Abstract

Strawberry fruit has a fast softening rate which affects its short shelf life. Fruit softening has been shown to be related to cell wall disassembling. As changes in the hemicellulosic fraction have been reported during ripening of Fragaria chiloensis fruit, it has been 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. Therefore, the aim of this work was to study the effect of glycosylation on FcXTH1 activity. For this, FcXTH1 was cloned and heterologous expressed in P. pastoris. The recombinant purified protein is active and displays both XEH and XET activities, with 10-12 times higher XET than XEH activity. The optimal pH and temperature for both activities was 5.5 and 37ºC. After the treatment of the protein with PNGase F, the deglycosylated protein retained only 50% XET activity, while XEH activity was lost. Additionally, the stability of the recombinant purified protein at 4ºC was tested: XET activity is relatively stable at 4ºC retaining 50% of activity after 5 days, while XEH activity retains 78% initial activity after 5 days. Nevertheless, XET activity of deglycosylated protein is rapidly lost at 4ºC, with less than 15% initial activity after 4 days. The data provided allow us to propose that the glycosylation in FcXTH1 is necessary for optimal enzyme activity, and that FcXTH1 should be glycosylated during the disassembling of the cell wall required for fruit softening.