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

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Objective: This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated micro organism.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 Ca 2+ , K + , Ni 2+ , Mg 2+ , and Fe 2+ increased the endoglu canase 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 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.