Endoglucanase Produced by Bacillus subtilis Strain CBS31: Biochemical Characterization, Thermodynamic Study, Enzymatic Hydrolysis, and Bio-industrial Applications

Microbial cellulases have become the mainstream biocatalysts due to their complex nature and widespread industrial applications. Here, homogeneous endoglucanase GluCB31 from Bacillus subtilis subsp. inaquosorum CBS31 was studied. GluCB31 was purified to 17.68-fold with an 8.33% yield and a specific activity of 1066.37 U/mg. Biochemical properties of GluCB31 were performed and the results are as follows; molecular mass of 35 kDa with an optimum pH at 7.5 and temperature at 50°C. GluCB31 was immobilized in calcium alginate gel and it exhibited the highest activity at 10°C higher temperature than soluble enzyme, as the entrapment in alginate gel made GluCB31 more stable. Kinetic studies showed the V_max of 1293.33 ± 2.51 U/mg and K_m of 0.0183 mg/mL. Enzymatic activity was activated by Tween-20 (106.7%), Tween-80 (111.6%), Triton X-100 (142.3%), SDS (135.5%), Mg⁺⁺ (185.7%), Cu⁺⁺ (167.6%), Zn⁺⁺ (153.7%), Mn⁺⁺ (106.3%), Ba⁺⁺ (181.9%), Ni⁺⁺ (107.2%) while inhibited by Fe⁺⁺ (15.8%), β-mercaptoethanol (46.8%), EDTA (54.5%). Enthalpy, free energy, and entropy of activation were calculated to be 38.526 kJmol^-1, 44.187 kJmol^-1, and -17.518 Jmol^-1K^-1 respectively. Also, ΔG_E-S and ΔG_E-T were found to be -10.75 kJmol^-1 and -45.92 kJmol^-1 respectively. A low ΔS, ΔG_E-S, and ΔG_E-T values were signified enzyme-catalyzed reaction occurs at a fast rate and the existence of the enzyme in its stable state. Cellobiose was the major end product of hydrolysis. These attributes of GluCB31 demonstrated the diversity of catalytic activities and serve in various biotechnological processes, thus deserve to be developed as a bio-industrial agent.