RESULTS AND DISCUSSION
The structural integrity and activity of the urease were retained due to mild
and precise physical condition during the immobilization procedure. The calcium
alginate beads were chemically inert to the entrapped enzyme. The porosity of the
gel was such that it allowed easy movement of the substrate molecules through
the beads. The beads were quite stable in tris buffer (pH 7) and in phosphate buffer
with pH > 7.8. However at higher pH values, the beads showed softening and
stickiness of the surface. The entrapment of urease in alginate gel was approximately
60 %. The immobilized enzyme retained 41 % of its activity. Urease is a
thermostable enzyme that is denatured at/above 80 °C23 and, therefore, it could
be easily immobilized on paraffin wax. Alginate beads are hydrophilic in nature
while paraffin and lac are hydrophobic so only the surface molecules react. The
reinforced films of paraffin were durable; the enzyme embedded could catalyze
and retained 45–50 % of its activity.24,25
As the above immobilization procedures were not applicable for thermolabile
enzymes, immobilization on lac films was a step refinement as it involved
extremely mild physical conditions. The lac films were tougher, thermostable and
the enzyme could easily catalyze and retained 28–30 % of its activity.
The results of the studies performed with these preparations were compared
with those of native enzymes to determine the utility of immobilized urease in
the hydrolysis of urea.
Storage stability and reusability
The native enzyme retained the same activity for about seven days, after which
it slowly decreased and was lost completely after a month. The enzyme immobilized
on calcium alginate retained the same activity for a fortnight, then decreased
and was lost in 30 days. The beads darkened (slightly brown) and deteriorated
(beads were stored at room temperature in CaCl2 solution).
The enzyme immobilized on paraffin wax films preserved in buffer retained
the same activity for a week and had retained about 73 % of its activity after a
month. When the films were preserved in distilled water, 70 % activity of the immobilized
enzyme was observed even after a month. When preserved under dry
condition, the enzyme retained 88 % of its activity. During wet preservation, the
film softened somewhat and the protein leached out slightly (Fig. 1).
The storage stability of the enzyme immobilized on lac films when preserved
in dry condition was the greatest and the immobilized enzyme had retained 98 %
of its activity even after a month.