Molecular cloning and characterization of an alpha-amylase occurring in the pulp of ripening bananas and its expression in Pichia pastoris

Abstract

Alpha-amylases (EC 3.2.1.1) are glycosyl hydrolases with endoglycolytic activity on the alpha-1,4-d-glucosidic linkages in starch. In bananas, the mobilization of starch accounts for sugar accumulation during ripening, and among several hydrolytic enzymes, alpha-amylase is the only enzyme argued to be able to attack the intact granules, indicating a pivotal role for this enzyme. A 1953 bp full-length banana alpha-amylase cDNA (MAmy), encoded for a sequence of 416 amino acids, was cloned and used for heterologous expression in Pichia pastoris. The cloned MAmy presented the highly conserved motifs common to alpha-amylases, and the amylolytic activity of the extracts from yeast transformed with MAmy demonstrated that it encodes for a functional alpha-amylase, suggesting a putative role for this gene in starch degradation during fruit ripening.