Researching for activities stability and reaction kinetic of laccase enzymes immobilized by chitosan derivatives

Laccase enzymes (EC 1.10.3.2; p-diphenol:dioxygen oxidoreductases) are involved in multi-copper blue protein class due to their inclusion of 3 type cupper ions which are spectrpscopically different. Laccase enzymes catalyze the oxidation of various substituted phenolic compounds, aromatic amines and even some inorganic compounds by using molecular oxygen as the electron acceptor. The ability of laccase enzymes to act on a wide range of substrates makes them highly useful biocatalysts for various biotechnological applications such as textile dye decolorisation, paper pulp biobleaching and bioremediation.In this study, chitosan was modified by glutaraldehyde (GA), dopamine (DA), clay, Fe3O4, ECH and histidine. Therefore chitosan-GA-DA, chitosan-clay-GA-DA, chitosan-Fe3O4-GA-DA-, Fe3O4-clay-GA-DA-chitosan and chitosan-ECH-histidine was obtained. Modified chitosan beads was used as a carrier for the immobilization of laccase (benezenediol:oxygen oxidoreductase EC 1.10.3.2). % immobilization yields and enzyme activities per gram carrier was recorded % 82 and 2,63 U/g for chitosan-GA-DA, % 86 and 2.73 U/g for chitosan-clay-GA-DA, % 75 and 3.01 U/g for chitosan-Fe3O4-GA-DA, %59 and 2.84 U/g for chitosan-ECH-histidine.Optimum pH was found pH 4.0 for free laccase and found pH 5.0 for immobilized laccase. Also optimum temperature was found as 40 oC for free and immobilized laccase. It was observed that resistance of immobilized laccase to changes in temperature is more than free laccase. It was observed that immobilized laccase was more resistant than free laccase to temperature changes.When ABTS was used as a substrat, KM and Vmax values of free laccase was found as 0,297mM and 26,96 U/mg, respectively. , KM and Vmax values of immobilized laccase was recorded 0,397 mM and 11,24 U/mg for chitosan-GA-DA, 0,410 mM and 12,03 U/mg for chitosan-clay-GA-DA, 0,365 mM and 11,76 U/mg for chitosan-Fe3O4-GA-DA, 0,426 mM and 12,26 U/mg for chitosan-clay-Fe3O4-GA-DA and 0,385 mM and 10,89 U/mg for chitosan-ECH-histidine, respectively.In the storage studies, free and immobilized laccase enzyme was stared during 16 weeks at the 4 and 25 oC. Enzyme activities were measured at regular times. As a result of storage studies, immobilized laccase was found to be more stabile than free laccase. Reusability of laccase immobilized on the different carries was also studied. As a result of repeated 100 times activity, the enzyme activity was still observed.