Production of a halotolerant endo-1,4-β-glucanase by a newly isolated Bacillus velezensis H1 on olive mill wastes without pretreatment: purification and characterization of the enzyme.

Facing the critical issue of high production costs for cellulase, numerous studies have focused on improving the efficiency of cellulase production by potential cellulolytic microorganisms using agricultural wastes as substrates, extremophilic cellulases, in particular, are crucial in the biorefinery process because they can maintain activity under harsh environmental conditions. This study aims to investigate the ability of a potential carboxymethylcellulose-hydrolyzing bacterial strain H1, isolated from an Algerian saline soil and identified as Bacillus velezensis, to use untreated olive mill wastes as a substrate for the production of an endo-1,4-β-glucanase. The enzyme was purified 44.9 fold using only two steps: ultrafiltration concentration and ion exchange chromatography, with final recovery of 80%. Its molecular mass was estimated to be 26 kDa by SDS-PAGE. Enzyme identification by LC-MS analysis showed 40% identity with an endo-1,3-1,4-β-glucanase of GH-16 family. The highest enzymatic activity was significantly measured on barley β-glucan (604.5 U/mL) followed by lichenan and carboxymethylcellulose as substrates, confirming that the studied enzyme is an endo-1,4-β-glucanase. Optimal enzymatic activity was at pH 6.0-6.5 and at 60-65 °C. It was fairly thermotolerant, retaining 76.9% of the activity at 70 °C, and halotolerant, retaining 70% of its activity in the presence of 4 M NaCl. The enzyme had a V of 625 U/min/mL and a high affinity with barley β-glucan resulting a K of 0.69 mg/mL. It also showed a significant ability to release cello-oligosaccharides. Based on such data, the H1 endo-1,4-β-glucanase may have significant commercial values for industry, argo-waste treatment, and other biotechnological applications.