An Integrated Workflow for Improving Thermostability: Mechanism and Engineering of GH11 Xylanases via Ser/Thr Substitutions.

Thermostability is critical yet challenging to engineer in industrial enzymes. Here, we present an integrated workflow for enhancing the thermostability of GH11 xylanases through systematic Ser/Thr optimization. Gibbs free energy calculations on XynCDBFV identified 169 stabilizing single-site mutations involving Ser/Thr residues, which were combinatorially integrated via rational design and a machine-learning model (ESM2-RF). Both strategies generated multisite variants with superior thermal tolerance. Top performers included RD-4M3 (ΔTm = 7.10 °C) and ER-4M6 (+56.19% residual activity). Molecular dynamics simulations revealed that R148-D57 competition in the WT at high temperature disrupts the R148-E109 interaction, and its elimination in Ser/Thr variants induces 150-loop rigidification, thereby enhancing thermostability. This finding was validated by transplanting two Ser/Thr substituted combinations to a second GH11 xylanase, PjxA. This work establishes a systematic Ser/Thr optimization strategy for engineering thermostable xylanases and underscores the efficiency of multisite combinatorial design.