Expression and characterization of Apis dorsata α-glucosidase III

α-Glucosidase III plays a major role in producing the monosaccharides in honey, mainly glucose and fructose, from sucrose in plant nectar. The honey from Apis dorsata is different and somewhat acidic compared to that of A. mellifera. The transcript expression level of the α-glucosidase III gene was evaluated by RT-PCR on total RNA extractions from eggs, larvae, pupae, and foraging imagoes. The highest expression level was found in foragers, which concurs with its biological function in honey bees as only foragers make honey. Using primers designed from the sequence of the A. mellifera α-glucosidase III gene, the full length ORF of the cDNA (1704 bp including the stop codon and 567 predicted amino acids) was obtained by RT-PCR of a total RNA preparation from foragers, and direct sequenced. BLASTn analysis revealed that the cDNA sequence showed the highest sequence similarity to the A. mellifera α-glucosidase III gene (96%). Enrichment of the native α-glucosidase III protein from forager bees was performed by ammonium sulfate precipitation at 95% saturation followed by diethylaminoethyl-cellulose ion exchange column chromatography of the unprecipitated fraction (yield and specific activity of 3.95 U/mg and 118.5%) followed by Superdex 75 ion exchange column chromatography to give a main protein and sole α-glycosidase activity band of ~ 63 kDa, with a yield of 5.57 U/mg and a specific activity of 19.5%. This enriched fraction had optimal pH, temperature, and substrate concentration for α-glucosidase activity of 4.5, 35 °C and 55 °C, and 50 mM maltose, respectively.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► The highest expression level of the α-glucosidase III gene was found in foragers. ► The full length ORF of the cDNA (1704 bp including the stop codon and 567 predicted amino acids) was obtained. ► After DEAE and Superdex columns, its yield and specific activity were 5.57 U/mg and 19.5%. ► The molecular weight of α-glycosidase was ~ 63 kDa. ► Optimal pH, temperature and substrate concentration were 4.5, 35 °C and 55 °C and 50 mM maltose.