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

Objective: This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene () 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 () Ce2 (Accession number: AB163733). The from isolated sp. was cloned using the published genome sequence and expressed through the BL21.

Results: The maximum activity of recombinant () 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, K, Ni, Mg, and Fe increased the endoglucanase activity but the addition of Mn, Cu, and Zn decreased. The Km and Vmax value of were 14.05 mg/mL and 45.66 μmol/min/mg. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of was 96.69 (s) and 6.88 (mL/mg/s), respectively.

Conclusion: Our results indicated that of 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.