Expression of a secretory α-glucosidase II from Apis cerana indica in Pichia pastoris and its characterization

• Published in: BMC biotechnology Vol. 13; no. 1; p. 16
• Main Authors: Kaewmuangmoon, Jirattikarn, Kilaso, Manlika, Leartsakulpanich, Ubolsree, Kimura, Kiyoshi, Kimura, Atsuo, Chanchao, Chanpen
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 18.02.2013; BioMed Central
• Subjects: alpha-Glucosidases - chemistry; alpha-Glucosidases - genetics; alpha-Glucosidases - metabolism; Amino Acid Sequence; Amino acids; Animals; API; Apis cerana indica; Apis mellifera; Bees - enzymology; Encoding; Enzymes; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Nucleotides; Optimization; Pichia - metabolism; Pichia pastoris; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Sequence Alignment; Substrate Specificity; Temperature
• ISSN: 1472-6750; 1472-6750
• DOI: 10.1186/1472-6750-13-16

α-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. The 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. Like 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.