Enzyme Design and Catalytic Function for Production of NovelMaterials

该提案描述了一个全面的计划，旨在开发生产特种生化和先进生物材料的新方法。这些物质将通过逆转两种蛋白酶的正常水解作用而形成。这些蛋白酶将被修饰以改变它们的选择性并增强它们在反应条件下的稳定性。胰蛋白酶或凝乳胰蛋白酶的特异性会被几种技术改变；位点定向诱变，筛选产生具有作用于目标底物能力的酶的突变体，在新底物周围重新折叠部分变性的酶，以及允许在酶的活性位点插入合成氨基酸的新方法。组织蛋白酶C将通过再折叠程序进行修饰，并通过各种技术进行固定化。在非天然氨基酸合成二肽和低聚物的过程中，研究提高水解反应逆转的方法。这些包括使用非水溶剂（水混溶和不混溶）来增加底物的溶解度，以及使用酶固定的定向方法来提高酶在反应条件下的稳定性。非水溶剂和固定化引起的酶构象变化将通过包括EPR在内的几种技术进行探讨。

This proposal describes a comprehensive program aimed at developing new methods for the production of speciality biochemicals and advanced biomaterials. These materials will be formed by reversing the normal hydrolytic action of two proteases. These proteases will be modified to alter their selectivity and to enhance their stability under reaction conditions. Trypsin or chymotrypsin specificity will be altered by several techniques; site-directed mutagenesis, screening for mutants which produce enzymes with the ability to act on the substrates of interest, refolding partially denatured enzyme around novel substrates and a new approach which will permit the insertion of synthetic amino acids at the active site of the enzyme. Cathepsin C will be modified by a refolding procedure, and immobilized by a variety of techniques. Methods for enhancing the reversal of the hydrolytic reaction for dipeptide and oligomer synthesis with non-natural amino acids will be developed. These include the use of non-aqueous solvents (both water-miscible and immiscible) to increase substrate solubility and the use of directed methods for enzyme immobilization to enhance enzyme stability under reaction conditions. Changes in enzyme conformation induced by non-aqueous solvents and by immobilization will be probed by several techniques, including EPR.