Dataset Information

Molecular cloning, purification, expression, and characterization of β-1, 4-endoglucanase gene (Cel5A) from Eubacterium cellulosolvens sp. isolated from Holstein steers' rumen.

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°C and pH 4.0. The enzyme was constant at the temperature range of 20°C to 40°C but also, at the pH range of 3 to 9. The metal ions including Ca²⁺, K⁺, Ni²⁺, Mg²⁺, and Fe²⁺ 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 the beneficial characteristics of this enzyme for possible industrial application.

SUBMITTER: Park T

PROVIDER: S-EPMC5838334 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Publications

Molecular cloning, purification, expression, and characterization of β-1, 4-endoglucanase gene (<i>Cel5A</i>) from <i>Eubacterium cellulosolvens</i> sp. isolated from Holstein steers' rumen.

Park Tansol T Seo Seongwon S Shin Teaksoon T Cho Byung-Wook BW Cho Seongkeun S Kim Byeongwoo B Lee Seyoung S Ha Jong K JK Seo Jakyeom J

Asian-Australasian journal of animal sciences 20170918 4

<h4>Objective</h4>This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (<i>Cel5A</i>) from the isolated microorganism.<h4>Methods</h4>To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with <i>Eubacterium cellulosolvens</i> (<i>E. cellulosolvens</i>) Ce2 (Accession number: AB163733). The <i>Cel5A</ ...[more]

PMID: 28920412

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