Cloning and expression of the endoglucanase gene from cattle rumen in Escherichia coli

A high cellulose degradation efficiency is vital in industrial settings. In this study, we constructed an Escherichia coli strain overexpressing recombinant endoglucanase that can efficiently hydrolyze cellulose. Using the genome of bovine rumen juice microorganisms as a template, the endoglucanase eg gene was amplified by PCR. The PCR product was ligated to pET-28a to yield the expression vector pET-28a::eg. pET-28a::eg was transformed into E. coli strain BL21 (DE3), and the expression of the EG protein in E. coli BL21/pET-28a::eg was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Finally, the enzymatic activity of the recombinant endoglucanase was measured using the 3,5-Dinitrosalicylic acid (DNS) method, and its enzymatic properties were determined. The results showed that the expression vector pET-28a::eg was successfully constructed. EG expression was induced with IPTG in E. coli BL21/pET-28a::eg at 28 ℃ for 14 h. The molecular weight of the recombinant EG protein was approximately 50 kDa after purification, with a hydrolysis circle visible with Congo red staining. The enzyme activity of EG is 12.60 U·mL⁻¹ and the total enzyme activity of filter paper is 3.53 U·mL⁻¹. Among the reactions of recombinase with different substrates, the enzyme activity towards sodium CMC-Na was the highest, and the activity towards absorbent cotton was the lowest. The optimal temperature for the recombinant enzyme was 40 ℃, and the optimal pH was 7.0. Under these conditions, Ca²⁺, Mg²⁺, Fe²⁺, K⁺, and Mn²⁺ plasma all contributed to the enzymatic activity of EG. While Zn²⁺ also contributed to EG activity, the contribution was not significant, whereas Hg²⁺ and Cu²⁺ inhibited enzyme activity. The construction of a bacterial strain harboring recombinant endoglucanase capable of efficiently degrading cellulose lays the foundation for the industrial application of endoglucanases.