Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

摘要

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in IN KOH. The beads were activated with 1 % glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 C (alginate). The activation energies were found to be 3.68 kcal mol~(-1),5.02 kcal mol~(-1), 6.45 kcal mol~(-1) for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 C and the t_(1/2) of decay in urease activity was 12min, 43min and 58min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7,6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_(max) for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 x 10~2 μmol NH3 min~(-1) mg~(-1) protein, 2.65 x 10~2 μmol NH3 min~(-1) mg~(-1) protein and 2.85 x 10~2 μmol NFh min~(-1) mg~(-1) protein, respectively. By contrast, reusability studies showed that chitosan-urease beads can be used almost 14 times with only 20% loss in original activity while alginate-urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 C and t_(1/2) of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer~R. The immobilization studies have a potential role in haemodialysis machines.