Mutagenesis confers endo-xylanase inhibitory competence to L. albus seed γ-conglutin

γ-Conglutin, a glycoprotein accounting for about 5% of the total seed proteins, shares high amino acid sequence identity and the conserved array of cysteines with XEGIPs and TAXI-I proteins. Expression of γ-conglutin during germination can be elicited by chitosan, suggesting an actual role of the protein in plant defence mechanisms. However, γ-conglutin failed to inhibit fungal endo-glucanases and other cell wall-degrading enzymes. Based on bioinformatic analysis, the lack of inhibitory activity of γ-conglutin was attributed to a sequence deletion in the inhibitor interaction domains. To investigate this hypothesis a gene mutagenesis approach has been carried out. Two mutants have been created by PCR gene fusion to insert an amino acid stretch and two or three amino acid substitutions, respectively, with the aim to simulate the corresponding region of XEGIP proteins and TAXI-I. The two mutants and the wild-type genes version have been expressed in Pichia pastoris cells and the three γ-conglutins have been purified, biochemically characterized and assessed for the inhibitory capacity against Thermomyces lanuginosus and Trichoderma longibrachiatum GH11 xylanases. All the mutants were able to inhibit the enzymes, although with different efficiencies and conditions, while the wild-type version was not. The obtained results support the hypothesis that γ-conglutin could be considered a defence-related protein which has lost the functional competence or, alternatively, it possess an enzyme specificity different from those of the homologous proteins.