Purificación y caracterización de las isoenzimas de la pectina metilesterasa en frutos de níspero (Manilkara zapota var. Prolific)
The Sapodilla (Manilkara zapota) is a very desired fruit by its aroma and pleasant flavor. In Venezuela the production of the sapodilla is limited to small and medium producers and the cause that has affected more the increase of the production and the supply in the market of this fruit is the accel...
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2013
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| Online Access: | http://hdl.handle.net/10872/2296 |
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| Summary: | The Sapodilla (Manilkara zapota) is a very desired fruit by its aroma and pleasant flavor. In Venezuela the production of the sapodilla is limited to small and medium producers and the cause that has affected more the increase of the production and the supply in the market of this fruit is the accelerated maturation process and the inadequate handling postharvest. The process of softening or loss of firmness during the maturation of the fruits is consequence of the biochemical changes produced in the cell wall. This study was realized in order to kinetically purify and to characterize isoforms of the PME in fruits of sapodilla (Manilkara zapota var. prolific). Fruits of sapodilla in state of physiological maturity were collected and they were stored under freezing -20ºC. For the extraction of the enzyme 20 g were taken from the pulp of the fruit, added liquid nitrogen and it pulverized in a mortar, soon (0,1M-pHs 8) were re-suspended in 20 ml of Tris-HCl and was centrifuged to 10000 rpm 30´.The measurement of the enzymatic activity was realized according to the methodology of Hagerman and Austin (1986). The obtained supernatant (20 mL) was filtered with glass wool and then stored to -20 ºC. Next the precipitation of the enzyme with liquid ammonium sulphate was realized. Pellet obtained was put under filtered chromatography with Sephadex G-50. The glycosylation of the enzyme by means of chromatography of affinity with the ConA-Sepharosa and the Kit of glicosylation of Sigma was determined. The kinetic constants of Km and Vmáx, the temperature, the optimal pH and the stability to the heat were determined. The results indicate the existence of two isoforms of the PME, which denominated PMEI and PMEII. The PMEI and the PMEII have equal km, Vmáx and molecular weight but defer in their pH and optimal temperature. Km of the PMEI and PMEII km μg/μL is of 1.15 μg/ μL and the 0.078 Vmáx is of μmol, acid/min. The optimal temperature of PME I is of 50 ºC and of PME II is of 25 ºC. The optimal pH of PME I is of 4 and of PME II he is 10. Molecular weight of the PME and the PME is between 15 and 20 kDa. Both isoforms are heat-resisting, nevertheless PME II is more stable than PME I. Isoforms of the PME in sapodilla fruits are glycosylated. This carbohydrate group is compound partly of manosydics groups. The following aspects are recommended to deepen in the knowledge on the PME: to realize the sequencing of isoenzimas, to lyophilize isoforms of PME I and PME II of sapodilla fruits, to test at agro-industrial level to determine the applicability of isoforms of the PME of sapodilla and to realize the corresponding analyses to determine the percentage and the composition of carbohydrates of isoforms PME and PME II. |
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