金属螯合双水相亲和分配技术分离纳豆激酶的研究

利用金属螯合亲和双水相分配技术对纳豆激酶的分离纯化进行了研究。考察了双水相系统、聚合物的分子量和浓度、亲和配基加入量、pH值、相比以及生物质加入量等因素对亲和分配的影响。结果表明，双聚合物系统比聚合物／无机盐系统更有利于纳豆激酶亲和分配；pH值和亲和配基加入量是影响分配的关键因素。优化的分配条件为：2．6％聚乙二醇，20．2％羟丙基淀粉，5％亲和配基PEG-IDA—Cu(Ⅱ)，相比12，pH8．2，发酵液加入量15％。分配系统放大到100g，仍保持一致的酶活收率(90％)和纯化因子(2.0)。设计了两次分配分离流程，纯化因子达到3．52，总收率为81％。

Separation of Nattokinase by Immobilized Metal Ions Affinity Partitioning in Aqueous Two-phase Systems

Nattokinase is a novel fibrinolytic enzyme with strong and specific thrombolytic activity. Isolation and purification of nattokinase directly from Bacillus subtilis fermentation broth by immobilized metal ions affinity partition (IMAP) in aqueous two-phase systems (ATPS) were studied. Effeet of various factors on ATPS, such as type of ATPS systems, molecular weight and concentration of phase-forming polymers, concentration of affinity ligand, pH value, phase ratio and the amount of fermentation broth were investigated. The results indicate that affinity partitioning in polymer/polymer ATPS is better than in polymer/salt ATPS. The control of pH and the amount of affinity ligand added are most important for affinity partitioning of nattokinase. The optimum separation conditions were obtained as: 2.6% polyethylene glycol (PEG 4000), 20.2% hydroxypropyl starch (PES100), 5% PEG-Iminodiacetic acid (IDA)-Cu(II), phase ratio of 1.2, 15% fermentation broth and pH 8.2. The separation was successfully scaled up to 100g system with similar enzyme yield of 90% and purification factor of 2.0. In addition, a twice partitioning process was developed, and the enzyme could be recovered with a yield of 81% and a purification factor of 3.52.