Preparation of Magnetic Chitosan Beads as Carriers for Papain Immobilization

Papain, a well-known cysteine protease, is predominantly found and extracted from papaya fruit. Its broad hydrolyzing activity and stability at different temperature and pH, make it a widely used enzyme in food, and pharmaceutical industries. For example, it is used in various food processing procedures and in the production of bioactive peptides. However, under extreme industrial conditions, the activity of this and other enzymes, are significantly decreased. This loss of enzyme activity could be overcome by immobilizing the enzymes on a solid support. The immobilization of enzymes also allows them to be reused. Chitosan is a non-toxic, biodegradable polymer derived from the deacetylation of chitin, the second most abundant polysaccharide in nature. Chitosan offers multiple functional groups for binding enzymes and other molecules and has myriad enzyme immobilization applications on a wide variety of materials, including nanoparticles, nanocomposites, and nanofibers. In this study, we have prepared mechanically stable magnetic chitosan beads as carriers for immobilized papain. Chitosan beads were prepared using a phase inversion technique. Chitosan (2.0 gm) was dissolved in 50mL diluted acetic acid and mixed thoroughly with 0.3M FeCl3 solution. The homogenous mixture was placed in a syringe and extruded dropwise into a beaker with 0.5M sodium hydroxide with contestant stirring. The resulting beads were stirred for one hour and washed with water. The washed beads were incubated with 0.5% glutaraldehyde solution for 10 hours with constant shaking. This step was carried out to stabilize the beads by crosslinking the chitosan beads while also creating additional free aldehyde groups for papain binding. Following washing, the beads were immersed in 20mL of 10% ammonium hydroxide and 0.2M FeCl2 solution and stirred for 2 hours to create magnetic chitosan beads. The beads were separated using an external magnet and washed thoroughly. The beads were stored in 50mM phosphate buffer (pH 7.5). For immobilization of papain, the beads were incubated at room temperature in 0.1% papain solution for 20 hours with constant shaking. The immobilized papain was washed and stored in buffer. The enzyme activity of free and immobilized papain was determined by measuring the acid soluble peptides generated by hydrolyzed casein. The thermal stability studies shows that the immobilized papain retained 93% activity after 60 minutes incubation at 80o C while the soluble enzyme retained only 40% of its initial activity. There was no loss of immobilized papain activity after six weeks of storage at 4o C. In addition, a significant amount of immobilized papain activity is retained after multiple usage. These results show that attachment of papain on the magnetic chitosan beads could broaden its application in industrial use. Moreover, immobilization on magnetic chitosan beads can impede losses in activity of other industrially important enzymes.