Characterization of a Thermostable Endoglucanase Involved in Cellulose Degradation from a Newly Isolated Bacillus subtilis SCUEC7 Strain

With the aim of screening highly efficient cellulose-degrading microorganisms and enhancing the efficiency of cellulose degradation, a strain with a high ability to degrade cellulose was isolated from bovine rumen fluid, which was classified and named as Bacillus subtilis SCUEC7 strain on the basis of the morphological observation, physiological and biochemical characteristics and 16S rDNA sequence analysis. However, the mechanism for cellulose degradation in this strain remains unclear, therefore, the function and biological properties of the ba7gA gene in the strain SCUEC7 were analyzed. The ba7gA gene was cloned by PCR with total DNA of B. subtilis SCUEC7 as template and used to construct the recombinant plasmid pET28a(+)-ba7gA, and expressed in Escherichia coli BL21(DE3). The overexpression of the Ba7gA protein was detected by SDS-PAGE analysis, and the effects of temperature, pH, and metal ions on the biological activities of the Ba7gA protein were studied. The endoglucanase gene ba7gA, which is composed of 1482 bp and encodes 493 amino acids, was cloned and sequenced from this strain. The Ba7gA protein contains two primary functional domains: the GH5 family catalytic domain and the CBM3 family cellulose-binding domain, and exhibited a molecular weight of approximately 54.5 kDa by SDS-PAGE, in agreement with a molecular weight of 54.53 kDa based on the amino acid sequence. The optimal reaction conditions for the biological activities of Ba7gA protein with CMC-Na as the substrate were determined to be 60°C and pH 6.0. Meanwhile, Fe2+, Mg2+, Mn2+, Ca2+, and Ni2+ enhanced its biological activity, whereas Cu2+, Na+, K+, Zn2+, Fe3+, and Li+ exhibited inhibitory effects. The suitable storage conditions for the Ba7gA protein were 0°C and pH 6.0. Using CMC-Na as the substrate, the KM and Vmax of the Ba7gA protein were 2.35 mM and 54.56 U/mg, respectively. These properties highlight the potential application of the Ba7gA protein from B. subtilis SCUEC7 strain as an endocellulase in the pretreatment of biomass.