Armored Urease: Enzyme-Bioconjugated Poly(acrylamide) Hydrogel as a Storage and Sensing Platform.

Jack bean urease is an important enzyme not only because of its numerous uses in medical and other fields but also because of its historical significance-the first enzyme to be crystallized and also the first nickel metalloenzyme. This enzyme hydrolyzes urea into ammonia and carbon dioxide; however, the stability of this enzyme at ambient temperature is a bottleneck for its applicability. To improve urease stability, it was immobilized on different substrates, particularly on polymeric hydrogels. In this study, the enzyme was coupled covalently with poly(acrylamide) hydrogel with an yield of 18µmol/cm3. The hydrogel served as the nanoarmor and protected the enzyme against denaturation. The enzyme immobilized on the polymer hydrogel showed no loss in activity for more than 30 days at ambient temperature, whereas free enzyme lost its activity within a couple of hours. The Michaelis-Menten constant (Km) for free and immobilized urease were 0.0256 and 0.2589mM, respectively, on the first day of the study. The Km of the immobilized enzyme was approximately 10 times higher than that of the free enzyme. The hydrogel technique was also used to prepare light diffracting polymerized colloidal crystal array in which urease enzyme was covalently immobilized. This system was applied for the detection of mercury (Hg2+) with the lower limit as 1ppb, which is below the maximum contaminant limit (2ppb) for mercury ions in water. The experimental details of these studies are presented in this chapter.

Urease immobilized polymer hydrogel: Long-term stability and enhancement of enzymatic activity.

A method has been developed in which an enzyme namely urease was immobilized inside hydrogel matrix to study the stability and enzymatic activity in room temperature (∼27-30°C). This urease coupled hydrogel (UCG) was obtained by amine-acid coupling reaction and this procedure is such that it ensured the wider opening of mobile flap of enzyme active site. A systematic comparison of urea-urease assay and the detailed kinetic data clearly revealed that the urease shows activity for more than a month when stored at ∼27-30°C in case of UCG whereas it becomes inactive in case of free urease (enzyme in buffer solution). The aqueous microenvironment inside the hydrogel, unusual morphological features and thermal behaviour were believed to be the reasons for unexpected behaviour. UCG displayed enzyme activity at basic pH and up to 60°C. UCG showed significant enhancement in activity against thermal degradation compared to free urease. In summary, this method is a suitable process to stabilize the biomacromolecules in standard room temperature for many practical uses.

Polystyrene-graphene oxide (GO) nanocomposite synthesized by interfacial interactions between RAFT modified GO and core-shell polymeric nanoparticles.

Here we report simple and robust one-pot method for the preparation of polystyrene (PS)/graphene oxide (GO) nanocomposite using reversible addition fragmentation chain transfer (RAFT) modified GO in surfactant free emulsion polymerization (SFEP). The results suggested that ionic comonomer, styrene sulfonate sodium salt (SS-Na), concentration plays vital role in forming PS/GO nanocomposite. X-ray and electron diffraction studies suggest that there is no recombination of GO sheets when moderate SS-Na concentration is used, resulting complete exfoliation of GO sheets in the PS/GO nanocomposite. The formation of core-shell particles in which PS is the core and polystyrene sulfonate sodium salt (PSS-Na) is the shell, and the specific interactions between functional groups of GO and PSS-Na are attributed as the driving forces for the PS/GO nanocomposite formation.