characteristics of this enzyme for possible industrial application.
Keywords: Rumen bacteria; Eubacterium cellulosolvens sp.; Endoglucanase; Optimal condition


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