Isolation, purification and characterization of glucosidases from three honey bee species (Apis mellifera, A. cerana and A. dorsata)

Honey is a natural food product recognized for its taste, nutritive and biological value. The presence of oligosaccharides in honey has been determined and they have been used as a fingerprint for authenticity. Some of these oligosaccharides have been shown to be synthesized via enzymes secreted by honey bees during nectar collection and nectar/honey transfer in the hive. Two alpha-glucosidases have been purified and partially characterized from honey and two from whole honey bees (Apis mellifera). Beta-glucosidase was found in honey, and this activity was shown to be associated with alpha-glucosidase. Based on this information, research was initiated to isolate and purify beta-glucosidase from honey bees. In addition, studies were undertaken to investigate the similarities/differences between alpha- and beta-glucosidases in three honey bee species ( Apis mellifera, A. cerana and A. dorsata).;Both alpha- and beta-glucosidase activities have been detected in the hypopharyngeal glands, honey sac, ventriculus and hindgut of three honey bee species (A. mellifera, A. cerana and A. dorsata). Purification protocols for alpha- and beta-glucosidases from the ventriculus and honey sac of A. mellifera, have been established employing gel filtration, anion and cation exchange, and hydroxyapatite chromatography. Purification protocols for alpha- and beta-glucosidases from the ventriculus of A. cerana have also been established employing ammonium sulfate precipitation, gel filtration, hydfoxyapatite and cation and anion exchange chromatography, and native gel electrophoresis. Two alpha-glucosidases (alpha-glucosidase-1 and -2) and one beta-glucosidase have been purified from the ventriculus of A. mellifera and A. cerana. Only alpha-glucosidase-2 and beta-glucosidase were found in, and purified from, the honey sac of A. mellifera. These results show that alpha-glucosidase-1 is produced in the ventriculus of A. mellifera and is not a constituent of the honey sac.;The physicochemical properties of these purified enzymes have been determined, including relative molecular mass (Mr), isoelectric point (pI), temperature and pH stability, temperature and pH optimum, maximum reaction irate (Vmax) and Michaelis-Menten constant (Km). The primary structure of these enzymes has been studied employing matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS).;Based on the similarities observed in physicochemical properties and peptide mapping by MALDI-TOF-MS, the following observations were made: (1) alpha-glucosidase-1 from the ventriculus of A. mellifera and A. cerana were the same; (2) alpha-glucosidase-2 from the ventriculus of A. mellifera and A. cerana were the same; (3) alpha-glucosidase-2 from the ventriculus and honey sac of A. mellifera, were the same; (4) beta-glucosidase from the ventriculus and honey sac of A. mellifera were the same; and (5) beta-glucosidase from the ventriculus of A. mellifera and A. cerana were not the same.