关键词: α-半乳糖苷酶, cDNA克隆, 咖啡豆, B→O血型转换, P. pastoris

Abstract: α-D-galactosidase（α-Gal,E.C. 3.2.1.22）is an exo-glycosidase. The enzyme isolated from coffee beans has been well characterized. It has high activity in hydrolyzing the terminalα-D-galactoside residues from glycoconjugates on human blood group B erythrocytes，as well as in converting the blood group B into O . A different 1089 bp cDNA open reading frame(ORF) encoding Gal of Coffea liberica & C. canephora was cloned by homology-based RT-PCR. The cloned Gal most closely resembles the corresponding one from C. aribica (98.7% and 99.27% identity). Heterologous overexpression of the two 1.1 kb cDNA fragments was obtained by using one Pichia pastoris stain GS115 and two secret expression vectors， pPICZαA and pGAPZαA. The expressed protein from P. pastoris stain GS115 was concentrated by ammonium sulfate precipitation and SDS-PAGE assay showed a clear band in the gel. The highest activity of the recombinant enzyme was up to 48.22 U/mL.