Purification and Characterization of β-Glucanase Activity Protein from B. velezensis 2-6

Objective: The aim of this work was to purify and identify the β-glucanase active protein from the fermentation supernatant of Bacillus velezensis 2-6 which originated from mangrove, and to investigate the enzymatic characteristics of the purified β-glucanase active protein. Methods: The ammonium sulfate precipitation method and DEAE-Sepharose Fast Flow ion exchange chromatography were used to purify the β-glucanase active protein from the supernatant of Bacillus velenzensis 2-6. LC-MS/MS analysis was used to identify the purified protein. The enzyme activity of β-glucanase active protein was analyzed using DNS method. Thin layer chromatography was used to detect the degradation level of β-glucanase active protein on laminarin. Filter paper method was used to analyze the antibacterial activity of β-glucanase active protein on pathogenic microorganisms. Results: A protein with a molecular weight of approximately 45 kDa was obtained from strain 2-6 fermentation supernatant by 80% ammonium sulfate precipitation and DEAE-Sepharose Fast Flow ion-exchange chromatography, and the β-glucanase activity of this protein reached 36956.5 U/mg. The purified protein was deduced as glucuronic acid xylanase by LC-MS/MS which consisted of 423 amino acids and named as GxylE-v26. The optimal enzyme reaction temperature of GxylE-v26 was 60 ℃ and the optimal pH was 4. Low concentration of Fe²⁺ (1 mmol/L) could promote the enzyme activity of GxylE-v26 while medium (10 mmol/L) and high (100 mmol/L) concentration of Fe²⁺, Ca²⁺, Mg²⁺ and Ba²⁺ could inhibit the enzyme activity of GxylE-v26. GxylE-v26 could hydrolyze laminarin and exhibit inhibitory effects on the growth of Staphylococcus aureus and Pantoea ananatis. The inhibition zone diameters of 1 mg/mL GxylE-v26 against S. aureus and P. ananatis were 1 cm and 1.2 cm, respectively. Conclusion: A β-glucanase active protein, named GxylE-v26, was purified and identified from the fermentation supernatant of Bacillus velezensis 2-6 which derived from mangrove. GxylE-v26 was an acid enzyme which could tolerate a certain degree of high temperature and possess antimicrobial activity against Staphylococcus aureus and Pantoea ananas.