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

BMC Biotechnology

Research Article

Chanpen Chanchao¹  Atsuo Kimura²  Kiyoshi Kimura³  Ubolsree Leartsakulpanich⁴  Manlika Kilaso⁵  Jirattikarn Kaewmuangmoon⁵ 

[1]Department of Biology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, 10330, Bangkok, Thailand

[2]Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, 060-8589, Sapporo, Japan

[3]Honeybee Research Group, National Institute of Livestock and Grassland Science, 305-0901, Ibaraki, Japan

[4]National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Phaholyothin Road, 12120, Pathumthani, Thailand

[5]Program in Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, 10330, Bangkok, Thailand

关键词: Apis cerana indica;    α–glucosidase;    Expression;    Homology;    Recombinant enzyme;   

DOI  :  10.1186/1472-6750-13-16

 received in 2012-08-14, accepted in 2013-02-14,  发布年份 2013

来源: Springer

【 摘 要 】

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.

【 授权许可】

Unknown   
© Kaewmuangmoon et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.