Cloning and Expression of Levansucrase Gene of Bacillus velezensis BM-2 and Enzymatic Synthesis of Levan.

Levan is a versatile and valuable fructose homopolymer, and a few bacterial strains have been found to produce levan. Although levan products have numerous specific functions, their application and promotion were limited by the production capacity and production cost. Bacillus velezensis BM-2 is a levan-synthesizing strain, but its levan production is too low to apply. In this study, the levansucrase gene of B. velezensis BM-2 was cloned to plasmid pET-32a-Acma-zz, and the recombinant plasmids were transferred to Escherichia coli BL21. A transformed clone was selected to express and secrete the fusion enzymes with an Acma-tag efficiently. The expressed products were further purified by a self-developed separating material called bacterial enhancer matrix (BEM) particles. The purification efficiency was 93.4%, with a specific activity of 16.589 U/mL protein. The enzymatic reaction results indicated that the optimal reaction temperature is 50 °C, the optimal pH of the acetate buffer is 5.6, and the buffer system greatly influenced the enzyme activity. The enzyme activity was enhanced to 130% in the presence of 5 mM Ca²⁺, K⁺, Zn²⁺, and Mn²⁺, whereas it was almost abolished in the case of Cu²⁺ and Fe³⁺. The values of K_m, k_cat, and k_cat/K_m were 17.41 mM, 376.83 s⁻¹, and 21.64 mM⁻¹s⁻¹, respectively. The enzyme amount of 20 U/g sucrose was added to the system containing 400 g/L sucrose, and the levan products with a concentration of 120 g/L reached after an incubation of 18 h, which was 8 times that of the yield before optimization. The results of molecular docking analysis indicated that the Asp86 might act as a nucleophilic catalytic residue for sucrose, Arg246 and Asp247 act as transition state stabilizer of transfructosylation, and Glu340 and Arg306 were recognized as general acid donors. They formed the catalytic-groups triad. The unique properties and catalytic activity of the levansucrase suggest that it deserves further research and might have good industrial application prospects. [ABSTRACT FROM AUTHOR]