Construction of a flow-type of measuring system with functions for capturing and sensing heavy metal ions and its characteristics

将热溶酶（Thermolysin）作为最典型的锌酶和热稳定性中性蛋白酶，溶解在含40%甘油的硼酸缓冲液中，固定在孔径为24.2 nm的多孔玻璃微珠上。固定化收率约为30%。固定化的制剂被装入一个小聚合物柱中，然后连接到光度传感系统上。锌（II）离子范围从1。0 nM到1。可以测定0 mM，但由于暴露于螯合剂（0.1 M EDTA, pH 4.0），酶柱并不总是保持良好的操作稳定性。在酶-硼酸盐缓冲液中加入溴化钠作为热溶酶的良好溶剂，可以使酶以满意的产率固定。在相同尺寸的柱中包装的制剂应用于量热传感系统。酶柱在1.0 mL注射4.0 mM合成底物(N-（3-[2-呋喃]丙烯酰亮氨酸酰胺），pH为8.0时表现出较高的催化活性，温度响应约为4.0 mK。在柱中加入2.5 mL螯合剂后，催化活性显著降低。一旦螯合剂暴露柱很容易重新激活注入锌离子溶液。几乎无锌柱的再活化与添加的锌离子浓度有关。因此，在0.01 ~ 1.0 mM浓度范围内的锌（II）离子的量热测定是正确的。脱酶再激活法。

○ Construction of a photometric flow-injection biosensing system and its characteristics Thermolysin, one of the most typical zinc-enzymes and thermostable neutral protease, was dissolved in borate buffer solution containing 40 % glycerol and immobilized onto porous glass beads with controlled pore size(24.2 nm). The immobilization yield was about 30 %. The immobilized preparations were packed into a small polymer column and then, attached to a photometric sensing system. Zinc (II) ions ranging from 1. 0 nM to 1. 0 mM could be determined, but the enzyme column did not always retain excellent operational stability due to exposure to chelating agents (0.1 M EDTA, pH 4.0).○ Construction of a calorimetric flow-injection biosensing system and its performance Sodium bromide as good solvent for thermolysin was added to the enzyme-borate buffer solution, and thereby, the enzymes could be immobilized in a satisfactory yield. The preparations packed in the same size of the column was applied to a calorimetric sensing system. The enzyme column exhibited high catalytic activity for 1.0 mL-injections of 4.0 mM synthetic substrate (N- (3-[2-furyl]acryloyl-gly-leu amide), pH 8.0) and the temperature response was about 4.0 mK. Introduction of 2.5 mL of the chelating agent to the column caused significant decrease in the catalytic activity. The once chelator-exposed column was readily reactivated by injecting zinc)II) ions solution. Reactivation of the almost zinc-free column was related to concentrations of zinc)II) ions added. Thus, calorimetric detrmination of zinc(II) ions in a concentration range of 0.01 to 1.0 mM was positively achieved based on.the apoenzyme reactivation method.