Luo Quanzhou, Mao Xiqin, Kong Liang, Huang Xiaodong, Zou Hanfa
National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 161 Zhongshan Road, Dalian 116011, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Sep 5;776(2):139-47. doi: 10.1016/s1570-0232(02)00105-8.
Reactive continuous rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) were prepared within the confines of a stainless steel column. Then papain was immobilized on these monoliths either directly or linked by a spacer arm. In a further step, a protein A affinity column was used for the characterization of the digestion products of human immunoglobulin G (IgG) by papain. The results showed that papain immobilized on the monolithic rod through a spacer arm exhibits higher activity for the digestion of human IgG than that without a spacer arm. The apparent Michaelis-Menten kinetic constants of free and immobilized papain, K(m) and V(max), were determined. The digestion conditions of human IgG with free and immobilized papain were optimized. Comparison of the thermal stability of free and immobilized papain showed that the immobilized papain exhibited higher thermal stability than the free enzyme. The half-time of immobilized papain reaches about a week under optimum pH and temperature conditions.
在不锈钢柱的范围内制备了大孔聚（甲基丙烯酸缩水甘油酯 - 二甲基丙烯酸乙二酯）的反应性连续棒。然后将木瓜蛋白酶直接或通过间隔臂连接固定在这些整体柱上。在进一步的步骤中，使用蛋白A亲和柱来表征木瓜蛋白酶对人免疫球蛋白G（IgG）的消化产物。结果表明，通过间隔臂固定在整体棒上的木瓜蛋白酶对人IgG的消化活性高于没有间隔臂的情况。测定了游离和固定化木瓜蛋白酶的表观米氏动力学常数K(m)和V(max)。优化了游离和固定化木瓜蛋白酶对人IgG的消化条件。游离和固定化木瓜蛋白酶的热稳定性比较表明，固定化木瓜蛋白酶比游离酶表现出更高的热稳定性。在最佳pH和温度条件下，固定化木瓜蛋白酶的半衰期约为一周。
