Simultaneous improvement of catalytic efficiency and thermostability in GH11 xylanase via linker disulfide bond engineering

The industrial application of certain GH11 xylanases, including XynASP from A. saccharolyticus JOP 1030–1, is constrained by inadequate thermostability. Here, we engineered disulfide bonds at the linker-linker interface of XynASP. A single mutant S134C and a disulfide-bonded mutant S134C/N178C with improved thermostability or catalytic activity were successfully screened. Enzymatic characterization showed that mutant S134C increased the half-life at 50 °C by 3.6‑fold and exhibited a substantial 9.5 °C increase in melting temperature (T_m), though it decreased specific activity. In contrast, the disulfide-bonded mutant S134C/N178C exhibited a 4.5-fold increase in specific activity and a moderate 1.6-fold improvement in thermostability, with the T_m values remaining comparable to the wild-type XynASP. Structural analysis and molecular dynamics simulations revealed that S134C enhanced local rigidity of the linker region through formation of a hydrophobic cluster, whereas the disulfide bond between Cys134 and Cys178 in the double mutant created a "rigid‑yet‑tunable" molecular scaffold, which optimized the microenvironment of the substrate-binding pocket and the dynamic correlation network, leading to a 5.4‑fold increase in catalytic efficiency (k_cat/K_m). This study establishes linker-linker interface disulfide bonds as a regulator of GH11 xylanase catalysis, revealing a new strategy for mitigating the stability-activity trade-off.