Immobilization of urease on vermiculite

Abstract

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...

Advantages of Using Non-isothermal Bioreactors in Agricultural Waste Water Treatment by Means of Immobilized Urease. Study on the Influence of Spacer Length and Immobilization Method

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Production of biodiesel using lipase encapsulated in κ-carrageenan

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