Rational design of a self-assembly promoting fusion domain enhances high molecular weight levan synthesis by levansucrase SacB

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.