Purification, characterization and gene expression of α-glucosidase of giant honey bee apis dorsata

Abstract

Apis dorsata (Giant honey bee) which is native to Thailand is the largest bee. Its honey is the most popular for consumers. α-glucosidase (AG) classified in E.C. 3.2.1.20 is an enzyme directly involved in honey processing. It hydrolyses 1, 2–α-glucosidic bond in sucrose of nectar to be monosaccharide, fructose and glucose, in honey. For the expression level of AG III in A. dorsata, total RNA was extracted from eggs, larvae, pupae, and foragers. By RT-PCR, the expression of AG III was the highest in foragers (114.24 ng), high in pupae (107.1 ng), low in eggs (71.4 ng), and the lowest in larvae (47.6 ng). The full length of ORF of cDNA was 1,704 bp and was deduced to be 567 amino acids. By BLASTn and BLASTp, the most similarity was found to the sequences of AG III in A. mellifera at 96%. Phylogenetic trees of amino acid sequence of AG were constructed by UPGMA and NJ. It showed its own evolutionary lineage of A. dorsata. AG from A. dorsata foragers (500 g) was purified. Crude enzyme (0.01 U/mg) was precipitated by ammonium sulfate at 95% saturation. After precipitation, specific activity of AG was found in both pellet (1.70 U/mg) and supernatant (1.34 U/mg). After DEAE cellulose column, specific activity of 0.004 and 3.95 U/mg were obtained from pellet and supernatant, respectively. For pellet, pooled positive unbound and bound fractions were further purified by Superdex 200 column. Specific activity of 0.09 U/mg was recovered. For supernatant, positive unbound and bound fractions were separately purified by Superdex 75 column. Specific activity of 0.11 and 0.26 U/mg were obtained, respectively. Partial purified AG was determined by SDS PAGE. The optimal conditions of partial purified AG were at pH 6.0, incubation temperature of 35℃, maltose concentration of 50 mM, and incubation time of 30 min