Effect of N-linked glycosylation of recombinant windmill palm tree peroxidase on its activity and stability.

Plant secretory peroxidases are valuable commercial enzymes. The windmill palm tree Trachycarpus fortunei produces one of the most stable and fastest peroxidases (WPTP) characterized to date; however, an economical source is needed. Pichia pastoris has been used as an expression system for WPTP and other peroxidases. However, yeast and plants synthesize different types of N-linked glycan structures and may differ the level of glycosylation at each site. Such non-native glycosylation can have unwanted consequences. Glycosylation site N256 was under-glycosylated in the wild-type (1.5%) compared to the native enzyme (55%); therefore, we mutated WPTP to promote glycosylation at this site (WPTP E254G). Glycosylation increased four-fold, as measured by liquid chromatography-tandem mass spectrometry. The mutation did not change the substrate specificity and optimal pH- and thermo-stability ranges, but it increased the catalytic activity 2-3-fold. In comparison with wild-type WPTP, WPTP E254G showed a shift of the most stable pH from 7 to 9, making it suitable for applications under alkaline conditions.