Improving the inhibitory resistance of xylanase FgXyn11C from Fusarium graminearum to SyXIP-I by site-directed mutagenesis.

The aim of this study was to improve the inhibitory resistance of xylanase FgXyn11C from Fusarium graminearum to XIP in cereal flour. Site saturation mutagenesis was performed using computer-aided redesign. Firstly, based on multiple primary structure alignments, the amino acid residues in the active site architecture were identified, and specific residue T144 in the thumb region of FgXyn11C was selected for site-saturation mutagenesis. After screening, FgXyn11C was selected as the best mutant, as it displayed the highest enzymatic activity and resistance simultaneously compared to other mutants. The specific activity of FgXyn11C was 208.8 U/mg and it exhibited complete resistance to SyXIP-I. Compared with the wild-type, FgXyn11C displayed similar activity and the most resistant against SyXIP-I. The optimal temperature and pH of the wild-type and purified FgXyn11C were similar at pH 5.0 and 30 °C. Our findings provided preliminary insight into how the specific residue at position 144 in the thumb region of FgXyn11C influenced the enzymatic properties and interacted with SyXIP-I. The inhibition sensitivity of FgXyn11C was reduced through directed evolution, leading to creation of the mutant enzyme FgXyn11C. The FgXyn11C resistance to SyXIP-I has potential application and can also provide references for engineering other resistant xylanases of the GHF11.