Urease from the soil bacterium Bacillus pasteurii: immobilization on Ca-polygalacturonate
1996
Ciurli, S. | Marzadori, C. | Benini, S. | Deiana, S. | Gessa, C.
Urease purified from the soil bacterium Bacillus pasteurii was adsorbed and immobilized on a preformed network of Ca-polygalacturonate, a substrate which has a similar composition and morphology to the mucigel present at the root-soil interface. The adsorption proceeded with an essentially quantitative yield, and the immobilized enzyme showed no decrease of specific activity with respect to the free enzyme. The dependence of urease adsorption on NaCl concentration suggested that the enzyme is bound to the carrier gel through electrostatic interactions. The immobilized enzyme showed increased stability, with respect to the free enzyme, with increasing time or temperature, and in the presence of proteolytic enzymes. The pH activity profile revealed that the adsorbed enzyme showed no change in the optimum pH (8.0), but it was more active than the free form in the pH range 5-8. The Michaelis-Menten kinetic parameters Vmax and Km were measured for the free (Vmax = 1960 +/- 250 units ml-1, Km = 235 +/- 20 mM) and immobilized (Vmax = 1740 +/- 185 units ml-1; Km = 315 +/- 25 mM) urease. The substantial similarity of Vmax in the two cases suggests that there were no conformational changes involving the active site upon enzyme immobilization, while substrate partitioning effects between the bulk solution and the micro-environment surrounding the immobilized enzyme must be operating so as to partly increase its Km. These results suggest that bacterial urease present in plant root mucigel plays a large role in the mobilization of urea N. Its activity is in fact significantly maintained and protected by immobilization on hydrophilic gels such as those procured by root exudates.
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