Polycation-coated polyanion microspheres of urease for urea hydrolysis

Urease (EC 3.5.1.5) was immobilized within polyanionic carboxymethylcellulose/alginate (CMC/Alg) microspheres coated with a cationic polysaccharide, chitosan (C). Coating with chitosan improved the mechanically durability of the polyanionic microspheres, as well as increased enzyme immobilization yield [approximately 0.4 mg.mL-1 gel]. The effects of chitosan coating and CMC/Alg ratio on the water uptake and spherical morphology of the microspheres were investigated. The optimal pH of urease was not extensively affected by the immobilization procedure. However, the optimal temperature of urease activity increased upto 60 and 65 degrees C within CMC/Alg and C(CMC/Alg) microspheres, respectively, while the optimum for the free enzyme was 50 degrees C. The half life (t1/2) and deactivation rate constant (kd) of free urease were 79 min and 8.77 x 10(-3) min-1, respectively, whilst the t1/2 and kd values of urease within polyanion and polycation-coated polyanion microspheres were 142 min and 4.88 x 10(-3).min-1, and 179 min and 3.87 x 10(-3).min-1, at 80 degrees C, respectively. While the activation energy of the hydrolysis reaction of free urease was found to be 11.86 kJ.M-1.dm-3, it increased to 18.91 and 20.02 kJ.M-1.dm-3, for the immobilized urease within CMC/Alg and C(CMC/Alg) microspheres, respectively. The free enzyme exhibited K(m) and Vmax values of 2.85 mM.dm-3 and 31.9 mM.dm-3.s-1.g-1p-1, respectively, whilst the K(m) and Vmax for urease within polyanion and polycation-coated polyanion microspheres were 3.94 mM.dm-3 and 73.4 mM.dm-3.s-1.g-1.p-1, and 4.22 mM.dm-3 and 81.4 mM.dm-3.s-1.g-1.p-1, in the same order. C(CMC/Alg) microspheres showed a nearly stable urease activity of around 80-85% of the initial maximum activity, after the first 100 minutes.