The catalytic product of levansucrase from Bacillus subtilis (SacB) is mainly composed of 10 % high molecular weight levan (HMW, ~2000 kDa) and 90 % low molecular weight levan (LMW, ~7000 Da). In order to achieve efficient production of food hydrocolloid, high molecular weight levan (HMW), with the help of molecular dynamics simulation software, a protein self-assembly element, Dex-GBD, was found and fused with the C-terminus of SacB to construct a novel fusion enzyme, SacB-GBD. The product distribution of SacB-GBD was reversed compared with SacB, and the proportion of HMW in the total polysaccharide was significantly increased to >95 %. We then confirmed that the self-assembly was responsible for the reversal of the SacB-GBD product distribution by the simultaneous modulation of SacB-GBD particle size and product distribution by SDS. The hydrophobic effect may be the main driver of self-assembly as analyzed by molecular simulations and hydrophobicity determination. Our study provides an enzyme source for the industrial production of HMW and provides a new theoretical basis for guiding the molecular modification of levansucrase towards the size of the catalytic product.

枯草芽孢杆菌左聚蔗糖酶的催化产物(SacB) 主要由 10% 高分子量果聚糖 (HMW, ~2000 kDa) 和 90% 低分子量果聚糖 (LMW, ~7000 Da) 组成。为了实现食品亲水胶体高分子量果聚糖（HMW）的高效生产，借助分子动力学模拟软件，找到了一种蛋白质自组装元件Dex-GBD，并与SacB的C端融合，构建了一种新型融合酶，SacB-GBD。SacB-GBD的产物分布与SacB相比发生了逆转，且HMW占总多糖的比例显着提高至>95%。然后，我们通过 SDS 同时调节 SacB-GBD 粒径和产物分布，证实自组装导致了 SacB-GBD 产物分布的逆转。通过分子模拟和疏水性测定分析，疏水效应可能是自组装的主要驱动力。我们的研究为HMW的工业化生产提供了酶源，并为指导果聚糖蔗糖酶的分子修饰向催化产物的尺寸方向提供了新的理论基础。