Membrane-Supported, Highly-Ordered Biomaterials for Bioreactors

摘要固定化酶的方法往往会由于构象改变、不利取向等因素导致活性降低。本课题将在膜生物反应器上探索一种更特异、更均匀附着间隔物的新技术。定点诱变用于修改半胱氨酸残基的遗传密码，然后表达变异酶（枯草素）。间隔化合物一端的官能团将与聚合物膜中的氨基或羟基部分反应，而另一端将包含一个硫醇，在完成固定过程中被氧化成二硫。固定化酶和中间结构将通过核磁共振、x射线荧光、自旋标记研究和其他方法进行表征。催化行为将在包括水溶剂和有机溶剂在内的各种反应条件下进行检验。酶的固定化促进了操作的连续模式，并有助于生物制品的纯化。这项基础研究可以帮助改善许多生物技术公司的生产过程。

CTS-9307518 Kentucky Research Foundation D. Allen Butterfield ABSTRACT Methods for the immobilization of enzymes often lead to reduced activity due to conformational changes, unfavorable orientations and other factors. A new technique for more specific and uniform attachment with spacers will be explored in this project on membrane bioreactors. Site-directed mutagenesis is to be used in modifying the genetic code for a cysteine residue and then expressing the variant enzyme (subtilsin). Functional groups at one terminus of the spacer compound will be reacted with amino or hydroxyl moieties in a polymeric membrane while the other terminus will contain a thiol to be oxidized to the disulfide in completing the immobilization. The immobilized enzyme and intermediate structures will be characterized by NMR, x-ray fluorescence, spin-label studies, and other methods. Catalytic behavior is to be examined under various reaction conditions including aqueous and organic solvents. Immobilization of enzymes facilitates a continuous mode of operation and aids the purification of bioproducts. This fundamental research could help improve the production processes in many biotechnology companies.