Design extraction method pectin methylesterase (EC 3.1.1.11) in sapodilla fruit

Abstract

Sapodilla (Manilkara zapota [L.] ‘Prolific’) is a fruit whose difficulties of post-harvest management have limited its commercialization. During the ripening of these fruits, the most dramatic change is the loss of firmness, which causes a decrease in quality. This softening is associated with the action of the pectin methylesterase (PME, E.C. 3.1.1.11), which is an enzyme that induces degradation of the cell wall. The objective was to design a method for efficient extraction of PME, with high quality, fast and using few reagents. Physiologically ripen Sapodilla fruits from the Germplasm Bank INIA- CENIAP, Maracay, Aragua state, were used. They were stored at -20 °C. Five methods of extraction Buffer Tris HCl; Buffer PBS; Liquid nitrogen (N₂L); Buffer Tris HCl + N L and NaCl, were designed. Methods were evaluated according to enzyme quality, amount (mg mL^-1  protein) and extraction. The analysis of variance showed highly significant differences (P<0.01) among the methods and Duncan mean comparisons determined that the liquid nitrogen method extracted more protein (231 mg ml^-1) with a catalytic activity of 1.97 µmol acid min^-1, excelling the other methods and the control with values ranging from 0.13 µmol acid min^-1  up to 0.6 µmol acid min^-1. Therefore, the C method is the most efficient, with higher protein yield and lower time (two hours). However, purification and characterization of PME for either agroindustrial purposes or biotechnological applications are recommended.