[Cloning and expression of alpha-D-galactosidase from coffee bean (Coffea liberica & Coffea canephora)].
Alpha-D-galactosidase (alpha-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 terminal alpha-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, pPICZalphaA and pGAPZalphaA. 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.
Liang
Su-Yu
SY
The institute of Bioscience and Biotechnologe, Chinese Academy of Tropical Agriculture Sciences, Haikou, China. sylsy@hotmail.com
Zheng
Xue-Qin
XQ
D001483:Base Sequence; D003001:Cloning, Molecular; D040503:Coffea / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D000519:alpha-Galactosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D018749:RNA, Plant; D011994:Recombinant Proteins; D000519:alpha-Galactosidase