MODULATING CATALYSIS AND CHEMOTACTIC DRIFT OF ENZYMES IN CONFINED AND CROWDED MEDIA

Abstract

Understanding enzymatic catalysis-cum-chemotaxis behavior is a matter of growing interest to the scientific community not only due to their biological significance but also their application in designing target-specific nanobiomachine. The cellular environment is entirely different from the aqueous buffered system as the vivo environment is more crowded and confined, and many enzymes are membrane-bound. 1 Thus, studying of catalytic-cum-chemotactic property of enzymes, both free and membrane-bound in crowded environment is more important as it mimics cellular environment. Also, it is essential to know how an enzyme drifts or moves toward its substrate while confined, which can find use in drug delivery applications. In this context, the following work investigates above stated factors and provides a basic understanding of change in catalytic activity and chemotactic drift of confined enzyme. It starts with introducing different systems of confining enzymes in Chapter 1 ranging from reverse micelle to Microemulsion based Gel and other systems like confining in crowding media to in microchannels. In Chapter 2, a simple method of confining enzymes in Carbohydrate mediated Microemulsion based Gel (MBG) was explored. The method is sui generis in the confinement techniques. Previously gelatin was used to make the MBG. The formed MBG has shown thermos- stiffening properties and the catalytic activity of entrapped HRP and alpha glucosidase (a thermophilic enzyme) in MBG was explored in the presence of their substrates. 2 In Chapter 3, The catalytic activity and inhibitory effect of Alkaline Phosphatase (AP), a physiologically and diagnostically important enzyme in context to our body, was studied in the presence of macromolecular crowders. Herein, we found that liposome-bound alkaline phosphatase had much higher activity in crowded environments, showing the importance and superiority of soft- deformable particles (i.e., vesicles) over hard spheres in macro-molecularly crowded media. We have also found a paradoxical behavior of inhibitors in terms of both their extent and pathway of inhibitory action. 3 Further, In Chapter 4, Acetylcholinesterase was confined, which catalyzes the hydrolysis of the neurotransmitter acetylcholine. An enzyme-based micropump was developed through a layer-by-layer assembly process. Its catalytic action, along with inhibitors (ranging from paraoxon to adenine-based nucleotides) was also studied. The same study was also done by immobilizing Human Plasma which contains Acetylcholinesterase, resulting in a prototype model of a plasma-based micropump. 4 Finally, in Chapter 5, the enzyme (AP) was confined in a microfluidic channel and its drifting behavior was studied in the presence of small crowders like sugar molecules, mainly sucrose, fructose and glucose which is pretty physiologically pertinent and catalytically relevant metal ions in purely non-catalytic conditions. 5 In the last chapter, the work summarizes the main insights of above performed experiments and provides a glimpse of attainable or explorable future perspectives.