Design and Synthesis of Novel Artificial Enzymes by Using Recombinant DNA Technique

利用重组DNA技术合成了假单胞菌KWI-56菌株C端10或20个酪氨酸残基连接的酯酶疏水突变酶。多聚酪氨酸链较长的突变蛋白比多聚酪氨酸链较短的突变蛋白更疏水性。用圆二色谱分析了突变蛋白的结构，发现突变蛋白的α-螺旋构象减少，β-折叠构象增加。由于多聚酪氨酸链与蛋白质疏水区相互作用引起结构变化，导致突变体酯酶的酶活性丧失。另一方面，合成了聚氧乙烯连接的疏水突变的枯草杆菌Carsberg酶，并研究了其在有机溶剂中的构效关系。该突变体酶在苯溶液中的酯交换反应中的活性是野生型的150倍。该突变体酶在二氯甲烷溶液中也有较高的活性，但在二甲基亚砜、乙腈和四氢呋喃等水相混溶的有机溶剂中活性较低。在这些溶剂中，突变蛋白的分子结构发生了显著变化，这可能是由于酶的必需水分耗尽所致。

Hydrophobilized mutant enzymes of esterase from Pseudomonas sp.KWI-56 by connection of 10 or 20 tyrosine residues at the C terminus were synthesized by using the recombinant DNA technique. The mutant protein with a longer poly (tyrosine) chain became more hydrophobic than that with a shorter chain. The structure of the mutant proteins was analyzed by CD spectroscopy, which revealed the decrease of alpha-helicalconformation and the increase of beta-sheet conformation. The enzymatic activity of the mutant esterase was abolished due to the structure change induced by connection of a poly (tyrosine) chain, which should interact with hydrophobic region of the protein. On the other hand, a hydrophobilized mutant enzyme of subtilisin Carsberg by connection of poly (oxyethylene) was synthesized and the structure-activity relationship was investigated in organic solvents. The mutant enzyme showed 150-fold higher activity than the wild type in the ester-exchange reaction in benzen solution. The high activity of the mutant enzyme was also observed in dichloromethane solution, but the activity was diminished in water-miscible organic solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran. In these solvents, the molecular structure of the mutant protein was change significantly probably due to depletion of the essential water from the enzyme.