基因工程酵母菌来源的D-氨基酸氧化酶分离纯化

目的以基因工程酵母表达的胞内D-氨基酸氧化酶(DAAO)为原料，通过破细胞，分离纯化DAAO。方法采用机械研磨，反复冻溶，超声波等多种方法破细胞，获得粗酶液，硫酸铵分段沉淀。沉淀透析后，经DEAE Sephadex A-50，DEAE-DE52，Q-sepharose等离子交换柱进行第一步分离，获得的含酶粗品，再经Sephacryl S-100分子筛进一步纯化获得电泳纯的DAAO。结果超声波破壁为最佳的破壁方法，得到的每毫升酶液所含的酶活力是其他破壁方法的两倍以上，最高达到12000U／mL。两步硫酸铵沉淀能粗分DAAO，50％硫酸铵沉淀酶回收率可达85％以上。用Q-sepharose、Sephacryl S-100分子筛柱层析两步纯化，酶活力回收率最高可达90％，SDS-PAGE显示单一蛋白条带。结论本实验方法能得到电泳纯的DAAO，总回收率约为40％。

Purification of D-amino Acid Oxidase from Pichia Pastoris

Purpose To extract and purify D-amino acid oxidase from recombinant Pichia pastoris. Methods First, the crude D-amino acid oxidase (DAAO) was extracted by disrupting cells from the recombinant Pichia pastoris. Several methods such as grinding,froze-melting and ultrasonic were effective. Second, the DAAO pellets was precipitated by ammonium sulfate. At last,DAAO pellets were applied to these ion-exchanged columns:DEAE Sephadex A-50,DEAE DE52 and Q-Sepharose to remove the most nonspecific proteins, then the sample containing DAAO was applied to Sephacryl S-100 to get more pure DAAO. Results Ultrasonic was the best method of disrupting cells. Using this method, DAAO activity per milliliter was two times more than that by other methods. The highest activity could reach 12 000 U/mL. Furthermore, 50%(NH4)2SO4 precipitation could achieve a recovery of 85%. Moreover, a Q-Sepharose column combined with a S-100 column could achieve the best purification. There was a single band on SDS-PAGE. Conclusions The result of SDS-PAGE indicates that, using the methods mentioned above, we can achieve pure DAAO and the total recovery is 40 % .