Enzymes in thermostable biosensors and nanomaterials

Thermophilic organisms live under extreme physico-chemical conditions. These microorganisms provide a valuable source for thermostable enzymes that can potentially lead to new advances in biosensors and biocatalysts. This thesis describes the use of thermostable asparaginase in the fabrication of a biosensor for asparagine. In order to monitor asparaginase activity, an ammonium-selective glass electrode was employed to detect the ammonium changes due to the reaction catalyzed by the asparaginase. The sensor demonstrated higher stability at elevated temperatures compared to mesophilic asparaginase enzymes. It has the advantage of tolerating temperatures as high as 135°C, compared to ammonia gas sensors which can tolerate no more than 60°C.; As Nanotechnology is becoming increasingly integrated with biological science, a new field is developing called bio-nanotechnology. The coupling of nanomaterials with biological molecules has become a way to fabricate nanoscale systems with new or improved biological properties. Alumina nanoparticles can be functionalized with biological molecules through condensation of surface hydroxyls between alumina with phosphoryl compounds. The enzyme pepsin has one phosphoryl group on its serine 68 residue. The alumina-pepsin nanoparticles are compared with the corresponding coupling with micro-sized particles. The capacity of the nanoparticles to binding pepsin on nanoparticles was about ten times larger than that on micro-sized particles. Both commercial γ-alumina nanoparticles and amorphous alumina nanoparticles derived from tetrametallic molecular precursors were used for the conjugation studies. Also, a recombinaut glutathione S-transferase (GST) was phosphorylated by a protein kinase. The conjugation of phosphorylated GST with alumina nanoparticles was compared with the coupling of native GST with nanoparticles. The coupling process is highly reversible, with over 93% bound enzyme being released after incubation with phosphate solution. Alumina nanoparticles can be assembled together with the dimeric recombinant phosphorylated GST to form higher order structures.