Isolation and Biochemical Characterization of a Robust Cellulase from Haloalkaliphilic Bacillus sp. E2 of Lonar Crater Lake

Abstract

A haloalkaliphilic bacterial strain, designatedE2, was isolated from the hypersaline and hyperalkaline Lonar Crater Lake and evaluated for itscellulase production potential. Among seven isolates screened on carboxymethyl cellulose (CMC) agar, E2 exhibited the largest hydrolysiszone, indicating superior cellulolytic activity. Morphological characterization suggested its affiliation with the genus Bacillus, which was furthersupported by its ability to thrive under alkaline and saline conditions. Enzyme activity was optimized with respect to pH, temperature, NaClconcentration, nitrogen source, organic solvents, and metal ions. The cellulase displayed broad pH tolerance (7–12) with an optimum at pH11, and maintained high activity over a wide temperature range (40–100°C), peaking at 70°C. Maximum enzyme yield was obtained at 3% NaCl, with substantial activity retained up to 7% NaCl, indicating moderate halotolerance. Among nitrogen sources, yeast extract and tryptonesupported the highest enzyme production, while ammonium chloride was the least effective. The enzyme showed high stability in the presenceof methanol, hexane, and chloroform, but reduced activity with acetone and formaldehyde. Mn²⁺ and Mg²⁺ significantly enhanced cellulaseactivity, whereas Cu²⁺ caused strong inhibition. Kinetic analysis yielded a Km of 0.46% CMC and a Vmax of 178.6 U/mL from Lineweaver–Burkplots, suggesting high catalytic efficiency and good substrate affinity. These results indicate that strain Bacillus Strain E2 produces a thermostable,alkali-tolerant, and salt-tolerant cellulase with potential applications in industrial processes such as textile desizing, detergent formulation,pulp biobleaching, and biomass hydrolysis under extreme conditions.