College of Food and Biological Engineering, Hefei University of Technology, Hefei, China.
College of Food and Biological Engineering, Hefei University of Technology, Hefei, China.
Int J Biol Macromol. 2023 Jul 31;244:125442. doi: 10.1016/j.ijbiomac.2023.125442. Epub 2023 Jun 15.
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.
来自枯草芽孢杆菌的蔗聚糖蔗糖酶（SacB）的催化产物主要由 10%的高分子量蔗聚糖（HMW，2000 kDa）和 90%的低分子量蔗聚糖（LMW，7000 Da）组成。为了实现食品水胶体的高效生产，借助分子动力学模拟软件，发现了一个蛋白质自组装元件 Dex-GBD，并将其融合到 SacB 的 C 末端，构建了一种新型融合酶 SacB-GBD。SacB-GBD 的产物分布与 SacB 相比发生了反转，HMW 在总多糖中的比例显著增加至>95%。我们随后通过 SDS 同时调节 SacB-GBD 颗粒大小和产物分布，证实了自组装是导致 SacB-GBD 产物分布反转的原因。通过分子模拟和疏水性测定分析，疏水力可能是自组装的主要驱动力。我们的研究为 HMW 的工业生产提供了酶源，并为指导蔗聚糖蔗糖酶向催化产物大小的分子修饰提供了新的理论依据。
