Surface interaction of glucose-6-phosphate dehydrogenase

This work focuses on the site-directed as well as reversible covalent immobilization of G6PDH from Leuconostoc mesenteroides. The following objectives are intended to aim a controlled immobilization process with a deep understanding of participating protein surface interactions. 1. Recombinant production of G6PDH as well as 6PGDH to present a potential application in an in vitro enzymatic reaction sequence. Besides activity and stability of the model enzymes, the necessity of a dimeric G6PDH is investigated. 2. G6PDH immobilization on glutaraldehyde (GDA) activated carriers. Resulting activities and stabilities are used as a reference for the alternative site-directed immobilization via thiol-disulfide interchange. 3. Genetic introduction of cysteine at different positions of the G6PDH surface to allow immobilization via thiol-disulfide interchange. In contrast to immobilization via GDA, a reduced number of immobilization points and a correlation between binding position and enzymatic activity are intended (Fig. 2.1). 4. Evaluations of the G6PDH variants after immobilization via thiol-disulfide interchange in view of enzymatic activity, stability and reversibility of the binding. The influence of enzyme orientation on G6PDH activity as well as the immobilization efficiency will be discussed in the context of standard immobilization via GDA.