Abstract

There are a multitude of situations in which several reactions are carried out outside the cell by a sereies of secreted enzymes to acheive the desired product. These processes can sometimes suffer decreased efficiency due to the unavoidable randomness of the enzymes involved in the given reaction. Clostridium Thermocellum utilizes an effecient and well characterized cellulosome scaffoldin molecule in conjuction with a anchoring protein to bind multiple catalytic subunits to its membrane. The subunits consists of two parts: a complete and functioning enzyme, and a docking domain which binds to one of several cohesion sites located on the cellulosome scaffoldin. There are a variety of docking sites which are compatible with a variety of cohesion sites. In C. Thermocellum, this allows multiple cellulose degrading enzymes to be optimally positioned for cellulose degredation. Our team proposes to express a modified version of a C. Thermocellum cellulosome in E. coli so that we may anchor a hybrid enzyme to the cell's surface. The hybrid enzyme will include the complete alkalane phophotase and one of the docking sites normally include in C. Thermocellum's own catalytic subunits. If the project is a sucess, it will be a step towards the standardization of cell surface enzyme anchoring and positioning of enzymes involved in multi- step processes.