Semi-rationally designed site-saturation mutation of Helicobacter pylori α-1,2-fucosyltransferase for improved catalytic activity and thermostability.

Helicobacter pylori HpfutC, a glycosyltransferase (GT) 11 family glycoprotein, has great potential for industrial 2'-fucosyllactose (2'-FL) production. However, its limited catalytic activity, low expression, and poor thermostability hinder practical applications. Herein, a semi-rationally designed site-saturation mutation was applied to engineer the catalytic activity and thermostability of HpfutC. The 6 single point mutants (K102T, R105C, D115S, Y251F, A255G and K282E) and 6 combined mutants (V1, V2, V3, V4, V5, and V6) with enhanced enzyme activity were obtained by mutant library screening and ordered recombination mutation. The optimal mutant V6, with an optimum temperature of 40 °C, was not a metal-dependent enzyme, yet the reaction was facilitated by Mn2+. Compared to wild-type HpfutC, mutant V6 exhibited a 2.3-fold increase in specific activity and a 2.18-fold increase in half-life at 40 °C, respectively. Kinetic parameters indicated that the Km values of mutant V6 were 34.5 % (lactose) and 25.0 % (GDP-L-fucose) lower than those of the wild enzyme, whereas the kcat/Km values were 1.20 and 1.25-fold higher than those of the wild enzyme. Further, 3D-structure analysis revealed that the highly rigid structure, formation of new hydrogen bonds, increased hydrophobic residues and redistribution of electrostatic charges on the surface may be responsible for the elevated enzyme activity and thermostability. The strategy adopted in this study is of great significance to the solution of the technical bottleneck of HpfutC and the industrial application of 2'-FL.