Characterization of apis cerana alpha-glucosidase cDNA

Abstract

Apis cerana is one of native honeybees in Thailand. It can be as well managed in an apiary as A. mellifera. Its honey and other products such as royal jelly, wax, propolis, etc. are popular among consumers. Alpha glucosidase (AG) as in E.C. 3.2.1.20 is an enzyme that specifically hydrolyses 1, 4-linked-alpha-glucosidic bond in sucrose to be fructose and glucose. It involves in honey production. Hypopharyngeal glands (HPGs) located in a head of worker bees were used as AG sources. Primers for RT-PCR were designed from conserved regions of AG in A. mellifera. Under the optimum condition, the cDNA sequence of AG (1,740 bp in length) was obtained and compared to that cDNA from other organisms. It is partially similar to the AG in A. mellifera at 96% (1,740 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). Due to UPGMA and Neighbor joining program, phylogenetic trees of amino acid support that AG of A. cerana was very similar to AG of A. mellifera. Later, AG of A. cerana was purified and characterized. Worker bees (430 g) were homogenized to be crude (0.696 u/ mg), precipitated with 95% ammomium sulfate (0.235 u/mg), and purified by DEAE cellulose (2.171 u/ mg), CM cellulose (0.154 u/mg), and Superdex 200 chromatography (1.804) u/mg). Specific activity increased to 0.34, 3.11, 0.22, and 2.59 fold, respectively. The optimum conditions of purified AG after Superdex 200 were at pH 5.0, at temperature of 50 degrees Celsius, and at incubation time of 50 min. The concentration of sucrose at 60 mM was used. MW of AG was estimated to be approximately 68 kDa. Furthermore, purified AG (both by in gel and in solution digestions of trypsin) was analysed by MALDI-TOF MS. The mass spectra confirmed the obtained amino acid sequence of AG (at least 4 matching masses with 4% coverage and at least 18 matching masses with 19% coverage).