Chanpen Chanchao1, Suwisa Pilalum2, Rumpalai Padoongsupalai2, Polkit Sangvanich3, Siriwat Wongsiri1, and MR Jisnuson Svasti4. (1) Biology, Chulalongkorn University, Bangkok, Thailand, (2) Biotechnology, Chulalongkorn University, Bangkok, Thailand, (3) Chemistry, Chulalongkorn University, Bangkok, Thailand, (4) Biochemistry, Mahidol University, Bangkok, Thailand
Apis cerana is native to Thailand. Alpha-glucosidase (AG) as in E.C. 3.2.1.20 specifically hydrolyses 1, 2–linked-alpha-glucosidic bond in sucrose to be fructose and glucose. It involves in honey production. Hypopharyngeal glands (HPGs) located in worker heads were used for AG sources. RNA was isolated and primers for RT-PCR were designed from the conserved region of AG in A. mellifera (accession# D79208). Under the optimum condition, the full length of cDNA was obtained and compared to that cDNA from other organisms. It is partially similar to the AG in A. mellifera at 96% (1,719 bp comparison), to maltase in A. mellifera at 53.44% (1,570 bp comparison), and to maltase in Culicoides sonorensis at 48.75% (1,600 bp comparison). Furthermore, the AG activity (by Momose’s method) was assayed from honeybee workers (500 g). They were homogenized to be crude (0.696 u/mg protein), precipitated with 95% Ammonium sulfate (0.235 u/mg protein), and purified by DEAE-cellulose (2.171 u/mg protein), CM-cellulose (0.154 u/mg protein), and Superdex-200 gel filtration chromatography (1.804 u/mg protein). Molecular weight of AG was estimated to be approximately 68 kDa by SDS-PAGE and the relationship between log M.W. and Rf value. Activity staining of renatured AG by Triton X-100 was observed. The activity band of MW between 50 – 75 kDa was coincided to the SDS-PAGE. The 68 kDa protein from SDS-PAGE was sequenced by MALDI-TOF MS. The obtained MALDI-TOF mass spectrum indicated the purified AG. Optimum conditions of AG are at pH 5.0 and at incubation time of 50 min. The optimum temperature is 50oC while the selective concentration of substrate (3, 6-Dinitropthalic acid) is 60 mM. Characters of AG in A. cerana are different from those in A. mellifera. This research is supported by Thailand Research Fund, grant# MRG4780007 and Graduate School of Chulalongkorn University.
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