Amberlite MB-1 was used to immobilize urease (EC.3.5.1.5). The thermal stability of the immobilized urease was better than that of the free urease. Its highest activity was obtained at 75°C and at pH 6.5 while the optimum temperature for the free urease was found to be 25°C. Urease immobilized on Amberlite MB-1 retained 65% of the original activity after 5 repeated uses and 62% of the activity after 60 days when stored at 4°C.

Urease (EC 3.5.1.5) of high activity was obtained when the enzyme was immobilized on vermiculite crosslinked with 2.5% glutaraldehyde in chilled EDTA-phosphate buffer (pH 5.5). The highest activity of the immobilized enzyme was at 65°C and pH 6.5 while the optimum temperature for free urease was found to be 25°C. The thermal stability of immobilized urease was observed to be much better than that of the free urease. When stored at 4°C, urease immobilized on vermiculite retained 69 to 81% of its activity after 60 days and 61 to 75% of its original activity was retained after 4 repeated uses. IntroductionVermiculite is a cheap, mica type mineral and is abundantly available in different countries. It is heated to obtain exfoliated vermiculite which is used for a variety of purposes because of its high porosity and good ion exchange properties. Vermiculite has been used successfully for the immobilization of neutral protease [1] and microbial protease [2]. Clays and minerals have been used for immobilization of urease [3,4].In the present studies, two different methods were used for the immobilization of urease. The effects of reaction parameters such as pH, enzyme concentration and enzyme coupling time were studied. The properties of the immobilized enzymes such as optimum pH, optimum temperature, thermal stability, storage stability and reusability were also investigated. Materials and methods MaterialsCommercial grade vermiculite was obtained from M/s Tamilnadu Mineral Corporation, India while urea, Nessler's reagent, urease (EC 3.5.1.5) type IV, glutaraldehyde, EDTA were purchased from Sigma Chemical Co., U.S.A. Preparation of vermiculiteCommercial grade vermiculite was treated as reported by Chellapandian and Sastry [1, 2] with 1 N sodium acetateacetic acid buffer of pH 5 and then the excess buffer was removed by centrifugation. The residual vermiculite was further treated with 30% hydrogen peroxide to remove organic matter present. Iron oxide present in vermiculite was removed by dithionite-citrate method. Vermiculite was further boiled in 2% sodium carbonate to remove free amorphous silica and alumina. Treated vermiculite was finally washed with distilled water. The cationic vermiculite was prepared by adding 1 M aluminium chloride solution to the treated vermiculite. The excess chloride was removed by repeated washing with 80% methanol till the supernatant liquid was free from chloride. Immobilization of urease Method 1: vermiculite + EDTA + urease100 mg of vermiculite was immersed in ice-cold buffer solution containing 1 mg/ml enzyme and the mixture was kept at 4°C overnight. Urease was coupled with vermiculite in 0.1 M sodium phosphate with 0.0078 g EDTA at pH 6.0. The vermiculite-bound urease was separated from the unbound urease by centrifugation and was washed several times with 1 M sodium chloride, distilled water and finally with a buffer solution of 0.1 M sodium phosphate (pH 6.5). Method 2: vermiculite + glutaraldehyde + EDTA + urease100 mg of vermiculite was treated with 2.5% (v/v) glutaraldehyde fo...

Urease (EC 3.5.1.5) was covalently attached through glutaraldehyde to partially hydrolysed nylon 6/6 tubes. The highest activity of immobilized enzyme was obtained at 65°C and pH 6.5, while the optimum temperature for free urease was found to be 25°C. Immobilized urease showed an improved thermal stability in comparison to free urease. It retained 76% of the original activity after 60 days when stored at 4°C and 78% of the activity after 5 repeated uses.