Abstract

Fruit softening is related to cell wall disassembling. Changes in the hemicellulose fraction have been reported during ripening of Fragaria chiloensis fruit and therefore we suggested that FcXTH1, a xyloglucan endotransglycosidase/hydrolase (XTH) enzyme, might play a key role in this process. XTHs may have two activities: transglycosidase (XET) and hydrolase (XEH). FCXTH1 contains a conserved N-glycosylation site adjacent to the predicted catalytic residues. The aim of this work was to study the effect of glycosylation on FcXTH1 activity. For this, FcXTH1 was cloned and heterologous expressed in Pichia pastoris. The recombinant purified protein was found to be active and displayed both XEH and XET activities, with 10-12 times higher XET than XEH activity. The optimal pH and temperatura for both activities were 5.5 and 37ºC. Both activities were stable at 4ºC as more than 50% initial activity remained after 5 days. A Km value of 29.5 M was determined for XGO (xyloglucan oligomer). The deglycosylation of FcXTH1 by the treatment with PNGase-F did not affect maximum activity but reduced the stability of the enzyme, and more importantly, induced changes in kinetic parameters of XTH activity (higher Km value). In conclusión, glycosylation of FcXTH1 affects its substrate affinity and stability.