Urease immobilized on nylon: preparation and properties

Bioprocess Engineering 17 (1997) 241±245 Ó Springer-Verlag 1997 Urease immobilized on nylon: preparation and properties A. Anita, C.A. Sastry, M.A. Hashim 241 Abstract 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. 2 Material and methods 1 Introduction Nylon 6/6 tubes are used as support material for the immobilization of Jackbean type 4 urease because they are non-porous, have mechanical strength, have good ¯ow through properties, have resistance to microbial attack and high relative hydrophilicity [1]. Enzymes such as alkaline protease [1], glucoamylase, glucose oxidase, b-Dgalactosidase [2] glucose oxidase [3] are immobilized on nylon. For the determination of urea [4], ureases immobilized on low molecular weight nylon powder, inside surface of nylon tubes and within the matrix of a nylon membrane have been separately employed. Further studies on the kinetic behaviour of multilayer urease-nonwoven nylon fabric reactor [5, 6] and the effects of substrate ¯owrate on immobilized urease [7] were reported. The present work describes the coupling of the urease on a cheap, non-biodegradable, commercial grade partially hydrolysed nylon tube 6/6 using glutaraldehyde as coupling agent. Together with this, the effects of reaction parameters such as pH, enzyme concentration and enzyme coupling time on the kinetics of immobilization were studied. Investigations were also carried out to determine the properties of the immobilized enzymes, such as the optimum pH and temperature, storage stability and reusability. 2.2 Preparation and activation of nylon Commerical grade nylon 6/6 tubes of one gram were treated as reported by Chellapandian and Sastry [1] with 20 ml of 3.5 M hydrochloric acid at 50 °C for 30 minutes. Then the partially hydrolyzed nylon was washed with distilled water. Before coupling the enzyme, the partially hydrolyzed nylon was treated with glutaraldehyde. Received: 9 December 1996 2.5 Estimation of urease activity The activity of urease was estimated by the method suggested by Jayaraman [9]. As the conversion of urea to ammonia by urease is stoichiometric, urease activity is estimated by measurement of ammonia formed using Nessler's reagent. The speci®c activity of urease was ex- A. Anita, C.A. Sastry, M.A. Hashim Institute of Advanced Studies University of Malaya 50603 Kuala Lumpur Correspondence to: C.A. Sastry 2.1 Materials Commerical grade Nylon 6/6 tubes were obtained from BDH Chemicals, England while urea, Nessler's reagent, urease (EC 3.5.1.5) type IV and glutaraldehyde were obtained from Sigma Chemical Co., U.S.A. 2.3 Immobilization of urease 100 mg of nylon was treated with 2.5% (v/v) glutaraldehyde for 15 minutes. The activated nylon was washed thoroughly with distilled water to remove excess glutaraldehyde. Then 1.5 ml of phosphate buffer (pH 6) and 1 ml of urease solution (7 mg) were treated with glutaraldehyde activated nylon for 3 hours with occasional shaking. The uncoupled enzyme was removed by washing with 1 M sodium chloride, distilled water and ®nally with phosphate buffer (pH 6.5). The immobilized enzyme was stored in phosphate buffer (pH 6.0) at 4 °C. 2.4 Protein estimation Protein determination of soluble urease was performed by the method of Lowry et al. [8] using Sigma protein assay kit. The quantity of protein bound on the support was calculated by subtracting the protein recovered in the combined washings of the nylon-urease complex from the protein used for immobilization. Bioprocess Engineering 17 (1997) pressed in mM ammonia liberated per minute per mg protein while the relative activity was taken as the amount of ammonia (mm) liberated and is expressed in percentage. 242 2.6 Immobilized urease The activity of immobilized enzyme was determine by incubating 100 mg of immobilized enzyme in 1 ml of phosphate buffer (pH 6.5) with 1 ml of 4% urea solution (pH 6.5) at 65 °C for 15 minutes. From the above mixture, the ®ltrate was taken and colorimetrically measured for activity at 500 nm after developing colour with Nessler's reagent. A calibration curve with different concentrations of ammonia was drawn and this was used for the estimation of ammonia formed. 3 Kinetics of urease immobilization 3.1 Effect of pH on immobilization of urease The nylon-glutaraldehyde conjugate was treated as described earlier. The buffer pH was varied from 5.5 to 10.0 by the addition of 0.1 M sodium hydroxide or hydrochloric acid and kept for 3 hours. 4.3 Determination of optimum pH The assays for free and immobilized urease activities were carried out under standard conditions and by varying the substrate pH from 6.0 to 10.0 by the addition of 0.1 M sodium hydroxide or hydrochloric acid. 4.4 Storage stability The immobilized urease was stored in phosphate buffer of pH 6.0 at 4 °C and 25 °C for 60 days. At frequent intervals the activity was measured. 4.5 Determination of optimum substrate concentration The assays of free immobilized urease activities were carried out under standard conditions and by varying the substrate concentration. The substrate concentration was varied from 1% to 6%. 4.6 Reusability The immobilized urease was repeatedly used for the hydrolysis of urea. 5 Results and discussion The optimum pH of immobilized urease obtained by the nylon-glutaraldehyde-urease conjugate was found to be 3.2 6.0. Similar results were obtained by Jiugao et al. [10] with Effect of urease concentration on immobilization The nylon-glutaraldehyde conjugate was mixed with 1 ml urease immobilized on periodate oxidised starch. As of phosphate buffer (pH 6.0) containing urease of various shown in Fig. 1, a small shift from the optimum pH was found to cause signi®cant reduction in the activity of the concentrations and was allowed to react for 3 hours. immobilized urease. This effect is in agreement with a general observation [11±13] that positively charged sup3.3 ports displace pH activity curves of enzymes attached to Effect of time on urease immobilization them towards lower pH values. The extent of adsorption The nylon-glutaraldehyde conjugates were treated with 1 ml of phosphate buffer of pH 6.0 containing 7 mg of urease and was allowed to react for different periods of time. 4 Properties of immobilized urease 4.1 Determination of optimum temperature The activity of free and immobilized urease was assayed as previously described at increasing temperatures using urea as the substrate. 100 mg of immobilized enzyme and 1 ml of free enzyme were used for each assay. 4.2 Determination of thermal stability Thermal stability of the free and immobilized urease was evaluated by measuring the residual activity of urease exposed to various temperatures in 0.1 M phosphate buffer of pH 7.0 for 15 minutes. After heating, the sample were quickly cooled to 25 °C and assayed immediately for enzymatic activity. The remaining activities were expres- Fig. 1. Effects of immobilization pH on relative activity of urease sed as relative to the original activities assayed at 25 °C. immobilized using nylon tubes and glutaraldehyde A. Anita et al.: Urease immobilized on nylon: preparation and properties levelled off as the pH was increased from 7.0 to 9.0. These results agree with the ®ndings of Gianfreda [14] and other workers on the adsorption of many different proteins [15]. When the concentration of urease was increased during immobilization, the relative activity of the immobilized enzyme was found to increase (Fig. 2). Urease concentrations of 7 mg was found to provide the optimum level of activity of immobilized urease. Above these concentrations for immobilized urease, a reduction in the relative activity was observed. Similar observations were made by Chellapandian and Sastry [1] with alkaline protease immobilized on nylon. The decrease in relative activity for higher concentrations of the enzyme may be due to enzyme-enzyme interactions and stearic hindrance. As shown in Fig. 3, nylon tubes used were found to saturate within 3 hours of incubation at 4 °C, beyond which there was no further increase in the relative activity. Tarafdar et al. [16] found that when urease was quickly adsorbed on clay surfaces, equilibrium was attained within one hour of incubation. Urease has a high molecular weight and its rapid adsorption can be attributed to this characteristic. However, Jiugao et al. [10] found that immobilization of urease on starch was most effective when the reaction time was 24 hours. As shown in Fig. 4, the highest relative activity of immobilized urease was obtained at 65 °C. Activity of free urease was found to be maximum at 25 °C. The signi®cant increase in the optimum temperature when urease was bound to nylon indicated that immobilized urease resisted denaturation due to temperature rise. Similar observation was made by Iyengar and Rao [17] using urease bound to chitin and by Lai and Tabatabai [18]. Results obtained by Pozniak et al. [19] using urease bound to modi®ed polysulphone membrane showed that the free urease (Type III) was immobilized on the membrane. The increased adsorption of protein with increasing temperature was reported to be due to the unfolding of the protein molecules, having more functional groups available for binding to the support [20]. The optimum temperature for immobilized urease activity increased with the increase in temperature up to a certain point after which, due to the denaturation of the enzyme, a decline in the activity was observed (Fig. 4). The effect of temperature on the stability of urease immobilized on partially hydrolysed nylon tubes and free urease is shown in Fig. 5. The activity of free enzyme retained was found to be 30% at 60 °C while that of immobilized urease was 61% at the same temperature. The immobilized urease was found to have higher thermal stability when compared to the free enzyme. Results re- Fig. 2. Effect of enzyme concentration on relative activity of urease immobilized using nylon tubes and glutaraldehyde Fig. 4. Optimum temperature of free urease and urease immobilized using nylon tubes and glutaraldehyde 243 Fig. 3. Effect on enzyme coupling time on urease immobilized using nylon tubes and glutaraldehyde Bioprocess Engineering 17 (1997) 244 Fig. 5. Thermal stability of urease immobilized using nylon tubes and glutaraldehyde and free enzyme ported by Gianfreda [14] showed that the immobilized urease had a higher sensitivity to temperature than the free enzyme. These results agree with those reported by Body and Wortland [21] for urease bound on HDTA-smectite and Pozniak et al. [19] but are in contrast to the ®ndings of Sundaram and Crook [22]. The latter found that jackbean urease adsorbed on kaolinite was as thermally stable as the free enzyme. Krajewska [13], Yoshiki and Yasumi [23] found that immobilized urease had a higher thermal stability when compared to the free urease. The pH effect on the activity of urease in immobilized and free state is shown in Fig. 6. Immobilization did not shift or alter the optimum pH of the enzyme. Similar ob- servations were made by Sundaram and Crook [22], Tarafdar [24], Lai [18] and Boyd and Mortland [21]. As illustrated in Fig. 6, the optimum pH for both the immobilized urease and free urea was 6.5. It was reported that urease had different pH optima ranging from 6.4 to 7.4 depending on the type of buffer used [25]. As reported by Pozniak [19], Iyengar and Rao [17] and Weetall and Hersh [26], when bound to a support, the optimum pH of urease shifted towards the more acidic side. However, when urease was activated with formaldehyde [27], the pH optimum shifted towards the alkaline side. From Fig. 7, it can be seen that immobilized urease retained 76% and 51.7% of its original activity after 60 days when stored at 4 °C and at 25 °C respectively. Observations by Jiuago et al. [10] showed that immobilized urease can retain its activity when stored at room temperature. Figure 8 shows that the maximum activity of immobilized urease and free urease were obtained at a substrate urea concentration of 4%. The reusability of the immobilized urease for urea hydrolysis is important. The immobilized urease retained 78% of its original activity after 5 repeated uses. However, results by Pozniak et al. [19] showed that after 17 reuses, the immobilized urease retained 60% of its initial activity. Urease immobilized on nylon tubes retained activity of 12.11% of free urease. In this study, 0.635 mg of urease was found to be bound on 100 mg of carrier. It was reported that immobilized enzyme activity can vary from almost zero [28] to values above 90% [13, 23, 29, 30]. The extent of protein binding and its retention of activity were found to vary according to the size and nature of the enzymes, support and the exact conditions employed for coupling of the enzyme. In studies conducted by Epton et al. [31], the bound urease retained on different carriers was 29.6% and 19.9% respectively of the free enzyme. Fig. 6. Effect of substrate pH on relative activity of urease imFig. 7. Storage stability of urease immobilized by nylon tubes mobilized using nylon tubes and glutaraldehyde and free enzyme glutaraldehyde at different temperatures A. Anita et al.: Urease immobilized on nylon: preparation and properties Fig. 8. Effect of substrate concentration on relative activity of urease immobilized by nylon tubes and glutaraldehyde and free enzyme 6 Conclusion The present study showed that except for low retained activity, the properties and kinetics of urease were improved after immobilization. The immobilized enzyme is more stable thermally. It was also more stable during storage and can be reused several times continuously. Matrices such as nylon coupled with glutaraldehyde can be effectively used in a packed bed reactor since it is non biodegradable and has good ¯ow through properties. This is of practical importance in urea hydrolysis. References 1. Chellapandian, M.; Sastry, C.A.: Immobilization of alkaline protease on nylon Bioprocess Bioeng. 11 (1994) 17±21 2. Inman, D.J.; Hornby, W.E.: Preparation of immobilized linked systems and their use in the automated determination of disaccharides. Biochem. J. 137 (1973) 25±32 3. 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