Investigation of enzymatic levan production in thermophilic bacteria

Exopolysaccharides (EPS) are branched structures found either as a capsule structure tightly bound to the cell surface or as a mucous (slime) structure loosely attached to the surface of the cell. There are many types of EPS. One of them is the levan polysaccharide. The formation of levan is carried out through the transfructosylation activity of levansucrase. The β-(2-1) bonds in sucrose are broken, and fructosyl groups are transferred to acceptor molecules such as sucrose and free glucose. Through this activity, levansucrases (EC 2.4.1.10) catalyze the formation of levan and fructooligosaccharides (FOS). The levan molecule is formed by the linking of fructose units via β-(2-6) and β-(2-1) bonds. Levan, a completely biocompatible polysaccharide, is considered one of the most promising microbial EPS for industrial sectors due to its antioxidant, anticancer, antibiotic, and prebiotic properties. In this study, levansucrase production, which is important for levan synthesis, was investigated using the thermophilic Bacillus zhangzhouensis 2CA and Bacillus licheniformis 2CS strains, isolated and identified from a hot spring in Çermik district of Diyarbakır province. Enzyme activity was determined by the 3,5-dinitrosalicylic acid (DNS) method, and protein concentration was determined by the Lowry method. For the optimization of levansucrase production, different sucrose concentrations (0.5–3.0%), time-dependent (3–60 hours) enzyme production, different pH levels (4.0–11.0), and different temperatures (40–70 °C) were used. The enzyme was partially purified using salt precipitation, ion exchange, and gel filtration chromatography methods. The temperature and pH stability of the purified enzyme, as well as the effects of different metals and chemicals, were investigated. It was determined that enzyme production for the B. zhangzhouensis 2CA strain occurred at 24 hours in the presence of 1% sucrose, and the optimum enzyme activity was at pH 6.0 and 50 °C; while for the B. licheniformis 2CS strain, maximum enzyme production occurred at 36 hours in the presence of 2% sucrose, and the optimum enzyme activity was at pH 7.0 and 60 °C. It was determined that the purified enzyme maintained its stability at the optimum pH and temperature values for both strains and that enzyme activity increased in the presence of Ca²⁺. The study revealed optimized conditions that can serve as a model for efficient levansucrase production for various commercial and industrial applications for both strains.