Levan is widely used as food additives. Its utilization is significantly influenced by its molecular weight. Bacillus subtilis levansucrase (Bs-SacB) and Priestia megaterium levansucrase (Pm-SacB) yield levan of different weights. To delve deeper into the molecular underpinnings of the molecular weight disparity between the products of these two enzymes, we conducted a focused study on the eight loops surrounding the active sites of Bs-SacB and Pm-SacB and identified Loop3 and loop4 as critical determinants in changing the molecular weight of Bs-SacB 's products. Subsequently, leveraging mutation energy analysis and non-homologous substitution strategies, we crafted tailored modifications in loop3 and loop4, yielding a spectrum of mutant enzymes that exhibit diverse molecular weight profiles including F182Y (3698 Da), CYTI (3093 Da), 3-Pbl (2776 Da), 4-Bml (1845 Da), and F182K (1571 Da). This research provide a novel comparative molecular dynamics approach to change product molecular weight and it is successfully applied in the modification of levansucrase.
Keywords: Levan; Levansucrase; Product spectrum; Rational design.
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