Immobilization of urease onto membranes of modified acrylonitrile copolymer

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Journal of Membrane Science 135 (1997) 93-98

Immobilization of urease onto membranes of modified acrylonitrile copolymer Ts. Godjevargova*, A. Dimov Department of General Chemical Technology, University "Prof. Dr. A. Zlatarov", 8010 Bourgas, Bulgaria

Received 13 January 1997; received in revised form 12 May 1997; accepted 13 May 1997

Abstract Urease was immobilized onto membranes prepared from acrylonitrile (AN) copolymer (powder) modified preliminarily with 2-dimethylaminoethyl methacrylate (DMAEM) and diacrylamido-2-methylpropanesulfonic acid (AMPSA). The results obtained were compared with those from commercial acrylonitrile copolymer membranes surface modified with DMAEM and AMPSA under the same conditions. The preliminary treatment was found to give higher amount of active groups and higher modification degree compared to surface modified acrylonitrile copolymer membranes. The modified membranes were used as carriers for immobilization of urease. The basic characteristics of the immobilized urease (amount of bound protein and relative activity) were studied. Temperature and pH optima and thermal stability of the immobilized urease were also studied. The membranes prepared from AN copolymer (powder) modified with AMPSA and DMAEM were used to manufacture diagnostic test-strips for analysis of urea in blood. Keywords: Immobilization; Urease; Acrylonitrile copolymer

1. Introduction Polymer membranes are often used for immobilization of enzymes [1-4]. An additional modification of polymer membranes can increase their suitability as enzyme carriers. In our previous work [5] we have discussed the immobilization of glucose oxidase onto acrylonitrile (AN) copolymer membranes surface modified by grafting dimethylaminoethyl methacrylate (DMAEM) and 2-acrylamido-2-methyl-propanesulfonic acid (AMPSA). By this modification, high *Corresponding author. Fax: +359 56 686141; e-mail: [email protected]. 0376-7388/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. P I I S0376-7388(97)001 2 1-X

content of active groups cannot be achieved without changing the pore structure of the initial membranes. The aim of the present work is to study the immobilization of urease onto membranes prepared from acrylonitrile copolymer (powder) modified with D M A E M and AMPSA, which has high degree of modification and pore structure identical to that of the commercial membranes of AN copolymer. The results are compared with matrices obtained by surface modification of commercial AN copolymer membranes under the same conditions. The former were used for preparation of diagnostic test-strips for analysis of urea in biologic liquids.

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2. Experimental 2.1. Materials Polyacrylonitrile-methyl methacrylate-sodium vinylsufonate membranes, molecular cut-off 10000, supplied by Spartak, Bulgaria, were used. The ternary copolymer (91.03 wt% acrylonitrile, 7.3 wt% methyl methacrylate and 1.4 wt% sodium vinylsulfonate) supplied by Neftochim, Bulgaria, were also used. The following agents were used for the modifications: dimethylaminoethyl methacrylate, 2-acrylamido-2methylpropanesulfonic acid, benzyl chloride, purum, product of Fluka, Switzerland; sodium hydroxide, hydrogen peroxide and nitric acid, purum, from Bulgaria; ferrous ammonium sulfate, purum, from Reachim, Russia. Urease used was with specific activity of 112 U/mg, product of Fluka Chemie AG, Switzerland.

2.2. Modification of acrylonitrile copolymer (powder) 2.2.1. By grafting of DMAEM Acrylonitrile copolymer was partially hydrolized with 3 wt% aqueous solution of HCI for 30 min at 40°C with intensive stirring. The hydrolyzed copolymer was treated subsequently with 1 wt% solution of NaOH and washed with 2 1 distilled water. Then they were immersed in a 0.01 M aqueous solution of ferrous ammonium sulfate (pH 5) for 10 min at room temperature and the excess of Fe 2+ ions washed with distilled water (pH 5.5). Further, the copolymer was immersed in 10 wt% aqueous solution of DMAEM for 15 min. The monomer was neutralized by acid prior to the reaction. Hydrogen peroxide (0.6 wt% - insufficient to initiate polymerization without Fe 2+) was added to the reaction mixture and the pH dropped to about 4. The graft copolymerization was carried out for 4 h at 40°C with intensive stirring. Homopolymerization is negligible under these conditions [5]. Modified acrylonitrile copolymer was thoroughly washed with distilled water to remove soluble homopolymer. Copolymer modified by grafting of DMAEM was immersed in 50 wt% solution of benzyl chloride in ethanol at 50°C. for 7 h to quaternize the tertiary amino groups. Then, the copolymer was washed with distilled water and dried at 90°C.

2.2.2. By grafting of AMPSA AMPSA was grafted onto partially hydrolyzed AN copolymer (3 wt% HC1, 40°C, 30 min) by immersing the latter in 0.5 wt% aqueous solution of ferrous ammonium sulfate for 10 min at room temperature, followed by washing with distilled water. The AN copolymer was immersed in a mixture containing 0.3 wt% hydrogen peroxide, 0.9 M water solution of AMPSA and 0.5 M solution nitric acid (pH 4) for 4 h at 30°C with intensive stirring. Then the copolymer was washed with distilled water to remove the soluble homopolymer and dried at 90°C. Membranes were cast from the AN copolymer modified with AMPSA and DMAEM by the phaseinversion method [6].

2.3. Surface modification of AN copolymer membranes by grafting of DMAEM and AMPSA Surface modification of commercial AN copolymer membranes was carried out under the same conditions as the modification of AN copolymer (powder).

2.4. Immobilization of urease The modified membrane (2 cm 2) was immersed in 0.6 mg/ml urease dissolved in 0.06 M phosphate buffer (pH 6.8) for 17 h at 4°C. It was then washed with double distilled water, 0.1 M solution of NaC1 and 0.06 M phosphate buffer (pH 6.8). All solutions used for the immobilization were prepared with double distilled water.

2.5. Analyses The contents of quaternary ammonium groups and sulfonate groups were proved by residual potentiometric titration in heterogeneous medium [7]. A Radelkis pH-meter (Hungary) was used for the measurements. Membrane hydrophilicity was expressed by the weight difference between the water-swollen and dry membrane (water content) per unit membrane weight [8]. The amount of protein bound to the membrane was determined by the method of Lowry et al. [9]. The free and immobilized urease activities were measured

Ts. Godjevargova, A. Dimov/Journal of Membrane Science 135 (1997) 93-98

spectrophotometrically (Specol 11, Carl Zeiss Jena) at 480 nm [10].

2.6. Preparation of diagnostic (dry) test-strips for analysis of urea in blood A reagent mixture containing urease and bromphenolblau dissolved in phosphate buffer with pH 5.5 was prepared. To improve membrane elasticity and wettability, as well as to preserve membrane pore structure, polyvinylpyrrolidone (I reagent mixture) or glycerin (II reagent mixture) were added to the initial mixture. The modified membranes were immersed in the reagent mixture for 24 h at room temperature and then dried at 30°C. Diagnostic tests (6 × 6 mm by size) were cut from the dried membranes and then glued at one end of 6 × 80 mm poly(vinyl chloride) bands.

3. Results and discussion The AN copolymer does not have active groups necessary for immobilization of enzymes, so it must be additionally modified to create them. Therefore, AN copolymer was modified with DMAEM and AMPSA to achieve high modification degree and high amount of active groups. Membranes with cut-off 10000 were cast from modified AN copolymer by the phase inversion method [6]. They have practically the same pore characteristics as the commercial AN copolymer membranes. The maximum of the two differential pore size distribution curves was at 1000 ,~ (the pore size was determined by mercury porosimetry). The polymer film thickness was 0.15mm. The flux with respect to water was

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243 1/m2 h. The flux was determined with a Lab Unit 20 supplied by DDS (Denmark). For comparison, commercial AN copolymer membranes were surface modified with AMPSA and DMAEM under the same conditions. Thus, sulfonate groups were introduced in the first case and tertiary amino groups (further subjected to quaternization) in the second case. Both were proved qualitatively by IR spectroscopy and quantitatively by residual potentiometric titration in heterogeneous medium. Table 1 shows some basic characteristics of the modified membranes - active groups content and degree of hydrophilicity. The results show that the preliminary treatment of the initial AN copolymer gave higher content of active groups and higher degree of modification, compared with the surface modified AN copolymer membranes, under the same conditions. This is probably due to the larger reaction surface area of the copolymer powder compared with that of the membrane. Besides, the increase of active groups content increased the degree of hydrophilicity of the modified membranes. The highest amount of active groups and highest hydrophilicity shows the membrane obtained from AN copolymer modified with AMPSA. The degree of hydrophilicity of unmodified AN copolymer membrane is only 53.9%. The modified membranes were used as carriers for immobilization of urease. For comparison, the same experiments were carried out with unmodified membrane. The basic characteristics of the immobilized urease were studied - amount of bound protein and relative activity. The relative activity of immobilized urease was determined as a ratio of immobilized enzyme activity to total activity of the free enzyme used for the immobilization. It can be seen from

Table 1 Basic characteristics of modified membranes of AN copolymer Membrane no.

Modifying agent

Amount of active groups (meq/g)

Degree of hydrophilicity (%)

1 2 3 4

AMPSA a DMAEM a AMPSA b DMAEM b Standard deviation

1.21 0.84 0.64 0.55 0.02

70.2 66.1 64.8 62.0 0.1

aMembranes from modified AN copolymer. bSurface modified membranes of AN copolymer.

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Table 2 Basic characteristics of urease bound onto membranes of AN copolymer Membrane No.

Modifying agent

Bound protein b (mg/cm2)

Relative activityc (%)

1 2 3 4 5

-AMPSA a DMAEM a AMPSA b DMAEM b Standard deviation

0.016 0.066 0.027 0.021 0.018 0.003

40.7 74.8 92.6 41.9 65.1 0.2

aMembranes from modified AN copolymer. bSurface modified membranes of AN copolymer. CActivity was measured at pH 6.8 and 30°C.

Table 2 that the membranes prepared from modified AN copolymer have higher amount of bound protein compared with the surface modified membranes. The highest amount of bound protein was measured in membranes of AN copolymer modified with AMPSA. These results are relevant to the high amount of active groups (binding the enzyme) in membranes prepared from modified AN copolymer and especially in these modified with AMPSA. The urease immobilization onto unmodified membrane is accomplished by physical adsorption, while onto modified membranes it is effected by electrostatic forces between the active groups of the carrier and the carboxylic and amino groups of the enzyme. Table 2 shows that urease immobilized onto membranes prepared from modified AN copolymer has high relative activity and it differs substantially from that of urease bound onto unmodified and surface modified membrane. Moreover, the urease immobilized onto membrane from AN copolymer modified with DMAEM showed higher relative activity compared with that of the same membrane modified with AMPSA. This is probably due to the higher amount of protein bound on the latter which leads to local aggregation of protein and steric disturbances for the penetration of the substrate to the active centers of urease [ 11 ]. The properties of the immobilized urease (temperature and pH optima, thermal stability) were studied. It was found that pH optimum of the free urease is 7 (Fig. l(a)) which corresponds to the results reported by other authors [2,3]. pH optimum of the urease bound on AMPSA modified membranes shifted to more alkali values compared to pH optimum of the native urease (from 7 to 7.5) (Fig. l(b)). It can be

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Fig. 1. Effect of pH on urease activity: (a) free (O); immobilized onto unmodified membrane of AN copolymer ( 0 ) ; onto membrane of AN copolymer surface modified with DMAEM (A); (b) immobilized onto membrane prepared from AN copolymer modified with AMPSA (O); onto AN copolymer membrane surface modified with AMPSA ( 0 ) ; onto membrane of AN copolymer modified with DMAEM (A).

explained by the negative electric charge present on membrane surface (due to excess of sulfonate groups not bound to the enzyme). The sulfonate groups attract the protons in the space surrounding the urease, so pH measured in free solution is higher. Vice versa, for DMAEM modified membranes the shift of pH optimum is in the other direction (Fig. l(a) and (b)). However, the reason for this is similar - the positive electric charge of the carrier (resulting from quaternary amino groups which did not react with the enzyme) attract the hydroxylic groups and pH measured in free solution is lower. The temperature optima (Topt) of native and immobilized urease were determined. For native urease it was found to be 30°C. For urease bound onto unmodified membrane of AN copolymer and onto carriers modified with AMPSA (by both types of modifica-

Ts. Godjevargova, A. Dimov /Journal of Membrane Science 135 (1997) 93-98

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80

~75 i~ 50

._-2 60

25 40 20 25 30 35 40 45 .Eempe~,Eure,aC

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2o,5F Js& temperakare,°C

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Fig. 2. Effect of temperature on urease activity: (a) free (O); immobilized onto unmodified membrane of AN copolymer (O); onto membrane of AN copolymer surface modified with DMAEM (/k); (b) immobilized onto membrane prepared from AN copolymer modified with AMPSA (O), onto AN copolymer membrane surface modified with AMPSA (Q); onto membrane of AN copolymer modified with DMAEM (/k).

tion), Topt w a s higher than that of the free urease (Fig. 2(a) and (b)). The highest Topt showed urease bound onto AN copolymer membrane surface modified with AMPSA (45°C). It should be noted also that the curve of the dependence temperature-enzyme activity for urease immobilized onto membrane of AN copolymer modified with AMPSA and onto membrane surface modified with AMPSA is rather wide (i.e. high enzyme activity was measured in wide temperature range). The thermal stability of free and bound urease was studied at 50 and 70°C for 2 h. Urease bound membranes used were prepared from AN copolymer modified with AMPSA and DMAEM. They were selected because of their best combination of characteristics amount of bound protein, relative activity, pH optimum and Topt. The thermal inactivation at 50°C of the native and bound urease was found to be identical for 2 h and the activity was preserved to at least 80%. At 70°C, the activity of urease bound onto membranes of AN copolymer modified with AMPSA and DMAEM dropped to 30% and 15%, respectively, while free urease showed no activity at all (Fig. 3). Recently, diagnostic enzyme express tests are widely used for qualitative and semi-quantitative analysis of some components in water, soil and agricultural plants, as well as in biological liquids. The results are usually read visually, by the intensity of the color obtained, sometimes by measuring with reflective

0.5

1.0

1.5

2D time,h

Fig. 3. Thermal inactivation at 70°C of free ( A ) and bound urease onto membranes of AN copolymer modified with DMAEM (/k) and AMPSA (O).

colorimeters. Such tests for analysis of urea in blood based on immobilized urease, have been discussed earlier [12,13]. The type of the carrier of the reagent mixture is one of the most important factors. It should have good porosity, hydrophilicity and amount of active groups to ensure satisfactory immobilization of the reagent mixture. Polymer membranes are suitable carders for this purpose. Besides, they have the additional and very important advantage to retain blood cells and let plasma through. The retained cells are washed off by thorough washing with water. Total washing is a crucial procedure which cannot be accomplished with all polymer membranes. Modified membranes from AN copolymer are washed perfectly from the retained cells. Another advantage of AN copolymer membranes is the intense coloring observed by the interaction of the reagent mixture with urea in blood, which persists after the test for a long time. The best carriers proved during the experiments membranes obtained from AN copolymer modified with AMPSA and DMAEM - were used for the preparation of diagnostic test strips for analysis of urea in blood. The reagent mixture immobilized onto them contained urease, chromogene (bromphenolblau) and phosphate buffer with pH 5.5, as well as glycerin (reagent mixture I) or polyvinylpyrrolidone (reagent mixture II) to improve wettability and preserve pore structure of the membranes. Membrane of unmodified AN copolymer membranes was studied for comparison. The initial color of the test strip is yellow and in presence of urea changes from light green to dark green-blue. The results are read visually by comparing the test strip color with color scale representing solutions with exact urea concentration

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Ts. Godjevargova, A. Dimov /Journal of Membrane Science 135 (1997) 93-98

Table 3 Study of the concentration interval, sensitivity of diagnostic test-strips for analysis of urea in blood prepared with reagent mixtures I and II Urea concentration (g/100 ml)

Unmodified AN membrane

Membrane from AN copolymer modified with AMPSA

1

II

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I

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0.02 0.05 0.1 0.15 >0.2

o o o x x

o o o o x

o x x x x

o o o x x

x x x x x

o o x x x

Membrane from AN copolymer modified with DMAEM

o - Test-strip was not colored after 60 s. x - Test-strip was colored after 60 s.

(0.02, 0.05, 0.1 and >0.2 g/100 ml). Sensitivity, reproducibility and measuring concentration interval of the diagnostic test-strips were studied to determine the best carrier for the immobilization (Table 3). Better results (sensitivity and concentration interval) were obtained with reagent mixture I. The best carrier was determined to be the membrane prepared from AN copolymer modified with DMAEM. The test-strips manufactured from this carrier showed the best sensitivity and the largest concentration interval (0.020.20 g/100 ml). The minimum concentration measured with these strips was 0.008 g/100 ml. The better sensitivity of this test is due to the higher relative activity of urease bound onto membrane prepared from AN copolymer modified with DMAEM. Furthermore, these membranes produce the most intensive coloring during the reaction enzyme-substrate. The results obtained with the diagnostic test strips are fully comparable to the well known 'Azostix' strips produced by AMES, USA [14]. Sodium oxalate and sodium citrate were used as blood anticoagulants. No fluorides or ammonium salts were employed since they reduce the activity of the bound urease. So far no substances present in blood were found to affect the specificity of the test.

References [1] B. Krajewska, M. Lezko and W. Zaborska, Urease immobilized on membranes for dialysate regeneration, Chem. Stosow., 34 (1990) 77-85.

[2] S. Kumaran, H. Maier and A. Parma, Immobilization of thin enzyme membranes to construct glass enzyme electrodes, Anal Chem., 63 (1991) 1914-1918. [3] M. Sakamoto, Effect of enzyme concentration on the dynamic behaviour of a membrane-bound enzyme system, J. Membrane Sci., 70 (1992) 237-248. [4] W. Kyowa, H. Kogyo, Enzyme membrane, Jpn. Pat., 1560691 (1987). [5] T. Godjevargova and A. Dimov, Grafting of acrylonitrile copolymer membranes with hydrophilic monomers for immobilization of glucose oxidase, J. Appl. Polym. Sci., 57 (1995) 487-491. [6] A. Dimov, St. Petrov, Preparation of ultrafiltration membranes with different permeability from PAN, Synthetic Polymeric Membranes, Waiter de Gruyter, Berlin, 1987. [7] K. Dimov, V. Sarmadjieva and P. Pavlov, Laboratory Practice on Technology of Synthetic Fibres, Technica, Sofia, 1983. [8] V. Chantora and R. Huang, Separation of liquid mixtures by using polymer membranes, J. Appl. Polym. Sci., 26 (1981) 3223-3243. [9] H. Lowry, N. Rosenbrough and A. Farr, Protein measurement with folin phenol reagent, J. Biol. Chem., 193 (1951) 265275. [10] T. Chochlova, M. Yanina, Yu. Nikitin and B. Kurganov, Mikrometod opredeleniya mocheviny v plasme i syworotke krovi s pomoshtyu ureasy immobilizowarmoi na Silochrome, Z. Anal. Chimii (Russ.), 18 (1988) 1875-1877. [11] M. Triven, Immobilizovannye fermentov, Mir, Moscow, 1983. [12] K.H. Hildebrand, H. D6hren, Test device and method for the detection of a component of a liquid sample, US Pat., 4824639. [13] K.H. Hildebrand, K. Wehling, Analysis method for determining substances from biological liquids, Eur. Pat., 0407800 A2. [14] M. Vassilev, Laboratory diagnostics. Express tests, Medicina I fizkultura, Sofia, 1990.