【摘要】 从大粒种咖啡(Coffea liberica)和中粒种咖啡(Coffea canephora)中分离克隆到了α-半乳糖苷酶(α-D-galactosidase)cDNA的开放阅读框架即编码区,分别记为Gal-D与Gal-Z,长度与已发表的小粒种咖啡cDNA编码序列相同均为1 089 bp,同源性与已发表的小粒种咖啡cDNA编码序列相比分别为98.7%和99.27%。将克隆到的Gal-D与Gal-Z用巴斯德毕赤氏酵母Pichia pastoris表达载体pPICZαA(分泌甲醇诱导型)和pGAPZαA(分泌组成型)成功地构建了如下酵母表达载体:pPICZαA/Gal-Z,pPICZαA/Gal-D和pGAPZαA/Gal-D,并转入酵母宿主菌GS115进行了发酵表达研究。实验得出工程菌株pPICZαA/Gal-D/GS115的重组表达产物酶活最高可达48.22(U/mL),对发酵产物进行了SDS-PAGE电泳,得到一条清晰的主条带。更多还原

【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 terminal α-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.更多还原