The banana starch degradation depends on the combined action of α-amylase and β-amylase in regions of different degrees of crystallinity of the granules

Author(s)

Renata Shitakubo

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2015-12-03

Examining board members

Beatriz Rosana Cordenunsi; Maria Angela Machado de Carvalho; Célia Maria Landi Franco; Eduardo Purgatto

• Advisor: Beatriz Rosana Cordenunsi; João Roberto Oliveira do Nascimento

Abstract

Banana is considered a good model to study the starch-sucrose metabolism, since it accumulates a high starch content during development, which is degraded during fruit ripening. It have been detected in banana pulp some proteins and activities of several enzymes supposedly involved in starch degradation process. Among them, α-amylase, β-amylase, starch phosphorylase and glucan-water-diquinases (GWD). These enzymes are involved in two separate processes of starch degradation in plants: the initial action of α-amylase dependent, and the starch granule phosphorylation by GWD and PWD enzymes and subsequent action of β-amylase. The difficulty of establishing the effective participation of each enzyme in the starch degradation process is associated with many factors, including the lack of correlation between real activity and involvement in the process, and accessibility of the enzyme to its substrate. Allied to study the morphology of the starch granule and its modifications suffered during the process of degradation, which occurs during the fruit ripening, in vitro studies that simulate the action of the enzyme on its substrate could be more effective in establishing the real action of a given enzyme on the argued substrate. However, attempts to obtain the proteins related to the degradation process were unsuccessful. Thus, assays of isolated starch granules versus enzymes were made with commercial α-amylase and β-amylase enzymes. The degree of phosphorylation of amylopectin in the Gluc-6 and Gluc-3 positions was determined, a necessary condition for the start of degradation by β-amylase enzyme. The results showed that the starch granules isolated from freshly harvested bananas, or green, are already phosphorylated and the enzymes responsible for this phosphorylation is associated with the starch granules surface. After 72 h incubation of the starch granules with the hydrolytic enzymes, the granules were separated from the buffer containing the enzymes and the hydrolysis products. The supernatants were analyzed by liquid chromatography coupled with amperometric detector and the granules were visualized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The results showed that the α-amylase preferentially hydrolyzes amorphous regions of the granule, especially amylose, exposing more crystalline regions of the growth rings, whereas β-amylase appears to act preferentially on crystalline regions of the granule, degrading blocklets that consist of amylopectin. It can be concluded that both enzymes appear to be important in the banana starch degradation process, with different specificities. (AU)

• FAPESP's process: 11/09978-8 - Morphological, chemical and structural properties of banana starch granules subjected to an in vitro model of degradation from assays with recombinant enzymes alfa-amylase and GWD
• Grantee: Renata Shitakubo
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)