Expression of the eg gene of cellulase from cattle rumen in Lactobacillus and analysis of enzymatic properties

In order to construct highly expressing endoglucanase genetically engineered bacteria, the whole genome of the microorganism in bovine rumen juice was used as a template in this study, and the eg fragment was obtained by PCR amplification In order to construct genetically engineered bacteria with high endoglucanase expression, we used the whole genomes of microorganisms in bovine rumen juice as a template. We obtained a fragment containing the eg gene of cellulase by PCR amplification, which was cloned into the expression vector pMG36e to yield the expression vector pMG36e::eg. Recombinant plasmids containing the pMG36e::eg construct were electrotransduced into lactic acid bacteria (Lactococcus lactis NZ9000) to obtain an L. lactis NZ9000/pMG36e::eg recombinant strain, and the fermentation supernatant of the recombinant strain was concentrated using the 10% trichloroacetic acid/acetone precipitation method. Enzyme activity of the recombinant endoglucanase was determined using the 3, 5-dinitrosalicylic acid (DNS) and Congo red staining methods, total enzyme activity was determined using the filter paper enzyme activity (FPA) method, and enzymatic properties were examined. A gene of approximately 1 500 bp was cloned from a bovine rumen microorganism and the molecular weight of the encoded enzyme was approximately 50 kDa. Congo red staining analysis revealed that the recombinant enzyme caused a clear 2.32 cm zone of hydrolysis. The enzyme activity of the recombinant protein was 12.401 9 U·mL⁻¹ based on the DNS method and 12.246 9 U·mL⁻¹ using the FPA method. Furthermore, the recombinant protease has enzymatic activity on CMC-Na, filter paper, microcrystalline cellulose, and absorbent cotton. Enzyme activity was found to be optimal at a temperature of 90 °C and pH 6. Metal ions, including Cu^{2 +} , Mn^{2 +} , Ba^{2 +} , Zn^{2 +} , and Co^{2 +} , were found to promote activity of the recombinant enzyme, whereas Fe^{2 +} inhibited recombinant endoglucanase activity. In this study, we thus demonstrated the stable high-efficiency expression of the cellulase eg gene in L. lactis NZ9000, the practical utilization of which will contribute to improving the nutritional value of silage and digestibility of cattle feed.