Understanding the Endo- and Exo-mechanisms Involved in the Enzymatic Hydrolysis of Levan and Inulin Polymers

This study explores the endo-levanase from <i>Bacillus licheniformis</i> (LevB1), providing new insights into how this enzyme selectively hydrolyzes levan chains. By analyzing the first resolved crystal structure of LevB1, conducting detailed simulations, and comparing it to other endo- and exo-fructanases, we identified key factors underlying its specificity. Experiments designed to explore this specificity revealed the critical role of three minus and three plus subsites in determining the enzyme's endo-specificity. We identified six specific subsites essential for the enzyme's ability to cleave levan chains at random internal linkages (endo-specificity) rather than at defined fructosyl nonreducing ends (exo-specificity). This research underscores the importance of enzyme-fructan interaction stability during the catalytic reaction in this process, highlighting the need for dynamic modeling to fully capture enzyme specificity, as conventional docking alone cannot fully explain the stability and motion of carbohydrate chains in the catalytic site. These findings contribute to a deeper understanding of the factors that influence endo- and exo-cleavage specificity in levan and inulin polymers, with broader implications for fructan metabolism and, eventually, the industrial production of fructose and/or fructo-oligosaccharides.