Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and...

To reduce the inhibition sensitivity of a thermoresistant xylanase AusM to xylanase inhibitor protein (XIP)-type in wheat flour, the site-directed mutagenesis was conducted based on the computer-aided redesign. First, fourteen single-site variants and one three-amino acid replacement variant in the thumb region of an AusM-encoding gene (AusM) were constructed and expressed in E. coli BL21(DE3), respectively, as predicted theoretically. At a molar ratio of 100:1 between SyXIP-I/xylanase, the majority of mutants were nearly completely inactivated by the inhibitor SyXIP-I, whereas AusM^N127A retained 62.7% of its initial activity and AusM^PKK retained 100% of its initial activity. The optimal temperature of the best mutant AusM^PKK was 60 °C, as opposed to 60-65 °C for AusM, while it exhibited improved thermostability, retaining approximately 60% of its residual activity after heating at 80 °C for 60 min. Furthermore, AusM^PKK at a dosage of 1000 U/kg was more effective than AusM at 4000 U/kg in increasing specific bread loaf volume and reducing hardness during bread production and storage. Directed evolution of AusM significantly reduces inhibition sensitivity, and the mutant enzyme AusM^PKK is conducive to improving bread quality and extending its shelf life.