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

酶是非常好的催化剂，因为它们在温和的条件下有效地和特异性地催化多种反应。它们不会产生副产品，也不会消耗大量的能源。当酶在化学过程中用作催化剂时，可以减少工艺步骤，提高产率，减少原料、废料、溶剂、能源、设备、公用事业、生产成本、环境污染，并增加利润和竞争力。然而，有时酶的一些优点变成了缺点。虽然酶在温和条件下有活性，但在极端条件下，如低pH、高pH和温度，以及在有机溶剂存在下，它们容易变性并失去催化活性。特别是，虽然大多数精细化学品不溶于水，但它们可溶于有机溶剂。然后，需要耐有机溶剂的酶作为催化剂来生产精细化学品。本研究旨在阐明酶的有机溶剂耐受性，并利用蛋白质工程技术开发新的有机溶剂耐受性酶。本研究的主要研究内容如下：1.了解有机溶剂-酶的耐受性。1 -1.有机溶剂耐受性相关领域的鉴定1 -2。各种酶和多肽在有机溶剂存在下的构象转变2。新型耐有机溶剂酶的开发2 -1。新的有机溶剂耐受酶的筛选2 -2。在异源宿主中的表达和有机溶剂耐受酶的活化2 -3。利用定点诱变技术开发新的有机溶剂耐受酶3。有效利用有机溶剂耐受性生物催化剂的工程研究3 -1。酶在有机溶剂中的反应动力学

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