• apparent Michaelis constant;
  • Eupergit® C and C 250 L supports;
  • free or immobilized acid urease;
  • internal effectiveness factor;
  • observable Thiele modulus;
  • particle grinding;
  • urea degradation kinetics;
  • thermal and storage stability


The adsorption capacity and immobilization rate of two Eupergit® supports for acid urease was studied by varying the ionic strength and enzyme preparation concentration in the immobilizing solution at pH 7. Eupergit® C250 L yielded a series of derivatives with enzyme loadings (YP/B) ranging from 48 to 171 mg of bovine serum albumin equivalent (BSAE) per gram of dry support (ds). Use of drastic postimmobilization conditions at pH 9 for 3–9 days yielded a slight decrease (8–14%) in the initial activity of immobilized enzymes and a limited increase in the stabilization factor (1.1–1.5), as assessed by accelerated aging tests at 65°C. Further storage tests at 4°C in the wet state showed that the activity of several derivatives either stabilized or not was practically constant for as long as 547 days. Both free enzyme and immobilized acid urease derivatives exhibited a kinetic pattern of the Michaelis–Menten type. Using the Eadie–Hofstee diagram, the specific ammonia formation rate constant for free (kcat) or immobilized (k′cat) enzyme resulted to be little affected by immobilization (kcat ≈ k′cat ≈ 18.86 ± 0.34 IU/mg BSAE), whereas the apparent Michaelis constant for immobilized enzymes exhibited a statistically significant increase at P < 0.05 from the intrinsic value (2.55 ± 0.14 mM) for free enzyme to 5.38 ± 0.87 mM as YP/B increased to 171 mg BSAE/g ds. By estimating the observable Thiele modulus (ϕobs), the activity of the biocatalyst with the greatest enzyme loading at the lowest urea concentrations tested (0.833 mM) was reduced by a factor of about 2 due to internal diffusional limitations. By operating in the pseudofirst-order regime with immobilized derivatives at YP/B about 126 mg BSAE/g ds, their activity after grinding was no more limited by intraparticle diffusion and approached the value for free enzyme. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012