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

近年来，智能分子系统的发展受到了强烈的要求。本文着重研究了通过与催化蛋白和调节蛋白形成分子信号响应性共轭物来控制酶活性的变构酶分子。本研究利用生物素或生物素类似物修饰的酶与亲和素分子的亲和结合和解离作用，设计并构建了分子信号响应的酶促反应体系，第一年制备了HABA（hydroxybenzeneazo-benzoic acid）修饰的β-半乳糖苷酶作为催化位点，与亲和素偶联作为调控位点。β-半乳糖苷酶活性在偶联物形成后被抑制了约60%，在适当的修饰比例下加入游离生物素后几乎完全恢复。生物素结合蛋白亲和素与重组酶结合，导致酶活性抑制。加入游离生物素后，酶-亲和素复合物发生解离，酶活性部分恢复，由此可见，通过分子信号调控酶活性的智能酶分子系统的发展方向是开放的。

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.