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Expression and characterization of a-glucosidase III in the dwarf honeybee, Apis florea （Hymenoptera： Apoidea： Apidae）

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Insect Science 2007年第4期14卷 283-293页

作者： CHANPEN CHANCHAO RUMPALAI PADOONGSUPALAI polkit sangvanich DepartmentofBiology ProgramofBiotechnology DepartmentofChemistry FacultyofScienceChulalongkornUniversityBangkokThailand

Alpha-glucosidase is synthesized in the hypopharyngeal glands located in the head of worker bees including Apisflorea. To analyze the developmental stage-specific expression of the a-glucosidase gene ***, total RNA was isolated from eggs, and the heads of nurse and forager bees. By reverse transcription polymerase chain reaction （RT- PCR）, it was shown that the highest expression levels of the a-glucosidase Ⅲ gene, in the three examined developmental stadia, were found in forager bees, with much lower expression levels in nurse bees and no detectable expression in eggs. A complete aglucosidase III eDNA was obtained by RT-PCR and sequenced. The 1 701 bp eDNA nucleotide sequence and the predicted 567 amino acids it encodes were assayed by BLASTn, BLASTp and BLASTx programs and revealed a 95% and 94% similarity to the A. mellifera aglucosidase III gene at the DNA and amino acid sequence levels, respectively. For purification of the active encoded enzyme, forager bee heads were homogenized in sodium phosphate buffer solution and the crude extract （0.30 U/mg） sequentially precipitated with 95% saturated ammonium sulfate （0.18 U/mg）, and purified by DEAE cellulose ion exchange chromatography （0.17 U/mg）, and gel filtration on Superdex 200 （0.52 U/mg）. After resolution through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single enzymically active band （73 kDa） was identified from renatured substrate gels. Excision of this band, elution of the protein and tryptic peptide digestives identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF MS） revealed six matching masses to the A. mellifera （Q17958） and predicted *** α glucosidase Ⅲ protein with 12% coverage, supporting the probable purification of the same α-glucosidase III protein as that encoded by the cloned cDNA.

关键词： α-glucosidase Apisflorea cDNA expression homology purification

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Purification and characterization of α-glucosidase in Apis cerana indica

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Insect Science 2008年第3期15卷 217-224页

作者： Chanpen Chanchao Suwisa Pilalam polkit sangvanich Department of Biology Faculty of Science Chulalongkom University Bangkok Thailand Program of Biotechnology Faculty of Science Chulalongkom University Bangkok Thailand Department of Chemistry Faculty of Science Chulalongkom University Bangkok Thailand

Apis cerana indica foragers were used for the isolation of a full-length α- glucosidase cDNA, and for purification of the active nascent protein by low salt extraction of bee homogenates, ammonium sulphate precipitation and diethylaminoethyl-cellulose and Superdex 200 c hromatographies. The molecular mass of the purified protein was estimated by polyacrylamide gel electrophoresis resolution, and the pH, temperature, incubation, and substrate optima for enzymic activity were determined. Conformation of the purified enzyme as α-glucosidase was performed by BLAST software homology comparisons between matrix assisted laser desorption ionization time of flight mass spectroscopy analysed partial tryptic peptide digests of the purified protein with the predicted amino acid sequences deduced from the α-glucosidase cDNA sequence.

关键词： α-glucosidase Apis cerana indica cDNA chromatography purification

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