Clarification of organic solvent-tolerance of enzymes and development of new organic solvent-tolerant enzymes using protein engineering

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Enzymes are very good catalysts, because they effectively and specifically catalyze many kinds of reactions under mild conditions. Then they do not produce by-products and not consume a lot of energy. When enzyme are used as a catalyst in a chemical process, it is possible to reduce process steps, to increase yield, to reduce raw materials, waste materials, solvents, energy, equipment, utilities, production costs, environment pollution, and to increase profits and competitiveness. However, sometimes some advantages of enzymes become disadvantages. Although enzymes are active under mild condition, they are easily denatured and lose catalytic activity under extreme conditions, such as low pH, high pH and temperature, and also in the presence of organic solvents. Especially, although most fine chemicals are insoluble in water, they are soluble in organic solvents. Then organic solvent-tolerant enzymes are required as catalysts to produce fine chemicals. In this research, clarification of organic solvent-tolerance of enzymes and development of new organic solvent-tolerant enzymes using protein engineering were performed. The examined item in this research is as follows.1. Understanding of organic solvent-tolerance of enzymes .1-1. Identification of domain concerning organic solvent-tolerance1-2. Conformational transitions of various enzymes and polypeptides in the presence of an organic solvent2. Development of new organic solvent-tolerant enzymes2-1. Screening of new organic solvent-tolerant enzymes2-2. Expression in a heterologous host and activation of organic solvent-tolerant enzymes2-3. Development of new organic solvent-tolerant enzymes by site-directed mutagenesis3. Engineering study for effective usage of organic solvent-tolerant biocatalysts3-1. Reaction kinetics of enzymes in the presence of organic solvents