Abstract
Backgroundα–glucosidase (HBGase) plays a key role in hydrolyzing α-glucosidic linkages. In Apis mellifera, three isoforms of HBGase (I, II and III) have been reported, which differ in their nucleotide composition, encoding amino acid sequences and enzyme kinetics. Recombinant (r)HBGase II from A. cerana indica (rAciHBGase II) was focused upon here due to the fact it is a native and economic honeybee species in Thailand. The data is compared to the two other isoforms, AciHBGase I and III from the same bee species and to the three isoforms (HBGase I, II and III) in different bee species where available.ResultsThe highest transcript expression level of AciHBGase II was found in larvae and pupae, with lower levels in the eggs of A. cerana indica but it was not found in foragers. The full-length AciHBGase II cDNA, and the predicted amino acid sequence it encodes were 1,740 bp and 579 residues, respectively. The cDNA sequence was 90% identical to that from the HBGase II from the closely related A. cerana japonica (GenBank accession # NM_FJ752630.1). The full length cDNA was directionally cloned into the pPICZαA expression vector in frame with a (His)6 encoding C terminal tag using EcoRI and KpnI compatible ends, and transformed into Pichia pastoris. Maximal expression of the rAciHBGase II–(His)6 protein was induced by 0.5% (v/v) methanol for 96 h and secreted into the culture media. The partially purified enzyme was found to have optimal α-glucosidase activity at pH 3.5 and 45°C, with > 80% activity between pH 3.5–5.0 and 40–55°C, and was stabile (> 80% activity) at pH 4–8 and at < 25–65°C. The optimal substrate was sucrose.ConclusionsLike in A. mellifera, there are three isoforms of AciHBGase (I, II and III) that differ in their transcript expression pattern, nucleotide sequences and optimal enzyme conditions and kinetics.
Highlights
Background αGlucosidases belong to the glycosyl hydrolase family (EC 3.2.1.20, HBGase) and catalyze the hydrolysis of non reducing terminals of substrates, such as sugars like sucrose and maltose and other glucosides including phenyl α–glucoside, to liberate α-glucose [1]
Three isoforms of HBGase have been reported in A. mellifera (AmHBGase I, II and III), which differ in their substrate specificity, mass, nucleotide and predicted amino acid sequences, and expression patterns in different tissues and developmental stages in the insect [7,8]
Under the previously determined optimum condition for room temperature (RT)-PCR, the highest expression of AciHBGase I transcripts was seen in adult forager bees since no detectable expression was found in eggs, larvae or pupae (Figure 1A), in keeping with the proposed role for this isoform in honey formation via hydrolysis of nectar derived sucrose in forager bees
Summary
Background αGlucosidases belong to the glycosyl hydrolase family (EC 3.2.1.20, HBGase) and catalyze the hydrolysis of non reducing terminals of substrates, such as sugars like sucrose and maltose and other glucosides including phenyl α–glucoside, to liberate α-glucose [1]. Three isoforms of HBGase have been reported in A. mellifera (AmHBGase I, II and III), which differ in their substrate specificity, mass, nucleotide and predicted amino acid sequences, and expression patterns in different tissues and developmental stages in the insect [7,8] These three enzymes showed different pH and temperature optima for enzyme activity, as well as pH and thermal stabilities, and sugar substrate preferences [9,10,11]. Homologs of these three AmHBGase isoforms (I, II and type II-like HBGases) have been reported in the yeast Sporothrix schenckii, where they were found to be located in the endoplasmic reticulum and to be involved in processing of the N-glycan core for glycoprotein biosynthesis [12]. These three yeast homologs differ in their molecular mass and biochemical characters
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