尼龙膜经稀盐酸水解、壳聚糖改性后，以木瓜蛋白酶为亲和膜的配基，通过戊二醛的活化处理后采用共价结合的方法将配基键合在尼龙膜上，从而得到有特异吸附性能的尼龙亲和膜.本实验考察了制备尼龙亲和膜的交联剂戊二醛的质量分数、pH值、温度、反应时间和酶用量对亲和膜上木瓜蛋白酶活力的影响，确定了最适合的制备条件：pH 9.0，质量分数为0.5%的戊二醛，反应温度为45℃，反应时间为6 h，酶用量为10 mg/mL。该优化条件下制备的尼龙亲和膜具备优良的色谱性能，可用于分离纯化半胱氨酸蛋白酶抑制剂.

 Nylon membranes are first hydrolyed with dilute HCl and then treated with chitosan  before being used as the affinity carrier. Papain as a ligand has been immobilized on the actived membranes with glutaraldehyde as crosslinking agent. The factors involving with the activity of immobilized papain, such as concentration of glutaraldehyde, pH, temperature, reaction time, and the amount of added papain have been studied. The results show that the optimum conditions for papain immobilization are as follows: pH 9.0, 0.5% glutaraldehyde solution, the concentration of added papain is 10 mg/mL, the reaction time is 6 h at 45℃. The nylon membrane prepared is then used to purify cystatin from potato juice, and showed that they are high efficiency affinity membranes base for cystatin separation.

[1] Fossum K, Whitaker J R. Ficin and papain inhibitor from chicken egg white[J]. Arch Biochem Biophys, 1968, 125(1):367－375.
[2] North M  J, Mottram J C, Coombs G H. Cysteine proteinases of parasitic protozoa[J]. Parasitology Today, 1990, 6(8)：270－275.
[3] Zeng Z K, Bao J K, Zhou H, et al. The molecular modification of cysteine proteinase inhibitor from wheat seed and its activity[J]. Scientia Agriculture Sinica, 1997, 30(1):38－43.
[4] Beeskow T C, Kusharyoto W, Anspach F B, et al.  Surface modification of microporous polyamide membranes with hydroxyethyl cellulose and their application as affinity membranes[J]. J Chromatogr A,1995, 715(1):49－65.
 [5] Lei H, Wang W, Chen L L, et al. The preparation and catalytically active characterization of papain immobilized on magnetic composite microspheres[J]. Enzyme Microb Tech, 2004, 35(1):15－21.
[6] 黄宜兵, 吴晓霞, 吴胜男, 等. 以卵清蛋白为载体的固定化木瓜蛋白酶的制备及其性质[J]. 吉林大学学报(理学版), 2004, 42(4):616-621.
[7] 贺枫,卓仁禧, 刘立建, 等. 固定化木瓜蛋白酶的制备和性质研究[J]. 高分子学报, 2000, 10(5):637－640.
[8] Wei S, Zhang F B, Zhang G L. Adsorption of bilirubin with polylysine carrying chitosan-coated nylon affinity membranes[J]. J Chromatogr B, 2005, 819(2):301－306.
[9] 苏赛男，聂华丽，朱利民，陈天翔. 尼龙亲和膜色谱法纯化半胱氨酸蛋白酶抑制剂[J]. 分析化学, 2008, 2(36): 231－234.