重组α-半乳糖苷酶的制备工艺研究  

The study on preparation of recombinant α-galactosidase

作　　者：高红伟[1] 高新[1] 鲍国强[1] 徐莉娟[1] 王颖丽[1] 田曙光[1] 李素波[1] 徐华[1] 李立立[1] 宫锋[1] 章扬培[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《生物技术通讯》2004年第6期566-569,共4页Letters in Biotechnology

基　　金：国家重点基础研究发展规划(2002CB713804);全军十五重点课题(01Z029)

摘　　要：α-半乳糖苷酶是B→O血型改造研究中的关键工具酶。在获得了可分泌表达α-半乳糖苷酶的基因工程毕赤酵母菌株的基础上,进行了工程菌株在5L发酵罐中的发酵。发酵液上清中α-半乳糖苷酶活性为80～150U/mL,蛋白浓度为3～4.5mg/mL,比活性约为20～30U/mg。发酵液采用超滤、阳离子交换层析、疏水层析和阴离子交换层析等纯化方法,建立起了规模化生产重组α-半乳糖苷酶的工艺。制备的重组酶纯度经鉴定达98%以上,符合新生物制品的纯度要求。制备的重组α-半乳糖苷酶可有效地将B型红细胞改造成O型红细胞,从而解决了应用此酶开展B→O血型改造研究的关键问题。Galactosidase is the key enzyme in the research of B to O blood conversion. Based on the previous study of construction of Pichia pastoris expressing recombinant α-galactosidase, this report described the optimal fermentation conditions for P. pastoris in a 5 L-working-volume fermentor. The results showed that the protein concentration in fermentation supernatant were 3～4.5 mg/mL and the activity of α-galactosidase were 80～150 U /mL, the enzyme activity ratio was 20～30 U/mg. The purification scheme by combining ultrafiltration, cation-exchange, hydrophobic and anion-exchange chomatography were constructed. The final purity of the recombinant enzyme was above 98%. The blood type B could be converted to O successfully using the purified enzyme without any structure and function destroyed.

关 键 词：Α-半乳糖苷酶 血型 毕赤酵母菌 红细胞 新生 菌株 蛋白浓度 比活性 重组酶 疏水层析 

分 类 号：Q786[生物学—分子生物学] R457[医药卫生—治疗学]