Design of Molecular-signal Responsive Enzyme Systems Using Bioaffinity Binding and Dissociation

近年来，人们对智能分子系统的发展提出了强烈的要求。变构酶分子的活性是通过与催化蛋白和调节蛋白的分子信号响应偶联来控制的。在本研究中，利用亲和素分子与生物素或生物素类似物修饰的酶之间的生物亲和结合和解离，设计并构建了分子信号响应型酶促反应体系。第一年制备HABA（羟基苯偶氮苯甲酸）修饰β -半乳糖苷酶作为催化位点，并与亲和素偶联作为调控位点。偶联后β -半乳糖苷酶活性被抑制约60%，以适当的修饰比例加入游离生物素后活性几乎完全恢复。第二年合成了生物素修饰的黄素辅酶衍生物，并重组为载脂蛋白d氨基酸氧化酶。一种生物素结合蛋白，亲和素结合到重组酶上，导致酶活性抑制。加入游离生物素后，酶-亲和素复合物解离，酶活性部分恢复。综上所述，开发由分子信号调控酶活性的智能酶分子系统的方向是开放的。

Recently development of intelligent molecular system is strongly requested. The author have paied much attention to allosteric enzyme moleules in which the enzyme activity is controlled by the molecular-signal responsive conjugate formation with catalytic protein arid regulatory protein. In this research, molecular signal-responsive enzymatic reaction systems was desingned and constructed by using bioaffinity binding and dissociation between avidin molecule and biotin- or biotin analogue-modified enzyme.In the first year, HABA (hydroxybenzeneazo-benzoic acid)-modified beta-Galactosidase was prepared as a catalytic site and conjugated with avidin as a regulatory site. The beta-Galactosidase activity was suppressed at about 60% after conjugate formation and was recovered almost completely by free biotin addition at the sutable modification ratio.In the second year, biotin-modified flavin coenzyme derivatives were synthesized and reconstituted into apo-D-amino acid oxidase. A biotin-binding protein, avidin bound to the reconstituted enzyme and it resulted in the enzyme activity suppression. Following addition of free biotin induced dissociation of the enzyme-avidin complexs and resulted in a partial recovery of the enzyme activity.In conclusion, the author would like to say the direction to develope intelligent enzyme molecular systems in which the enzymatic activity is regulated by molecular signal was open.