Immobilization of catalase on modify chitosan beads

Enzyme immobilization techniques usually provide, in addition to the desired reuse of the enzyme, unexcelled advantages such as product separation and continuous operation. For successful development and application of an immobilized biocatalyst, the enzyme support is generally considered as the most important component. Despite the great ease allowed by continuous immobilized enzymes reactors, the large-scale applications of immobilized enzymes are stil rare primarily owing to the unacceptable expense of the support materials in most cases. Accordingly, it is imperative to develop new techniques for enzyme immobilization on industrially available and cheaper carriers like chitosan that has been known as an ideal support material for enzyme immobilization because of its hyrophilicity, biocompatibility, biodegradability, and anti-bacterial property. This macromolecule, derived chemically by deacetylation of chitin the second most abundant biopolymer in nature next to cellulose, has widely been assumed to be a cheaper and versatile sorbent for transition metal ions and organic substances through the coordination and/or reaction sites composed of the amino (/NH2) and hydroxy (-OH) groups anchoring on chitosan chains.In this study, catalase immobilization was carried out covalently onto chitosan and modified chitosan beads. The physical modification of chitosan was fulfilled with clay and Fe3O4, the chemical modification was fulfilled with L-lysine. Chitosan, chitosan-clay (1:1), chitosan-Fe3O4 (1:0,2) were dissolved in asetic asid solution and these solutions were dropped through a syringe into cold TPP solution. The beads were stirred a certain time to become ripe. For preparing lysine modified chitosan beads, at first chitosan beads were prepared and than these beads were modifed with L-lysine. Optimization and characterization studies (optimum glutaraldehyde concentration, optimum Cross-linking time, optimum immobilization time) of catalase immobilized onto chitosan and modified chitosan beads were realized. Glutaraldehyde was used as a cross-linker and optimum glutaraldehyde concentration was determined %2 for lysine modified chitosan beads whereas it was determined % 3 for chitosan, chitosan-clay and Fe3O4 modified chitosan beads. The protein content of catalase solution was determined by the method of Bradford. the amount of protein bounded on chitosan, chitosan-clay, chitosan-Fe3O4 and chitosan-lysine beads were calculated % 57.14, % 60.71, % 78.57 and % 73.21.The activity of immobilized catalase was calculated as unite of 1 g carrier (U/g). The activity of chitosan, chitosan-clay, chitosan-Fe3O4 and chitosan-lysine were measured 60.83 U/g, 79.07 U/g, 88.50 U/g and 121.19 U/g, respectively. The measurement of activity of free and immobilized was carried out pH 4.0-9.0 at various pH, at optimum pH studies. Optimum pH of catalase immobilized on different carriers was determined pH 7.0. At optimum temperature studies, free and immobilized catalase activity were measured at various temperatures between 5 oC and 60 oC. Optimum temperature for free and immobilized catalase was determined 35 oC. At temperature and pH stability studies, free and immobilized catalase were incubated at various pH and temperatures. After 1 hour, the remaining activities of free and immobilized catalase were measured. We observed that immobilized catalase was more resistance than free catalase. The KM values of chitosan, chitosan-clay, chitosan-Fe3O4, chitosan-lysine and free catalase were found as 33,76 mM, 30,33 mM, 26,66 mM, 27,0 mM and 14,28 mM. The Vmax values of chitosan, chitosan-clay, chitosan-Fe3O4, chitosan-lysine and free catalase were found as 141,92 U/mg protein, 176,6 U/mg protein, 195,04 U/mg protein, 197,5 U/mg protein ve 3076,92 U/mg protein. These results were showed that the KM value of immobilized catalase was higher than free catalase and the Vmax value of immobilized catalase was lower than free catalase. At the end of the storage stability studies, it was determined that immobilized catalase was more stable than free catalase at 4 oC and 25 oC. For determining reusability of immobilized catalase, the activity of the same beads were measured 100 times and it was determined that the beads were stil active.