Influence of expansin carbohydrate binding module on in vitro polygalacturonase enzyme activity

CRISTINA F. NARDI; CRISTIAN ESCUDERO; NATALIA VILLARREAL; GUSTAVO MARTINEZ; MARCOS CIVELLO

Tucumán

Congreso; SAIB; 2009

Fruit softening is associated with cell wall disassembly mediated by the action of a set of enzymes and proteins. Expansins (Exps), a group of proteins with unknown enzymatic activity, and poligalacturonases (PGs) are proposed to be involved in this process. It has been suggested that Exps and PGs could cooperatively participate in ripening-associated cell wall degradation, though there is scarce experimental evidence that support such statement. FaEXP2 encodes a putative strawberry expansin with a predicted carbohydrate binding module (FaExp2-CBM) in its C-terminal and an “expansin domain” in its N-terminal. In order to evaluate the influence of expansin, we performed in vitro PG activity assays in the presence of full FaExp2 or FaExp2-CBM. Both molecules were cloned, over-expressed in E. coli, and the recombinant polypeptides were purified. In vitro PG activity was measured using enzymatic extract from tomato, citrus pectin as substrate and the addition of FaExp2-CBM or full FaExp2. The presence of FaExp2-CBM in the reaction mixture decreased PG activity, and a slighter effect was found in the case of full length FaExp2. These results suggest that FaExp2-CBM could interact with pectins and then interfere with the activity of polygalacturonase enzyme.