טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentTzor Omer
SubjectIntegrated Biochemical Systems: Spatial Arrangement and
Biochemical Function
DepartmentDepartment of Biomedical Engineering
Supervisor Professor Emeritus Noah Lotan
Full Thesis textFull thesis text - English Version


Abstract

This research was aimed at establishing procedures for the design and optimization of biochemical assemblies made of several components, all required for performing the task set forth while co- immobilized on a solid support.  An additional aim was to study the functional characteristics of these assemblies.

First, a biocatalytic system involving an apoenzyme and its coenzyme was considered. The apoenzyme is chemically attached to a carrier. The coenzyme is attached at one end of a flexible, linear polymer (the “leash”), the other end of which is tethered to the same carrier.  This system is active when the coenzyme reaches the apoenzyme.

The three dimensional conformation of the leash was analyzed in terms of the “freely rotating chain” concept. Using this model, the system’s efficiency was assessed as a function of the leash length and of the relative location of the apoenzyme and the leash.

            The study was subsequently extended to the more complex systems in which one apoenzyme is surrounded by a number of coenzyme molecules, each attached at the end of its own leash.  For such complex systems, the biochemical efficiency was also assessed as a function of the total number of surrounding coenzyme molecules and of their surface density.

            For both the basic system and the complex systems, the analytical models developed and the calculations performed indicate that an optimal surface configuration exists, the latter depending on the geometrical characteristics achieved. 

In parallel, experimental studies were carried out involving the biocatalytic model system composed of alcohol dehydrogenase (ADH) as the apoenzyme, nicotinamide adenine dinucleotide (NAD) as the coenzyme and poly(ethylene glycol) (PEG) as the coenzyme-carrying leash. These studies included synthesis of the required components, evaluation of the biochemical activity of the systems thus assembled, and development of an analytical procedure for assaying the system at hand.

            The main achievements of this research are:

-         Development of a computational method for evaluation of system parameters (chain length, distances between components and surface density) required for optimal activity of the system;

-         Development of an analytical procedure for detection and quantitation of immobilized and biochemically-active NAD;

-        Synthesis of a PEG-based polymeric reagent which allows one to attach NAD to the surface of a solid carrier;

The results obtained clearly indicate that the design, optimization and assembly of the multi-component systems considered is indeed feasible, but only when using the principles outlined and the computational tools developed.