Biosensors can be developed using different immobilization methods. Interest in immobilization methods have increased because biosensors have been important for science. Polyphenol oxidase (PPO) was used generally in biosensor applications. For this purpose, Polyphenol oxidase from banana was purified and covalently immobilized on chitosan-gelatin bio-composite. The properties of immobilized enzyme were investigated and compared to free enzyme. Various parameters were studied such as pH, temperature and storage stability on immobilized and free enzyme. Kinetic parameters were also evaluated by different substrates on immobilized and free enzyme. Catechol was determined the best substrate for immobilized enzyme with optimum condition. In vitro effects of metal ions were studied on immobilized enzyme. Concentration range of metal ions is 1.0-10.0 x10-6 mol/L. The activity of immobilized PPO was increased by Fe+2 and Ag+1 ion. Co+3 and Cu+1 had very strong inhibitory effects with IC50 values of 19.69x10-3 mol/L and 23.49 x10-3 mol/L, respectively. Inhibition constants (Ki) and inhibition types of metal ions were determined with immobilized enzyme. Zn+2 and Cr+3 ions were showed competitive inhibition and Pb+2 ions were determined non-competitive inhibition with immobilized enzyme. Mixed type inhibition was obtained with Co+3 ion using catechol as substrate with 3.33x10-5 mol/L Ki value on immobilized PPO. Immobilized PPO can be evaluated for biosensor for the purpose of measurements of metal ions.

Xanthine oxidase (XO), in purine metabolism is a flavoprotein containing molybdenum with a key role. It has biological functions such as regeneration of NAD+, iron absorption and mobilization, reduction of nitrates. In this study, xanthine oxidase enzyme was purified by Sepharose-4B-L-tyrosine-4-aminobenzamidine dihydrochloride gel according to affinity chromatography technique and immobilization on glutaraldehyde was investigated. XO purified by ammonium sulfate precipitation and affinity chromatography was obtained with an 11.5 % yield and 694.04 degrees of purity. The purity of XO was confirmed by SDS-PAGE and a single band of around 150 kDa was observed. Kinetic constants (KM and VMax) of the enzyme were determined 1.67x10-4 M and 0.56 U/mL.min respectively by using xanthine compound as a substrate. The in vitro effects of NH4F, NH4Cl, CaCl2, ZnCl2, HgCl2, Hg(NO3)2.H2O compounds and commercially named colchicum dispert, commonly used in the treatment of gout disease in the clinic, were investigated. The IC50 values of compounds showing inhibition effect were determined. Afterward XO was immobilized on glutaraldehyde, which was used as a solid support material. The highest XO activity was observed in the sample of the immobilized enzyme at a rate of 6 % glutaraldehyde. The kinetic constants (KM and VMax) of the immobilized enzyme were determined as 5.18x10-4 M and 0.73 U/mL.min respectively. These values revealed that the catalytic activity of the free enzyme was higher than the immobilized enzyme.