Thesis (Ph.D.)--Chulalongkorn University, 2012

α-Glucosidase (HBGase) catalyzes the non-reducing end of a substrate to liberate α-D-glucose and can be classified into three types of HBGase I, II and III according to the substrate specificity. Recombinant HBGase I, II and III from A. cerana indica (rAciHBGase I, II and III) were focused in this research in order to find a new source of enzyme. Also, this bee species is native and economic to Thailand. Previously, it was reported that the full length ORF cDNA and predicted deduced amino acid of AciHBGase III was 1,704 bp and 567 residues. In this research, the full length ORF cDNA and predicted deduced amino acid of AciHBGase I (1,734 bp and 577 residues) and AciHBGase II (1,740 bp and 579 residues) were obtained by RT-PCR. Later, the full length cDNA of AciHBGase II and III were cloned into pPICZαA expression vector and were successfully expressed in Pichia pastoris GS115. The highest expression of rAciHBGase II and III was induced by 0.5% (v/v) methanol for 96 h and 1.0% (v/v) methanol for 144 h, respectively. However, the full length cDNA of AciHBGase I was cloned into pEcoli expression vector and was successfully expressed in E. coli Rosetta (DE3) after induced by 1 mM IPTG for 3 h. The purified rAciHBGase I, II and III showed an optimum pH of 3.5, 3.5, 5.0 and was stable in the pH range of 3.5-5.0, 5.0-7.0, 5.0-7.5, respectively. In addition, rAciHBGase I and II showed a thermal optimum at 40 and 45°C, respectively. Theirs activity still remained active in an acidic environment and was acid tolerant. Also, they could work well at the relatively high temperatures (35-50 and 45-55°C, respectively). In contrast, rAciHBGase III showed an optimum activity at 37°C with no thermo tolerance. Moreover, substrate specificity of rAciHBGase I and II were sucrose but of rAciHBGase III was maltose and PNPG. In conclusion, rAciHGAases are useful and can be a new source for further application.