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

点击翻译按钮获取中文摘要

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.