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Enzyme textile for removal of urea with coupling process: enzymatic reaction and electrodialysis
DESALINATION Desalination
144 (2002) 163-l 66 www.elsevier.com/locate/desal
Enzyme textile for removal of urea with coupling process: enzymatic reaction and electrodialysis V. Magne”, M. Amounas”, C. Innocent”, E. Dejeanb, P. Setaa* “lnstitut Europeen des Membranes, UMR CNRS n”5635, 34293 Montpellier Cedex 5, France Tel. +33 (4) 67 14 91 11; Fax +33 (4) 67 I4 91 19; email: [email protected] “Institut Fraqais du Textile et de I’Habillement, BP 60, 69132 Ecully Cedex, France Received 6 February
2002; accepted 20 February
2002
Abstract Ion exchanging textile are used as organic supports for enzyme immobilisation in the aim to develop reactive fibrous materials able to be substituted to membrane systems in bioreactors or acting for instance as reactive bags
for waste treatment devices, thus extending the range of their potential applications. Integration of this modified textile in the electrodialysis cell allowed simultaneous removal of urea and ions produced by the hydrolysis reaction. The reactor-separator Keywords:
systems were studied under different current densities.
Ion exchange textile; Reactor-separator; Urea hydrolysis
Enzyme immobilization;
1. Introduction
Membranes are well known to be used for separation processes in various chemical and biochemical domains. In recent years, interest has been increasingly targeted in combining a membrane separation process to biocatalysis [I]. In the resulting membrane bioreactor, the chemical product obtained by the enzymatic reaction is separated from the reaction medium by the use of the membrane process. Moreover, fouling and poisoning phenomena which occur during the *Corresponding author. Presented at the International July 7-12, 2002.
Congress on Membranes
Molecular recognition;
filtration step are major limitations for the use of such technology. To improve the efficiency of the reactionfiltration coupling, the route which consists in immobilizing an enzyme in a porous material could avoid the post treatment of the solution after the biochemical reaction. The immobilization of a catalyst such as an enzyme, has particular attractions, since the sophistication of this process allows us to suppress the further separation step and also avoid many problems which occurred in some cases with catalysts. Different types of enzyme immobiliand Membrane
Processes
001 I-9 164/02/$- See front matter 0 2002 Elsevier Science B.V. All rights reserved PII: SO0 I 1-9 164(02)00306-5
Electrodialysis;
(ICOM),
Toulouse, France,
164
V Magrze et al. /Desalination 144 (2002) 163-166
zation have been used in order to fix a biological catalyst on a support. We have recently shown that the use of the enzyme molecular recognition process between avidin and biotin presents a very good efficiency to attach irreversibly and with high stability enzymes on a porous material, and as a consequence, we applied this technology to modify the reactivity of textile supports. The reason of this choice is that the textile can also be considered as a filtration membrane material thanks to its own porosity. In this paper we present an application of this concept to an enzyme modified textile based on urease immobilization in a coupling process (reaction-separation). Urea was shown to be efficiently removed from aqueous solutions, combining catalytic reaction and ionic migration through ion exchange membrane in an electrodialysis cell [2,3], As urease allows the hydrolysis of urea with production of ammonium and carbonate ions, these produced cations and anions were separated by electrodialysis across cation and anion exchange membranes, respectively. As an indication of the efficiency of this coupling process, we were able to totally remove urea and its by-products from the treated aqueous solutions. Moreover, the migration through the membrane of ionic reaction products has an additional advantage since it avoided the kinetic inhibition of the enzymatic reaction due to back reaction. The kinetic study of the related phenomena is in progress. In the prospect to achieving a complete removal of specific compounds such as organic pollutants by an enzymatic process, this work represents an interesting example where the coupling between a chemical reaction process and membrane separation technology allows complete treatment of a liquid phase. 2. Experimental 2. I. Textile fkzctionalization Textile supports are made of non-woven cellulose fibres modified by ion exchange group
(carboxylic, tertiary amine) and were a generous gift of the Institut Franqais du Textile et de 1’Habillement (IFTH, France). The grafting ratio is 20%, with an average polymerization degree of 50 to 200, the specific mass and the cut off being 450 g.me2 and 100 pm, respectively. Carboxylic textiles were modified according to the following procedure: a sample of textile (4 cm’) is immersed in NaOH and urea solution (1 M) during 12 h to graft amino groups on carboxylic acid functions. Biotin is attached to this amino group using N-hydroxy-succinimide biotin as coupling reagent (0.4 mgml-’ during 12 h at room temperature). Anion exchange textiles were modified directly by immersion in a sulfo-N-hydroxy-succinimide biotin (0.4 mg.ml-‘) aqueous solution for 12 h. The textiles were then thoroughly rinsed with distilled water and stored at room temperature prior to be used. 2.2. Enzyme biotinylation A biotinylated urease was prepared by adding a fixed amount (2 mg ml-‘) of urease solution (EC 3.5.1.5, type III) from Jack Bean, in phosphate buffer 100 mM, pH 7.5, to 100 times mole excess of biotin-amidocaproate N-hydroxysuccinimide ester. The reaction was carried out at room temperature in a vial under constant stirring for 3 h. At this time, 10 mg of glycine was added to react with the unreacted biotin. Removal of all small molecular weight reactants and products was achieved by gel chromatography through a Sephadex G 25 column from SIGMA (USA) (10000 MW/cut-off). 2.3. Fixation of enzyme on the textile The biotin modified textile support was rinsed with distilled water and immersed in a 2 ml avidin aqueous solution (1 mg ml-‘) for 1 h. The resulting modified porous material was washed with distilled water and immersed in a 2 ml biotinylated enzyme solution (2 mg ml-‘) for 1 h and then washed again with distilled water.
VI Magne et al. /Desalination
144 (2002) 163-166
165
2.4. Enzyme textile device
NN,CONH,
A laboratory cell used for assays of catalytic substrate transformation in a few experiences was described previously [4]. It was composed of three compartments separated by two ion-exchange membranes. The area of these membranes was 20 cm*. The thickness of the central compartment was 2 mm while the others were 8 mm, respectively. Modified textile was squeezed between the anion and the cation exchange membrane in the central compartment. The solution flowed continuously between both membranes across the textile enzyme membrane as in a frontal filtration system by means of a peristaltic pump (Waston Marlow 302) (flow rate: 100 mlmin-’ in each compartment) (Fig. 1). A urea (1 O-*M) solution circulated in the central compartment and molar sodium hydroxide solution and molar sulfuric acid solution circulated in anodic and cathodic compartments, respectively.
The determination of the activities of the free and immobilized urease was carried out by measuring of the ammonium production according to the procedure given by Sigma (Sigma catalog No. 535) with BUN acid reagent and BUN color reagent (Sigma product). For the establishment of a calibration curve, 3 ml of BUN acid reagent were mixed with 2 ml of BUN color reagent and 1 ml of sample solution (urea concentration between 2 to 60 mM) and precisely maintained for 10 min at 80°C. After cooling, the respective absorbances at 524 nm of these concentration varying samples were recorded [5].
2.5. Enzymatic
activity measurements
Urease catalyzes the hydrolysis of urea producing ammonium and carbonate ions according to the reaction:
+ AEM
CEM
urea + water Fig. 1. Scheme of electrodialysis enzyme modified textile.
cell incorporating
an
+ 2 H,O + 2 NH,+ + CO;-
3. Results and discussion Fig. 2 shows the specific activity of immobilized urease as compared to free enzyme. It appears clearly that immobilization via avidin-biotin technology on the textile support allows to keep catalytic activity of the enzyme. The electrical field was applied to the electrodialysis cell with the textile inserted in the central compartment and clipped between the two ion exchange membranes as indicated in the experimental section. Figs. 3 and 4 depict the ammonium concentration evolution in the cathodic and central compartments respectively. Three current densities were used (1,5 and 10 mA.cm-‘). In the central compartment, the ammonium concentration produced by the enzymatic reaction decreases as the current density increases. The cations are transported through the cation exchange membrane to the cathodic compartment owing to the driving force of electric field. In the central compartment ammonium and carbonate ions were produced by the enzymatic reaction. When the current is increased in the electrodialysis cell the ammonium ion concentration decreases as the concentration in the cathodic compartment increases because of the efficiency of the transport process across the cation exchange membrane due to the driving force of the electrical field.
V Magne et al. /Desalination
166
144 (2002) 163-166
0
Fig. 2. Specific activity of free urease and immobilzed urease on the textile support. (1 enzymatic unit corresponds to the degradation of 1 pM of urea at 20°C in phosphate buffer pH = 8.5).
18
c
16
i
ol 14 E c 12 0
‘0 ” E
.$
8-
64 initial time 0
A
I- ~~
;
4
4 Current
6 density
a
10
(mA.cm-‘)
Fig. 4. Ammonium concentration in central compartment vs. applied current density (initial time (0) and after 40 (A) and 90 min of electrodialysis (W)).
after 90 min of eleclrodialysis
1
‘E loc 8
!
.
.
q
2
I
6
1--
a
-or
10
compartment are changed (8.5 at the initial time and 7 after 90 min of process). In conclusion, a enzyme modified textile are used to remove the urea solution in the electrodialysis cell. The electrical migration associated with enzymatic reaction have been used successfully to hydrolyse urea solution and to remove ions produced. A kinetic study and optimisation of the experimental process are in progress.
Current density (mA.&)
Fig. 3. Ammonium concentration in cathodic compartment vs. applied current density (initial time (0) and after 90 min of electrodialysis (m)).
References
the efficiency of the transport of the ions produced by enzymatic reaction is also demonstrated since no ammonium are detected in the anodic compartment. A weak linkage of ammonium through the anion exchange membrane is detected at high current density after 90 min of working (only 1% of total ammonium concentration). The values of pH are maintained constant during the process in anodic and cathodic compartment owing to the high volume used and the weak current density. On the opposite the pH in central
PI
Moreover,
[II
D.M.F. Prazeres and J.M.S. Cabral, Enzymatic membrane bioreactors and their applications, Enz. Microb. Technol.,
16 (1994) 738-750. T.C. Huang and D.H. Chen, A study of removal of urea from aqueous solution with immobilized urease and electrodialysis, J. Chem. Tech. Biotechnol., 55 (1992) 191-199. [31 T.C. Huang and D.H. Chen, Coupling of urea hydrolysis ammonium removal in an electrodialyzer with immobilized urease, Chem. Eng. Commun., 1205 (1993) 191-201. [41 E. Dejean, E. Laktionov, J. Sandeaux, R. Sandeaux, G Pourcelly and C. Gavach, Electrodeionization with ionexchange textile for the production of high resistivity water: influence of the nature of the textile, Desalination, 114 (1997) 165-173. [51 M. Yakup Arica, Epoxy-derived pHEMA membranes for use bioactive macromolecules immobilization: covalently bound urease in a continuous model system, J. Appl. Polym. Sci., 77 (2000) 200&2008.
144 (2002) 163-l 66 www.elsevier.com/locate/desal
Enzyme textile for removal of urea with coupling process: enzymatic reaction and electrodialysis V. Magne”, M. Amounas”, C. Innocent”, E. Dejeanb, P. Setaa* “lnstitut Europeen des Membranes, UMR CNRS n”5635, 34293 Montpellier Cedex 5, France Tel. +33 (4) 67 14 91 11; Fax +33 (4) 67 I4 91 19; email: [email protected] “Institut Fraqais du Textile et de I’Habillement, BP 60, 69132 Ecully Cedex, France Received 6 February
2002; accepted 20 February
2002
Abstract Ion exchanging textile are used as organic supports for enzyme immobilisation in the aim to develop reactive fibrous materials able to be substituted to membrane systems in bioreactors or acting for instance as reactive bags
for waste treatment devices, thus extending the range of their potential applications. Integration of this modified textile in the electrodialysis cell allowed simultaneous removal of urea and ions produced by the hydrolysis reaction. The reactor-separator Keywords:
systems were studied under different current densities.
Ion exchange textile; Reactor-separator; Urea hydrolysis
Enzyme immobilization;
1. Introduction
Membranes are well known to be used for separation processes in various chemical and biochemical domains. In recent years, interest has been increasingly targeted in combining a membrane separation process to biocatalysis [I]. In the resulting membrane bioreactor, the chemical product obtained by the enzymatic reaction is separated from the reaction medium by the use of the membrane process. Moreover, fouling and poisoning phenomena which occur during the *Corresponding author. Presented at the International July 7-12, 2002.
Congress on Membranes
Molecular recognition;
filtration step are major limitations for the use of such technology. To improve the efficiency of the reactionfiltration coupling, the route which consists in immobilizing an enzyme in a porous material could avoid the post treatment of the solution after the biochemical reaction. The immobilization of a catalyst such as an enzyme, has particular attractions, since the sophistication of this process allows us to suppress the further separation step and also avoid many problems which occurred in some cases with catalysts. Different types of enzyme immobiliand Membrane
Processes
001 I-9 164/02/$- See front matter 0 2002 Elsevier Science B.V. All rights reserved PII: SO0 I 1-9 164(02)00306-5
Electrodialysis;
(ICOM),
Toulouse, France,
164
V Magrze et al. /Desalination 144 (2002) 163-166
zation have been used in order to fix a biological catalyst on a support. We have recently shown that the use of the enzyme molecular recognition process between avidin and biotin presents a very good efficiency to attach irreversibly and with high stability enzymes on a porous material, and as a consequence, we applied this technology to modify the reactivity of textile supports. The reason of this choice is that the textile can also be considered as a filtration membrane material thanks to its own porosity. In this paper we present an application of this concept to an enzyme modified textile based on urease immobilization in a coupling process (reaction-separation). Urea was shown to be efficiently removed from aqueous solutions, combining catalytic reaction and ionic migration through ion exchange membrane in an electrodialysis cell [2,3], As urease allows the hydrolysis of urea with production of ammonium and carbonate ions, these produced cations and anions were separated by electrodialysis across cation and anion exchange membranes, respectively. As an indication of the efficiency of this coupling process, we were able to totally remove urea and its by-products from the treated aqueous solutions. Moreover, the migration through the membrane of ionic reaction products has an additional advantage since it avoided the kinetic inhibition of the enzymatic reaction due to back reaction. The kinetic study of the related phenomena is in progress. In the prospect to achieving a complete removal of specific compounds such as organic pollutants by an enzymatic process, this work represents an interesting example where the coupling between a chemical reaction process and membrane separation technology allows complete treatment of a liquid phase. 2. Experimental 2. I. Textile fkzctionalization Textile supports are made of non-woven cellulose fibres modified by ion exchange group
(carboxylic, tertiary amine) and were a generous gift of the Institut Franqais du Textile et de 1’Habillement (IFTH, France). The grafting ratio is 20%, with an average polymerization degree of 50 to 200, the specific mass and the cut off being 450 g.me2 and 100 pm, respectively. Carboxylic textiles were modified according to the following procedure: a sample of textile (4 cm’) is immersed in NaOH and urea solution (1 M) during 12 h to graft amino groups on carboxylic acid functions. Biotin is attached to this amino group using N-hydroxy-succinimide biotin as coupling reagent (0.4 mgml-’ during 12 h at room temperature). Anion exchange textiles were modified directly by immersion in a sulfo-N-hydroxy-succinimide biotin (0.4 mg.ml-‘) aqueous solution for 12 h. The textiles were then thoroughly rinsed with distilled water and stored at room temperature prior to be used. 2.2. Enzyme biotinylation A biotinylated urease was prepared by adding a fixed amount (2 mg ml-‘) of urease solution (EC 3.5.1.5, type III) from Jack Bean, in phosphate buffer 100 mM, pH 7.5, to 100 times mole excess of biotin-amidocaproate N-hydroxysuccinimide ester. The reaction was carried out at room temperature in a vial under constant stirring for 3 h. At this time, 10 mg of glycine was added to react with the unreacted biotin. Removal of all small molecular weight reactants and products was achieved by gel chromatography through a Sephadex G 25 column from SIGMA (USA) (10000 MW/cut-off). 2.3. Fixation of enzyme on the textile The biotin modified textile support was rinsed with distilled water and immersed in a 2 ml avidin aqueous solution (1 mg ml-‘) for 1 h. The resulting modified porous material was washed with distilled water and immersed in a 2 ml biotinylated enzyme solution (2 mg ml-‘) for 1 h and then washed again with distilled water.
VI Magne et al. /Desalination
144 (2002) 163-166
165
2.4. Enzyme textile device
NN,CONH,
A laboratory cell used for assays of catalytic substrate transformation in a few experiences was described previously [4]. It was composed of three compartments separated by two ion-exchange membranes. The area of these membranes was 20 cm*. The thickness of the central compartment was 2 mm while the others were 8 mm, respectively. Modified textile was squeezed between the anion and the cation exchange membrane in the central compartment. The solution flowed continuously between both membranes across the textile enzyme membrane as in a frontal filtration system by means of a peristaltic pump (Waston Marlow 302) (flow rate: 100 mlmin-’ in each compartment) (Fig. 1). A urea (1 O-*M) solution circulated in the central compartment and molar sodium hydroxide solution and molar sulfuric acid solution circulated in anodic and cathodic compartments, respectively.
The determination of the activities of the free and immobilized urease was carried out by measuring of the ammonium production according to the procedure given by Sigma (Sigma catalog No. 535) with BUN acid reagent and BUN color reagent (Sigma product). For the establishment of a calibration curve, 3 ml of BUN acid reagent were mixed with 2 ml of BUN color reagent and 1 ml of sample solution (urea concentration between 2 to 60 mM) and precisely maintained for 10 min at 80°C. After cooling, the respective absorbances at 524 nm of these concentration varying samples were recorded [5].
2.5. Enzymatic
activity measurements
Urease catalyzes the hydrolysis of urea producing ammonium and carbonate ions according to the reaction:
+ AEM
CEM
urea + water Fig. 1. Scheme of electrodialysis enzyme modified textile.
cell incorporating
an
+ 2 H,O + 2 NH,+ + CO;-
3. Results and discussion Fig. 2 shows the specific activity of immobilized urease as compared to free enzyme. It appears clearly that immobilization via avidin-biotin technology on the textile support allows to keep catalytic activity of the enzyme. The electrical field was applied to the electrodialysis cell with the textile inserted in the central compartment and clipped between the two ion exchange membranes as indicated in the experimental section. Figs. 3 and 4 depict the ammonium concentration evolution in the cathodic and central compartments respectively. Three current densities were used (1,5 and 10 mA.cm-‘). In the central compartment, the ammonium concentration produced by the enzymatic reaction decreases as the current density increases. The cations are transported through the cation exchange membrane to the cathodic compartment owing to the driving force of electric field. In the central compartment ammonium and carbonate ions were produced by the enzymatic reaction. When the current is increased in the electrodialysis cell the ammonium ion concentration decreases as the concentration in the cathodic compartment increases because of the efficiency of the transport process across the cation exchange membrane due to the driving force of the electrical field.
V Magne et al. /Desalination
166
144 (2002) 163-166
0
Fig. 2. Specific activity of free urease and immobilzed urease on the textile support. (1 enzymatic unit corresponds to the degradation of 1 pM of urea at 20°C in phosphate buffer pH = 8.5).
18
c
16
i
ol 14 E c 12 0
‘0 ” E
.$
8-
64 initial time 0
A
I- ~~
;
4
4 Current
6 density
a
10
(mA.cm-‘)
Fig. 4. Ammonium concentration in central compartment vs. applied current density (initial time (0) and after 40 (A) and 90 min of electrodialysis (W)).
after 90 min of eleclrodialysis
1
‘E loc 8
!
.
.
q
2
I
6
1--
a
-or
10
compartment are changed (8.5 at the initial time and 7 after 90 min of process). In conclusion, a enzyme modified textile are used to remove the urea solution in the electrodialysis cell. The electrical migration associated with enzymatic reaction have been used successfully to hydrolyse urea solution and to remove ions produced. A kinetic study and optimisation of the experimental process are in progress.
Current density (mA.&)
Fig. 3. Ammonium concentration in cathodic compartment vs. applied current density (initial time (0) and after 90 min of electrodialysis (m)).
References
the efficiency of the transport of the ions produced by enzymatic reaction is also demonstrated since no ammonium are detected in the anodic compartment. A weak linkage of ammonium through the anion exchange membrane is detected at high current density after 90 min of working (only 1% of total ammonium concentration). The values of pH are maintained constant during the process in anodic and cathodic compartment owing to the high volume used and the weak current density. On the opposite the pH in central
PI
Moreover,
[II
D.M.F. Prazeres and J.M.S. Cabral, Enzymatic membrane bioreactors and their applications, Enz. Microb. Technol.,
16 (1994) 738-750. T.C. Huang and D.H. Chen, A study of removal of urea from aqueous solution with immobilized urease and electrodialysis, J. Chem. Tech. Biotechnol., 55 (1992) 191-199. [31 T.C. Huang and D.H. Chen, Coupling of urea hydrolysis ammonium removal in an electrodialyzer with immobilized urease, Chem. Eng. Commun., 1205 (1993) 191-201. [41 E. Dejean, E. Laktionov, J. Sandeaux, R. Sandeaux, G Pourcelly and C. Gavach, Electrodeionization with ionexchange textile for the production of high resistivity water: influence of the nature of the textile, Desalination, 114 (1997) 165-173. [51 M. Yakup Arica, Epoxy-derived pHEMA membranes for use bioactive macromolecules immobilization: covalently bound urease in a continuous model system, J. Appl. Polym. Sci., 77 (2000) 200&2008.