嗜热酰基氨肽酶非特异性催化机制的研究及改造

Most of chemical materials, chemical reagent, medicine, pesticides, dye, spice and detergents are mainly come form organic synthesis. Environment-friendly green chemistry is the development tendency and necessary of current organic synthesis. Enzymatic synthesis is the main method for green organic synthesis. Enzyme catalytic promiscuity means a given enzyme can catalyze different chemical transformation of non-natural substrates other than its natural substrates, it expanded the application of enzyme in organic synthesis, and was focused these days. We reported an acylaminoacyl peptidase ST0779 could catalyze Aldol reaction and Henry reaction, which are very important reactions in carbon-carbon formation. ST0779 showed superior activity at elevated temperature for Aldol reaction and high yield and high selectivity for Henry reaction. But the catalytic promiscuity mechanism of ST0779 is unclear. In this application, we will crystallize and resolve the structure of ST0779. And then use molecular docking to study the interaction between enzyme and substrate, use direct mutation and domain exchanging to explore the catalytic promiscuity mechanism of ST0779. The amino acid sequence homology of acylaminoacyl peptidase family members is rather low, but they apparently share a similar three-dimensional structure. The substrate specificity is quite different among different members. We will recombinant the propeller domain and the catalytic domain of acyl-amino peptidase family members from different organisms to cerate hybrid enzymes. With the rule of the substrate specificity, properties of hydrid enzymes and the catalytic promiscuity mechanism of the acyl-amino peptidase family, we can create new enzymes with different substrate specificity, high catalytic efficiency, high selectivity and different optimum temperature.

有机合成是各种化工原料、化学试剂、医药、农药、染料、香料和洗涤剂等的主要来源，环境友好的绿色化学是当前有机合成的发展趋势和要求，酶催化合成反应是实现合成绿色化的重要途径。酶非特异性催化拓展了酶在有机合成中的应用，是酶学领域的重大发现及研究热点。本课题组发现嗜热酰基氨肽酶ST0779能够高效催化Aldol和Henry反应，比之前报道的酶具有更高的转化率及立体选择性，但其非特异性催化机制尚不清楚。本申请拟从ST0779出发，通过蛋白结晶、结构解析、分子对接、多点突变和结构域改组来研究ST0779的非特异性催化机制。酰基氨肽酶成员间序列相似性差，但均由双结构域组成，成员间的底物特异性差异较大。我们将不同来源酰基氨肽酶的催化结构域和推进器结构域进行重组构建杂合酶，基于其非特异性催化机制，结合点突变，改造出不同温度耐受型的高催化效率、高选择性的新酶，丰富酶资源。

本研究对嗜热酰基氨肽酶ST0779进行了深入研究，通过计算机辅助虚拟突变和分子对接，确定了突变位点，经过突变后酶的活性明显提高，揭示酶关键位点残基对酶活性的影响，特定位置的氨基酸（两个结构域连接处）可以对酶的构象产生较大影响，从而影响酶的活性。通过对酶活性中心和相近氨基酸残基的突变发现，嗜热酰基氨肽酶的催化残基的改变对酶的非特异性催化能力没有影响，而其他位置的残基反而会有明显影响，说明该酶的非特异性催化能力同时取决于酶的结构和相关氨基酸残基。通过对不同酶的结构域的互换，实现了酶催化特异性改造和温度适应性的改造，高温酶的结构域和低温酶的结构性杂交可以得到中温酶。ST0779 催化Aldol 反应的速度是目前报道所有酶中最快的，正常4 个小时即可完成反应。我们将ST0779与纳米金结合，通过红外光照射纳米金激活，提高了酶催化非特异性反应的效率，在红外光照射下，反应时间缩短到0.5小时，这为该酶的催化应用打下了良好的基础。同时我们发现红外光激活策略对嗜热酶的效果远好于对常温酶的效果。我们还将ST0779通过特异性定向固定化固定在四氧化三铁磁性纳米粒子上，置于交变磁场中实现了酶的激活，但交变磁场对酶的激活更适合于中温酶。