| Summary: | 碩士 === 淡江大學 === 化學學系碩士班 === 101 === α-Galactosidase is capable of hydrolyzing terminal non-reducing galactosyl residues, and it is distributed in most organisms with many different biological functions such as metabolism and transportation of photoassimilates in plants. In application, α-galactosidase can be used to hydrolyze the galactosyl group of type B antigen on the red blood cell surface, and covert the type B red blood cell into type O. Thus, the Pichia patoris expression system was used to fastly produce large amount of taro α-galactosidase to proceed the seroconversion of erythrocyte.
In this work, the Pichia patoris cells harboring the taro α-galactosidase gene on the expression vector were grown to large scale. After the recombinant gene had been induced for one day, the highest intracellular enzyme activities were 3.6 units/mL. Lysis of yeasts was achieved by using Lyticase to extract the crude taro α-galactosidase.
From the crude extractsα-galactosidase was then purified to homogeneity by using four different types of column chromatography, include SephadexTM G-100, Q SepharoseTM Fast flow, HiTrap Phenyl FF (high sub), and SepharoseTM 6 10/300 GL. The purified enzyme showed single band on SDS-PAGE, and then identified by by MALDI-TOF MS analysis.
The purified α-galactosidase was characterized in terms of blood conversion, and a conversion rate of 80％ type B red blood cell into type O with use 2 units of purified α-galactosidase in 2h was achieved.
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