Immobilization of Urease onto Nanochitosan Enhanced the Enzyme Efficiency: Biophysical Studies and in Vitro Clinical Application on Nephropathy Diabetic Iraqi Patients

Abstract

Immobilization of enzymes is an effective method for improving the properties and applications of modern enzymes. There are several supports for enzyme immobilization. Because of its unique features, such as inertness and high surface area, chitosan was widely used to immobilize enzymes. Immobilization of urease onto chitosan is a promising approach to treating high urea levels in the blood, however, the immobilization conditions for the best kinetics and enzyme efficiency are still challenging. Herein, we tried to immobilize urease onto nanochitosan (chitosan NPs) through a cross-linker and study the kinetics (km and $v_{\max }$ values) and thermodynamics (Ea, ∆H, ∆S, and ∆G) parameters of the enzyme reaction before and after immobilization at different substrate concentration (50, 100, 150, 200, and 250 mg/dl) and incubation temperature (15, 20, 25, 30, 35, and 40°C) under selected optimum conditions. The immobilized urease chitosan NPs was characterized in our previous work using Fourier transform infrared $\text{infrared}$ (FTIR), Atomic force $\text{force}$ microscopy (AFM), and $\text{and}$ imaged here by scanning electron microscopy $\text{microscopy}$ (SEM). Results revealed that the highest efficiency % of immobilization (70.38%) was observed at 750 mg/ml chitosan NPs and phosphate buffer pH 7 at 40°C. With an increase of Km value for the immobilized enzyme, however, the efficiency of the enzyme was significantly higher than the free enzyme, $p<0.001$ . In addition, the activation energy of the reaction catalyzed by the immobilized enzyme was lower than that of the free enzyme, which suggests that the active site geometry of the immobilized enzyme was more favorable to accommodate the substrate and thus required less energy than that of the free enzyme. The reaction was endothermic by means of positive ∆H. The immobilized urease enzyme was in vitro applied to blood samples of Iraq nephropathy diabetic patients (n = 35) to investigate the effect on serum urease activity and urea level compared to healthy volunteers. Interestingly, the activity of serum urease significantly increased after adding the immobilized enzyme and the level of urea significantly decreased ( $p<0.0001$ ) by ∼1.5 folds. Thus, applying an immobilized urease $\text{urease}$ to remove urea from blood could be effective in the blood detoxification or dialysis regeneration system of artificial kidney machines.