Activity of enzymes immobilized on microspheres with thermosensitive hairs
Activity of enzymes immobilized on microspheres with thermosensitive hairs
复制标题
热敏毛微球固定化酶的活性
DOI：
10.1016/s0927-7765(96)01323-9
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
H. Kawaguchi
中科院分区：
文献类型：
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作者：
Mamoru Yasui;T. Shiroya;K. Fujimoto;H. Kawaguchi
以4，4-偶氮二（N，N，-氰基戊酸）（V-501）为引发剂，β-巯基丙酸（MPA）为链转移剂，通过聚合反应制备了一端或两端羧基化的聚N-异丙基丙烯酰胺（PNIPAM）。将两端羧基化的PNIPAM的一个端基与胶乳颗粒缀合，另一个端基使用碳二亚胺与胰蛋白酶缀合。差示扫描量热法（DSC）显示，PNIPAM的颗粒上表现出激烈的相变，和转变温度大大提高时，酶被固定在链端。因此，颗粒上的PNIPAM显示两个相变，因为酶结合和非共轭PNIPAM的共存。用PNIPAM间隔基固定在颗粒上的胰蛋白酶的活性表现出显着的温度依赖性。表观相对活性增加以上的非酶结合的PNIPAM的颗粒上的转变温度。其原因之一是衬底的扩散在转变温度附近不连续地变化。因此，温度依赖性的酶活性显着影响底物的分子大小。胰蛋白酶和PNIPAM在颗粒上的表面密度以及PNIPAM间隔物的分子量也影响酶活性。
Poly(N-isopropylacrylamide)s (PNIPAMs) carboxylated at one chain end or both ends were prepared by polymerization using 4,4-azobis(N,N,-cyanopentanoic acid) (V-501) as an initiator and β-mercaptopropionic acid (MPA) as a chain transfer reagent. One end group of PNIPAM carboxylated at both ends was conjugated with latex particles, and another with trypsin using carbodiimide. Differential scanning calorimetry (DSC) revealed that PNIPAM on the particles exhibited a drastic phase transition, and that the transition temperature was largely elevated when the enzyme was immobilized at the chain end. Therefore, PNIPAM on the particles showed two phase transitions because of the coexistence of the enzyme-conjugated and non-conjugated PNIPAMs. The activity of trypsin immobilized on the particles with the PNIPAM spacer showed significant temperature dependence. The apparent relative activity increased above the transition temperature of non enzyme-conjugated PNIPAM on the particles. One of the reasons for this is that the diffusion of the substrate changed discontinuously around the transition temperature. Therefore, the temperature dependence of the enzymatic activity was significantly affected by the molecular size of the substrates. The enzymatic activity was also influenced by the surface density of trypsin and PNIPAM on the particle, and the molecular weight of the PNIPAM spacer.