α-半乳糖水解酵素(EC.3.2.1.22)能水解B型紅血球表面抗 原的α-半乳糖基，使B型紅血球可以轉換成O型紅血球。為了要 大量表現α-半乳糖水解酵素做紅血球轉型以利輸血之用，我們將 稻米的α-半乳糖水解酵素基因分別重組到表現質體pPICZαA及 pPIC9K上。並分別轉殖到酵母菌GS115及SMD1168內，目前挑選出來 表現最多的菌株為pPIC9K-αgal in SMD1168。目前可以表現在細胞內 每公升10毫克的α-半乳糖水解酵素；而細胞外的酵素每公升為1毫克。 分泌在細胞外的蛋白質可以省去很多純化過程，我們目前試著再改變培養 條件以增加產率。包括使用YNB-最簡單的培養基，加必要胺基酸及 乳糖；或增加供氧量等方式。發酵槽的培養可得到約10倍的基因表現 ，我們接著應該一試。將來也可能試用昆蟲細胞、植物細胞及哺乳類 動物細胞，以達到大量表現酵素的目的。 α-galactosidase(EC. 3.2.1.22) is able to cleave the terminal α-galactose from surface oligosaccharide chain of B red blood cells, thus B type RBC can be converted into O type RBC. In order to gain large quantity of enzyme for this purpose, we tried to clone the rice α-galactosidase gene into each of the pPICZαA and pPIC9K vector(Invitrogen®), and to expression the enzyme in both GS115 and SMD1168 strain. The transformants from pPIC9K/SMD1168 was obtained. Expression the transformants in flask culture, most enzyme activity was found intracellularly (10 mg per liter culture);while there is about 1 mg per liter culture of the enzyme activity was secreted in the media. The secreted enzyme would be easier for further media purification. From SDS-PAGE, the secreted enzyme revealed as a major band. We have to try some other culture conditions to improve this results.
