Characterization of alpha-glucosidase gene in Apis florea

Abstract

Apis florea, dwarf honey bee is one of native species in Thailand. It is small in body size and builds a small rest consisting of a single comb. Honey is one of the important products from honeybee. The honey is mainly produced by the activity of alpha – glucosidase, AG. The AG (E.C.3.2.1.20) is an enzyme that can hydrolyze 1, 4 – linked – alpha – glucosidic bond in sucrose, nectar, to be fructose and glucose, honey. The AG is synthesized in hypopharyngeal glands (HPGs) located in a head of honeybee. Total RNA was isolated from eggs, heads of nurse bees, and heads of forager bees. They were amplified by RT – PCR and the results indicated that the expression of AG is highest in forager bees (760.589). In addition, it was highly expressed in nurse bee (386.633) but was not expressed in egg (16.082). The length (1739 bp) of AG cDNA was obtained by RT – PCR. The nucleotide and deduced amino acid (568 amino acids) sequences were aligned by Clustal W program. The results showed the amino acid identity of about 95% to the AG in A. mellifera. Phylogenetic trees were made by UPGMA and NJ programs. The AG was purified by column chromatography. Forager bees (500 g) in sodium phosphate buffer (pH 6.3) were homogenized to be crude (0.30 u/ mg), precipitated with 95% ammonium sulfate (0.18 u/ mg), and purified by ion exchange chromatography on DEAE – cellulose (0.17 u/ mg) followed by gel filtration on Superdex 200 (0.5 and 2.38 u/ mg) and Sephadex G – 150 (1.355 u/ mg). Protein in fractions containing AG activity, by Momose’s method, was separared by SDS – PAGE and renatured. The activity band of AG could be recovered. The partial purified and concentrated AG was separated by SDS – PAGE. The target band (73 kDa) was cut from a polyacrylamide gel and peptide identified by MALDI – TOF MS. The MALDI – TOF peptide mass maps of partially purified AG showed six matching masses of AG in A. mellifera (Q17958) with 12% coverage. The optimum condition for AG activity in partial purified enzyme was at pH 5, at temperature of 55℃, and at incubation time of 40 min. The concentration of sucrose at 80 mM was used