半乳糖水解酶可分解多醣，水解B型紅血球表面抗原的半乳糖基，使B型紅血球轉換成O型紅血球，可用於臨床輸血。本研究將稻米半乳糖水解酶基因整合到pPIC9K質體，在轉殖到重組酵母菌(Pichia pastoris)的染色體DAN上，並以4 mg/ml G418篩選出具有multicopy的菌株，以表現稻米半乳糖水解酶。重組酵母菌之培養條件測試，分別針對培養基種類、誘導溫度、誘導起始A600值、每日甲醇量及時間間隔、誘導培養基添加額外碳源檢測其對產量的影響，得到最佳表現量，細胞內酵素單位為5.124 units/ml，細胞外酵素單位為1.127 units/ml。大量培養之細胞內及分泌酵素，經由陰離子交換樹脂（DEAE-Sepharose FF）與疏水性管柱層析（Phenyl-Sepharose FF）分離純化，純化倍率分別為74及94。純化後重組半乳糖水解酶經SDS-PAGE分析，有2種主要蛋白質大小分別為40及60 kDa。重組半乳糖水解酶特性分析，最適反應溫度為37℃、熱穩定性為50℃以下、最適反應pH為4、pH穩定性為3.0～5.0。以HPLC分析重組半乳糖水解酶水解α1→6連結的多糖特異性，Melibiose>Raffinose>Stachyose。在實驗特定條件下，以1.5 units的純化酵素在2小時內可將約50%的B型紅血球轉為O型紅血球。 α-Galactosidase hydrolyzed the terminal galactosyl residues from oligosaccharides including blood group B substance on the B red blood cell surface. For the blood conversion purpose, We had cloned the rice-α-galactosidase in pPIC9K vector and transformed into Pichia pastoris SMD1168. We intended to find a optimal culture condition for α-galactosidase to be expressed. We had tested the type of the medium , the start pH of the medium, the induce temperature, the starting A600 of the culture, the concentration of methanol to induce and the effects of carbon source concentration for the expression on α-galactosidase. The enzyme activity inside the cells was 5.124 units/ml; in the medium was 1.127 units/ml. Both ion exchange(DEAE-Sepharose) and hydrophobic interaction (Phenyl-Sepharose) column chromatographies were used to purify intracellular and secreted protein. They were purified 74 and 94 fold, respectively. The purified enzyme showed two major band on SDS-PAGE. The molecular weight of recombinant α-Galactosidase was estimated about 60 and 40 kDa. The maximum activity occurred at a temperature of 37℃；however, inactivation was observed at the temperature above 50℃.The enzyme showed maximal activity at pH 4.4 and was slowly inactivated above pH 5.0 and below pH 3.0. The substrate specificities of the enzyme for α1→6 linked galactose were investigated by using galactose-containing oligosaccharides: melibiose, raffinose and stachyose. The enzyme specificity of these oligosaccharides was in the decreasing order: melibiose> raffinose> stachyose. For the blood conversion test, in the experiment condition, 1.5 units of purified enzyme converted 50% B RBC into O RBC in 2 hours.
