J . Ckem. Tech. Biorechnol. 1990,48, 337-350
Urease Immobilized on Chitosan Membrane: Preparation and Properties Barbara Krajewska, Maciej Leszko & Wieslawa Zaborska Faculty of Chemistry, Jagiellonian University, 30-060 Krakow, Karasia 3, Poland (Received 18 May 1989; revised version received 4 September 1989; accepted 20 September 1989)
ABSTRACT Urease was covalently immobilized on glutaraldehyde-pretreated chitosan membranes. The optimum immobilization conditions were determined with respect to glutaraldehyde pretreatment of membranes and to reaction of glutaraldehyde-pretreated membranes with urease. The immobilized enzyme retained 94 % of its original activity. The properties of free and immobilized urease were compared. The Michaelis constant was about five times higher for immobilized urease than for the jiee enzyme, while the maximum reaction rate was lower for the immobilized enzyme. The stability of urease at low p H values was improved by immobilization: temperature stability was also improved. The optimum temperature was determined to be 65°C for the free urease and 75°C for the immobilized form. The immobilized enzyme had good storage and operational stability and good reusability, properties that offer potentiul for practical application. Key words: chitosan membranes, immobilization of urease. 1 INTRODUCTION
Membrane-immobilized enzymes may serve as model systems for natural enzymes bound to in vivo membranes or find practical application in enzymatic reactors as less costly, more stable and reusable alternatives to soluble enzymes. There are numerous applications or potential applications of membrane-immobilized enzymes in biotechnology and biomedicine.' One such system, a membraneimmobilized urease can be used for removal of urea from blood in the artificial kidney, for blood detoxification' or in the dialysate regeneration system of artificial 337 J. Chem. Tech. Biotechnol. 0268-2575/90/$03.5001990 Society of Chemical Industry. Printed in Great
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kidney^.^ The introduction of urease to the artificial kidney is done in an attempt to reduce the size of the artificial kidney m a ~ h i n e . ~ Among the polymeric materials used as membranous enzyme carriers chitosan is is a derivative of chitin, of interest. Chitosan, (1-+4)-2-amino-2-deoxy-fl-~-g~ucan, (1+4)-2-acetamido-2deoxy-fl-~-glucan,chemically prepared by N-deacetylation of the latter. Chitin is a natural polymer which serves as a structural component of crustacean shells and fungal cell walls. It is available at a very low cost and in large quantities from wastes of seafood processing. Both chitin and chitosan are polysaccharides which can be formally considered as being derivatives of cellulose, where the C-2 hydroxyl groups are replaced by acetamido groups in the case of chitin and by amino groups in the case of chitosan. Natural chitin, however, is always deacetylated to a certain degree. Ndeacetylation of chitin leads to a mixture of chitins of various degrees of deacetylation. Such a mixture can be called chitosan when it becomes soluble in dilute aqueous solutions of organic acids. The solubility of chitosan in organic acids allows for gel and membrane fabrication.’ What makes chitin and chitosan attractive enzyme carriers is their low cost, their robustness, inertness and hydrophilicity, and the presence of hydroxyl and amino groups which facilitate immobilization of enzymes by both adsorption and covalent binding. Covalent binding to chitin and chitosan is mainly achieved by using dialdehydes such as glyoxal, malonaldehyde or glutaraldehyde which react with the free amino groups of chitin or chitosan and of protein. Attempts have been made to immobilize urease on chitin and chitosan. Iyengar and Rao6 immobilized urease by covalent binding of the enzyme to glutaraldehydetreated chitin powder, whereas Kasumi et al.’ achieved covalent binding of the enzyme to chitosan powder by catalytic reaction of water soluble carbodiimides. Hirano and Miura’ immobilized urease in chitosan gels but obtained inactive preparations. The present studies investigate the use of chitosan membranes as an immobilization matrix. The preparation and the properties of urease immobilized by covalent binding to glutaraldehyde-pretreated chitosan membrane are described. 2 EXPERIMENTAL 2.1 Materials
Chitosan was obtained from the Sea Fisheries Institute in Gdynia, Poland, where it is produced by deacetylation of chitin of Antarctic krill shells. A fraction of grade 0.43-0.75 mm was used. The urease was Sigma type 111. Its specific activity was 32 units mg-’ protein. One unit is the amount of enzyme that liberates 1.0pmol of NH, from urea per minute at pH 7 and 25°C. Glutaraldehyde (25% w/v solution in water) was from BDH Chemicals Ltd, Poole, England. Urea (analar grade) and all other chemicals (analar grade) were obtained from POCh, Gliwice, Poland.
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Urease and urea solutions were prepared as needed in phosphate buffers, ranging from pH 5.3 to 8.2, 22 mmol dm-, containing 1 mmol dm-, EDTA and NaCl added in such amounts that ionic strength of each of the buffers was I 0.07. Redistilled water was used throughout. 2.2 Determination of urease activity The activity of both free and immobilized urease was determined by measuring the amount of ammonia liberated from the ureasecatalyzed hydrolysis of urea per unit time:
and was expressed in pmol NH, min-' mg-' protein for the free enzyme, and in pmol NH, min-' cm-' membrane or pmol NH, min-' mg-' bound protein for the immobilized enzyme. Unless otherwise stated, the reaction was carried out in the phosphate buffer pH 7.0 (22 mmol drn-,, 1 mmol dm-3 EDTA, I 0.07)at 25°C. To make sure that the reaction was not urea limited, the concentration of urea used was 10 g drn-,, i.e. 10times higher than the estimated Michaelisconstants. Samples were removed from the reaction mixture at intervals for estimation of N H 3 by the phenol-hypochlorite m e t h ~ d Linear .~ fragments of rate plots were used for activity calculations.
2.3 Determination of immobilized protein The amount of immobilized enzyme protein was estimated by subtracting the amount of protein determined in supernatant after immobilization from the amount of protein used for immobilization. The protein content in the solutions was determined by the method of Lowry et aI."
2.4 Preparation of chitosan membranes5~' The solution of chitosan (1 % w/v) in acetic acid (0.8 % w/v) at 60°C was prepared. Membranes supported with a glass fabric were cast on polyethylene plates (034 cm3 of chitosan solution per 1 cm2 surface area). The membranes were then dried at 65°C for 30 hours, neutralized with NaOH, washed with water and dried again. The thickness of the membranes obtained was 0.08-0.09mm (glass fabric included).
2.5 Immobilization of urease on chitosan membranes Urease was immobilized according to the following general procedure: the membranes were conditioned in water, then treated with glutaraldehyde at room temperature and washed until the washings were free of glutaraldehyde. The membranes were then reacted with urease solution for 1 h a t room temperature with occasional stirring, left overnight at 4°C and finally washed until the washings were free of urease. To optimise preparation of ureasexhitosan membranes, the effects of the following basic factors of the applied immobilization procedure on the activity yields of the ureasexhitosan system were studied: waterconditioning,
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glutaraldehyde concentration, time of membrane-glutaraldehyde reaction, pH of glutaraldehyde solution, pH of urease solution and urease concentration. The study was performed in such a way that a chosen factor of the procedure under examination was changed while the remaining factors of the procedure were kept fixed. Unless otherwise stated, urease activity was determined as described in Section 2.2.
2.6 Determination of physicochemical properties of free and immobilized urease 2.6.1 Kinetic parameters KM,v,, Constant amounts of the free and immobilized urease were subjected to reaction of urea hydrolysis in solutions of increasing urea concentration. For each experiment the initial rate of the reaction was measured. The reactions of the free urease were stopped by inactivation of the enzyme with the chemicals of the phenolhypochlorite method. 2.6.2 Effect of pH on activity The effect of pH on activity of both the enzymes was studied in the phosphate buffers of pH ranging from 5.3 to 8.2. 2.6.3 Effect of temperature on activity The effect of temperature on activity of both the enzymes was studied between 10 and 95°C at the optimum pH of both the enzymes, i.e. 7.0. The samples of the ureases and of urea solutions were heated to a required temperature, then mixed and the reaction rate was measured at the same temperature. 2.6.4 Heat inactivation at 70°C The samples of both enzymes in the phosphate buffer pH 7.0 were incubated at 70°C for the desired times (between 0 and 250 min) and then their activities were assayed at 25°C. 2.6.5 Stability at 4°C To compare the storage stability of the examined enzymes at 4°C three types of samples were prepared: a solution of the free urease in the phosphate buffer pH 7.0 (0.1 mg ~ m - ~equal ) ; sized samples of the chitosan membrane with immobilized urease stored wet in the phosphate buffer pH 7.0; and equal sized samples of the chitosan membrane with immobilized urease stored dry (the membranes were dried for 3 h at room temperature after urease had been immobilized on them). All the samples were stored at 4°C. The activities of the consecutive fresh samples taken from the store were assayed periodically at 25°C. 2.6.6 Stability at 2YC The same types of urease samples as used for the storage stability assay at 4°C were prepared. All the samples were stored at 25°C. Activities of the samples newly taken from the store were assayed periodically.
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2.6.7 Reusability Two types of reusability assay were performed: one sample of the chitosan membrane-immobilizedurease was stored in the phosphate buffer pH 7.0, the other one dry, both at 4°C.The activities of the samples were measured every other day at 25°C. After each activity determination the samples were washed with the buffer pH 7.0and left stored till the next assay, the first sample in the buffer, the other one dried after the assay. 2.6.8 Operational stability The reaction of urea hydrolysis catalyzed by the chitosan membrane-immobilized urease was carried out continuously for 120 h in static conditions. The sample was kept in the urea solution (10 g dm- 3), which was exchanged every 1.5 h, and the activity of the membrane was assayed periodically.
3 RESULTS A N D DISCUSSION 3.1 immobilization of urease onto chitosan membranes
The proposed method of immobilizationof urease on chitosan using glutaraldehyde involves the following reactions: Ch-NH,
-
+ q\C-(CH,)3-Cp H ’
Chitosan Ch-N=CH-(CH,),--C
8)
Ch-N=CH-(CH,),-C
\H
H ‘
Glutaraldehyde
8)+
HZN-E
aCh-N=CH-(CH,),xH=N-E
(3)
H‘
Glutaraldehyde-pretreated chitosan Enzyme
Reaction (2) should introduce aldehyde groups onto the chitosan membranes to facilitate the reaction with the enzyme. It was found, however, that maximum activity was obtained with membranes on which urease was immobilized by simple adsorption. This suggests that reaction (3) alters the enzyme structure so as to diminish its activity. Membranes with adsorbed urease, however, lacked stability and lost enzymatic activity because of easy desorption of the enzyme. On the other hand, glutaraldehyde concentrations greater than 0.03 % (w/v) and reaction times greater than 1.5 h render the membranes brittle as a result of cross-linking of chitosan. As a compromise, a glutaraldehyde concentration of 0-01% (w/v) and a reaction time of 1.5 h were considered optimal. Reaction (2) was not pH dependent and so aqueous glutaraldehyde solution was chosen for further studies. The study of reaction (3) showed that maximum immobilized enzyme activity was achieved with the lower pH urease solution. This may result from the fact that an acidic pH favours reaction between amino and aldehyde groups, and also from the fact that in solutions of pH 6 chitosan exists in
-=
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TABLE 1 Activities of Selected Urease-Carrier Systems Activity of urease-carrier system
Carrier
(pmol NH, min- cm carrier)
(jimol NH, min-' g - ' carrier)
1.56
1100 1200 215
'
Chitosan membrane Chitin powder Chitosan powder Gelatin membrane Collagen-poly (glycidyl methacrylate) graft copolymer
3.0-6.0
AQ-nylon membrane
ImmunodyneTMnylon membrane
Activity of free urease used (jimol NH, min-' mg-' protein)
32 80 0.487
32 165
1.2
116
0.36
0.088
Ref.
32
6 7 12 13 14 15
a gel form which probably helps more urease molecules to bind the membrane via adsorption or gel inclusion. Consequently a urease solution at pH 5.3 was chosen for the procedure. Finally it was found that in order to achieve maximum bound urease activity a urease concentration of at least 0-5 mg cm-3 for reaction (3) at pH 5.3 should be used. Prior to chemical treatment chitosan membranes were waterconditioned, the determined optimum time of waterconditioning ranges from 8 to 14 days.
3.2 Activity of immobilized urease The membrane-immobilized urease had the following activity: 1.56 pmol NH, min- cm-' membrane, i.e. 1100 pmol NH, min- ' g-' chitosan membrane, which corresponds to 00490mg protein immobilized on 1 cm2 of the membrane. For comparison, activities of selected ureasecarrier systems are listed in Table 1. The protein content ofthe membrane was 0.0507 mg cm-'; which means that the immobilized urease retained 94% of the activity of the free urease. This represents an improvement over values reported by other investigators (51 % , I 6 2 ( r 3 O X 6 and for the retained activity of immobilised urease.
'
3.3 Physicochemical properties of immobilized urease 3.3.1 Kinetic parameters KM, vmax The kinetic results obtained for the free and immobilized urease are presented in Fig. 1 as Lineweaver-Burk plots, l/u versus l/[S]. The linear nature of the Lineweaver-Burk plots proves that in the ranges of urea concentration examined both the enzymes follow the Michaelis-Menten kinetics represented by the
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vma. * 0.337 mmolsa UREA/s q PROTEIN
3 10
K, = 5.01 mmol/dm5 vmqX * 0.236 mmoles UREA/s g PROTEIN
I
I
I
I
200
400
600
c
f/p] (moles UREA/dm,)-'
Fig. 1. Lineweaver-Burk plots, l/u versus l/[S], for free and chitosan membrane-immobilized urease. Reactions were carried out in phosphate buffer pH 7.0 (22 mmol dm-', 1 mmol dm-', EDTA, I (307)at 25°C.
following equation:
where v and v,,, are actual and maximum reactions rates, respectively, KM is the Michaelis constant and [S] is substrate concentration. For low concentrations of substrate the reaction is of the first order, for high concentrations it changes into zero order when the reaction rate is independent of substrate concentration. This range of concentration was used for enzyme activity determinations. The calculated values of the kinetic parameters: the Michaelis constants and the maximum reaction rates are listed in Fig. 1. The chitosan-immobilized urease exhibited K , values about 5 times higher than that of the free urease. This increase may be a consequence of either structural changes in the enzyme introduced by the applied immobilization procedure, or lower accessibility of the substrate to the active site of the immobilized enzyme. The latter may result either from diffusional resistances of stagnant solvent layers produced around the immobilized molecules, or from the confinement of enzyme molecules within the skin of the membrane during the process of immobilization (gelation of chitosan at pH 6.5). Consequently the maximum rate of reaction catalyzed by the immobilized urease was lower than that of the free urease. Increased K , of urease after immobilization has similarly been reported by various authors.' 7-20
-=
391
3.3.2 Effect of p H on activity Figure 2 shows that the immobilized urease was less sensitive to pH changes at
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I 0 IMMOBILIZED
1 5.0
1
I
I
6.0
zo
8.0
c
PH
Fig. 2. Effect of pH on activity of free and chitosan membrane-immobilized urease. Reactions were performed at 25°C in phosphate buffers (22 mmol dm-3, 1 mmol dm-3 EDTA, I 0.07) of various pH values.
pH c 7 and slightly more sensitive at pH > 7 than the free urease. Both the enzymes had optimum pH values of 7, but the optimum of the immobilized urease was broader at lower pH values. This effect is in agreement with a general observation that positively charged supports displace pH-activity curves of enzymes attached to them towards Lower pH values.21 Different pH-activity profiles of immobilized urease have been obtained depending on a support chosen and on an immobilization method a ~ p l i e d . ~3 .* 1' 6.1 7 * 9 , 2 2 * 2 3
'
3.3.3 Effect of temperature on activity Figure 3(a) shows the temperature optima curves for the free and immobilized urease preparations. The free urease had an optimum temperature of about 65T, whereas the temperature optimum of immobilized urease was shifted to 75°C. The results for the temperature range from 10°C to the optimum temperatures are also presented in the form of Arrhenius plots (Fig. 3(b)). The plots for both the enzymes were linear and the calculated values of activation energy were equal to 5.71 and 7.37 kcal mol-' for the free and chitosan-immobilized urease, respectively. The higher value of activation energy obtained for the immobilized urease indicates that the applied immobilization procedure introduced changes in the structure of urease molecules which impeded the enzymecatalyzed reaction. Similar results were reported previously.' 7.1 In contrast, cases where urease activation energy decrcased after immobilization have also been reported.' 9 , 2 2 3.3.4 Heat inactivation at 70°C As shown in Fig. 4 the immobilization of urease on chitosan protected the enzyme
Urease immobilized on chitosan membrane
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4
0 TREE 0 IMMOBILIZED
ioo
5
4.0-
20
1
1.0
I
I
I
1
1
10
20
30
40
50
I
1
I
60
4
I
70 00 90 TEMPERATURE
PC]
100
-
a5 -
60
50
I
I
1
i0
I
&I
40
30
10
10
I
I
I
1
I
I
s2
53
3.9
15
0 I I
c
3.6 T-’ 10’ [K-‘]
(b) Fig. 3. Effect of temperature on activity of free and chitosan membrane-immobilized urease: (a) temperature optima; (b) Arrhenius plots, log (activity) versus 1/T. Reactions were performed in phosphate buffer pH 7.0 (22 mmol dm-), 1 mmol d m - 3 EDTA, I 0-07).
against heat inactivation. The half-times for the decay of activity were about 120 and 250 min for the free and immobilized enzyme, respectively. 3.3.5 Stability at 4°C As shown in Fig. 5 the free urease retained activity for 8 days, but thereafter activity quickly decreased, the half-time of activity decay was 13 days. The chitosanimmobilized urease, whether stored wet or dry, remained very active over a long
B. Krajewska, M . Leszko, W . Zaborska
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t
IMMOBILIZED
\
I
1
50
100
'
I
A
150 200 250 PREINCUBATION TIME [min]
Fig. 4. Heat inactivation at 70°C of free and chitosan membrane-immobilized urease. Enzyme activity was determined at 25°C in phosphate buffer pH 7.0 (22 mmol 1 mmol dm-3 EDTA, I 007).
4°C
0 TREE 0 I M M O b I L I Z E D (MEMBRANE5 5TORED DRY) 0 IMMOBILIZED (MEMBRANE5 STORED WET)
I
I
I
1
I
1
8
16
LO
32
40
48
1
4
56 64 STORAGE T I M E [DAY51
Fig. 5. Storage stability (4°C) of free and chitosan membrane-immobilized urease (wet and dry). Enzyme activity was determined at 25°C in phosphate buffer pH 7.0 (22 mmol dm-', 1 mmol dm-3 EDTA, I 0.07).
Urease immobilized on chitosan membrane
347
U
0
0
0 TREE 0 I M M O I I L I Z E D (MEMBRANES STORED DRY) 0 IMMOBILIZED (MEMBRANES JTORED WET)
I
I
I
I
I
I
I
I*
8
i6
24
3.2
40
48
56
64
STORAGE T I M E [DAYS]
Fig. 6. Storage stability (25°C)of free and chitosan membrane-immobilizedurease (wet and dry). Enzyme activity was determined at 25°C in phosphate buffer pH 7.0 (22 mmol dm-3, 1 mmol dm-3 EDTA, I 0.07).
period of time: the membranes stored wet lost about 10%of their original activity over the period of 60 days, whereas membranes stored dry lost about 20% of their original activity over the same period of time. 3.3.6 Stability at 25°C As shown in Fig. 6 the free urease lost activity within 12 days, the half-time of activity decay was 3 days. The membranes stored wet lost 30% of their original activity within 10days and then remained active; the membranes stored dry slowly lost 20% of their original activity over the period of 60 days similarly to those stored at 4°C. Both these sets of data show that the chitosan-immobilized urease was considerably more stable than the free enzyme, and could be stored for extended periods, in both wet and dry forms, before use. Various other reports confirm that the storage stability of immobilized urease varies depending on the immobilization method applid.6.13.17.19.22-24 3.3.7 Reusability Figure 7 shows that the immobilized urease lost activity after 9 cycles of reuse. Drying of the membrane after each use considerably speeded up the process of activity decay. 3.3.8 Operational stability Figure 8 shows that the membrane lost about 30% of its activity within 5 h of the continuous process and then slowly decreased its activity to about 40% within 120 h.
B. Krajewska, M . Leszko, W.Zaborska
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OMEMBRANES STOREbWET 0 MEMBRANE5 STORED DRY
1
4
3
2
I
I
5
6
I
I
1 -
8 9 NUMBER O f REU5E5
1
Fig. 7. Reusability of chitosan membrane-immobilized urease. Enzyme activity was determined at 25°C in phosphate buffer pH 7.0 (22 mmol dm-3, 1 mmol dm-3 EDTA, I @07).
40
t
t
1
I
I
1
I
10
20
30
40
I
I
If
1
I
*
50 60" 110 110 TIME OF OPERATION [HOURS]
Fig. 8. Operational stability ofchitosan membrane-immobilized urease. Enzyme activity was determined at 25°C in phosphate buffer pH 7.0 (22 mmol dm-3, 1 mmol dm-3 EDRA, 1 007).
Urease immobilized on chitosan membrane
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4 CONCLUSIONS This study shows that urease can be successfully immobilized on glutaraldehydepretreated chitosan membranes. The ureasexhitosan membrane system offers various advantages: (1) chitosan is a relatively inexpensive material, it is inert and easy to cast in the form of membrane; (2) the proposed immobilization procedure is simple to carry out; and (3)the properties of the immobilized enzyme offer potential for use in production processes. The obtained material has a comparatively high enzymatic activity. The activity retention of urease after immobilization of 94 % is higher than others previously reported. The physicochemical properties of urease were changed after immobilization, but whilst the kinetic parameters are slightly less favourable than those of free urease the excellent storage stability, thermal stability, reusability and operational stability of the immobilized enzyme demonstrates the superior potential of the immobilized enzyme for practical application. ACKNOWLEDGEMENT This work was supported by the State Programme of Fundamental Research on Membranes and Membrane Processes (CPBP 02.11).
REFERENCES 1. Chang, T. M.S. In Immobilized Enzymes, Antigens, Antibodies, and Peptides, ed. H. H. Weetall. Marcel Dekker, Inc., New York, 1975, pp. 245-92. 2. Chang, T. M. S., Artificial cells: the use of hybrid systems. Artificial Organs, 4 (1980) 264-71. 3. Drukker, W., Parsons, F. M. & Gordon, A. In Replacement of Renal Function by Dialysis, ed. W. Drukker, F. M. Parsons & J. F. Maher. Martinus Nijhoff, The Hague, 1979, pp. 24458. 4. Krajewska, B. & Zaborska, W., Dialysate regeneration system of artificial kidney. Biocyb. Biomed. Eng., 9 (1989) 113-29. 5. Muzzarelli, R. A. A., Chitin. Pergamon, New York, 1977. 6. Iyengar, L. & Rao, A. V. S. P., Urease bound to chitin with glutaraldehyde. Biotechnol. Bioeng., 21 (1979) 1333-43. 7. Kasumi, T., Tsuji, M., Hayashi, K. & Tsumura, N., Preparation and some properties of chitosan bound enzymes. Agr. Biol. Chem., 41 (1977) 1865-72. 8. Hirano, S. & Miura, O., Alkaline phosphatase and pepsin immobilized in gels. Biotechnol. Bioeng., 21 (1979) 71 1-14. 9. Weatherburn, M. W., Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem., 39 (1967) 9 7 1 4 . 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J., Protein measurement with the Fohn phenol reagent. J . Biol. Chem., 193 (1951) 265-75. 1 1. Brzeski, M., Technologia Otrzymywania Chityny i Chitozanu z Pancerzy Kryla Antarktycznego oraz Zastosowanie Tych Polimerow d o Formowania Blon. PhD thesis, Szczecin Polytechnic, Szczecin, 1984.
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12. Tanny, G. B., Kedem, 0.& Bohak, Z., Coupling of transport and enzymatic reaction in a membrane composed of an anion exchanger and immobilized urease. 3. Membrane Sci., 4 (1979) 363-77. 13. Raghunath, K., Rao, K. P. &Joseph, K. T., Preparation and characterization of urease immobilized on to collagen-poly(glycidy1 methacrylate) graft copolymer. Biotechnol. Bioeng., 26 (1984) 1049. 14. Miyama, H., Kobayashi, T. & Nosaka, Y., Immobilization of enzyme on nylon containing pendant quaternized amine groups. Biotechnol. Bioeng., 26 (1984) 139C2. 15. Krajewska, B., Leszko, M. & Zaborska, W., Membrane-immobilized urease for possible use in dialysate regeneration system. Env. Prot. Eng., in press. 16. Karube, I. & Suzuki, S., Electrochemical preparation of urease-collagen membrane. Biochem. Biophys. Res. Commun., 47 (1972) 5 1 4 . 17. Madeira, V. M. C., Incorporation of urease into liposomes. Biochim. Biophys. Acta, 499 (1977) 202-11. 18. Onyezili, F. N. & Onitri, A. C., The effect of substrate flow-rate on immobilized urease assays. Biochim. Biophys. Acta, 659 (1981) 244-8. 19. Ramachandran, K. B. & Perlmutter, D. D., Effects of immobilization on the kinetics of enzymecatalyzed reactions. 11. Urease in a packedcolumn differential reactor system. Biotechnol. Bioeng., 18 (1976) 685-99. 20. Miyama, H., Kawata, M. & Nosaka, Y., Immobilization of enzyme on dimethylaminated nylon gels. Biotechnol. Bioeng., 27 (1985) 1403-10. 21. Zaborsky, O., Immobilized Enzymes. CRC Press, Cleveland, 1973. 22. Sundaram, P. V. & Hornby, W. E., Preparation and properties of urease chemically attached to nylon tube. FEES Letters, 10 (1970) 325-7. 23. Sundaram, P. V . &Crook, E. M., Preparation and properties ofsolid-supported urease. Can. J. Biochem., 49 (1971) 1388-94. 24. Riesel, E. & Katchalski, E., Preparation and properties of water-insoluble derivatives of urease. J . Biol. Chem., 239 (1964) 1521-4.
Urease Immobilized on Chitosan Membrane: Preparation and Properties Barbara Krajewska, Maciej Leszko & Wieslawa Zaborska Faculty of Chemistry, Jagiellonian University, 30-060 Krakow, Karasia 3, Poland (Received 18 May 1989; revised version received 4 September 1989; accepted 20 September 1989)
ABSTRACT Urease was covalently immobilized on glutaraldehyde-pretreated chitosan membranes. The optimum immobilization conditions were determined with respect to glutaraldehyde pretreatment of membranes and to reaction of glutaraldehyde-pretreated membranes with urease. The immobilized enzyme retained 94 % of its original activity. The properties of free and immobilized urease were compared. The Michaelis constant was about five times higher for immobilized urease than for the jiee enzyme, while the maximum reaction rate was lower for the immobilized enzyme. The stability of urease at low p H values was improved by immobilization: temperature stability was also improved. The optimum temperature was determined to be 65°C for the free urease and 75°C for the immobilized form. The immobilized enzyme had good storage and operational stability and good reusability, properties that offer potentiul for practical application. Key words: chitosan membranes, immobilization of urease. 1 INTRODUCTION
Membrane-immobilized enzymes may serve as model systems for natural enzymes bound to in vivo membranes or find practical application in enzymatic reactors as less costly, more stable and reusable alternatives to soluble enzymes. There are numerous applications or potential applications of membrane-immobilized enzymes in biotechnology and biomedicine.' One such system, a membraneimmobilized urease can be used for removal of urea from blood in the artificial kidney, for blood detoxification' or in the dialysate regeneration system of artificial 337 J. Chem. Tech. Biotechnol. 0268-2575/90/$03.5001990 Society of Chemical Industry. Printed in Great
Britain
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B. Krajewska, M . Leszko, W . Zaborska
kidney^.^ The introduction of urease to the artificial kidney is done in an attempt to reduce the size of the artificial kidney m a ~ h i n e . ~ Among the polymeric materials used as membranous enzyme carriers chitosan is is a derivative of chitin, of interest. Chitosan, (1-+4)-2-amino-2-deoxy-fl-~-g~ucan, (1+4)-2-acetamido-2deoxy-fl-~-glucan,chemically prepared by N-deacetylation of the latter. Chitin is a natural polymer which serves as a structural component of crustacean shells and fungal cell walls. It is available at a very low cost and in large quantities from wastes of seafood processing. Both chitin and chitosan are polysaccharides which can be formally considered as being derivatives of cellulose, where the C-2 hydroxyl groups are replaced by acetamido groups in the case of chitin and by amino groups in the case of chitosan. Natural chitin, however, is always deacetylated to a certain degree. Ndeacetylation of chitin leads to a mixture of chitins of various degrees of deacetylation. Such a mixture can be called chitosan when it becomes soluble in dilute aqueous solutions of organic acids. The solubility of chitosan in organic acids allows for gel and membrane fabrication.’ What makes chitin and chitosan attractive enzyme carriers is their low cost, their robustness, inertness and hydrophilicity, and the presence of hydroxyl and amino groups which facilitate immobilization of enzymes by both adsorption and covalent binding. Covalent binding to chitin and chitosan is mainly achieved by using dialdehydes such as glyoxal, malonaldehyde or glutaraldehyde which react with the free amino groups of chitin or chitosan and of protein. Attempts have been made to immobilize urease on chitin and chitosan. Iyengar and Rao6 immobilized urease by covalent binding of the enzyme to glutaraldehydetreated chitin powder, whereas Kasumi et al.’ achieved covalent binding of the enzyme to chitosan powder by catalytic reaction of water soluble carbodiimides. Hirano and Miura’ immobilized urease in chitosan gels but obtained inactive preparations. The present studies investigate the use of chitosan membranes as an immobilization matrix. The preparation and the properties of urease immobilized by covalent binding to glutaraldehyde-pretreated chitosan membrane are described. 2 EXPERIMENTAL 2.1 Materials
Chitosan was obtained from the Sea Fisheries Institute in Gdynia, Poland, where it is produced by deacetylation of chitin of Antarctic krill shells. A fraction of grade 0.43-0.75 mm was used. The urease was Sigma type 111. Its specific activity was 32 units mg-’ protein. One unit is the amount of enzyme that liberates 1.0pmol of NH, from urea per minute at pH 7 and 25°C. Glutaraldehyde (25% w/v solution in water) was from BDH Chemicals Ltd, Poole, England. Urea (analar grade) and all other chemicals (analar grade) were obtained from POCh, Gliwice, Poland.
Urease immobilized on chitosan membrane
339
Urease and urea solutions were prepared as needed in phosphate buffers, ranging from pH 5.3 to 8.2, 22 mmol dm-, containing 1 mmol dm-, EDTA and NaCl added in such amounts that ionic strength of each of the buffers was I 0.07. Redistilled water was used throughout. 2.2 Determination of urease activity The activity of both free and immobilized urease was determined by measuring the amount of ammonia liberated from the ureasecatalyzed hydrolysis of urea per unit time:
and was expressed in pmol NH, min-' mg-' protein for the free enzyme, and in pmol NH, min-' cm-' membrane or pmol NH, min-' mg-' bound protein for the immobilized enzyme. Unless otherwise stated, the reaction was carried out in the phosphate buffer pH 7.0 (22 mmol drn-,, 1 mmol dm-3 EDTA, I 0.07)at 25°C. To make sure that the reaction was not urea limited, the concentration of urea used was 10 g drn-,, i.e. 10times higher than the estimated Michaelisconstants. Samples were removed from the reaction mixture at intervals for estimation of N H 3 by the phenol-hypochlorite m e t h ~ d Linear .~ fragments of rate plots were used for activity calculations.
2.3 Determination of immobilized protein The amount of immobilized enzyme protein was estimated by subtracting the amount of protein determined in supernatant after immobilization from the amount of protein used for immobilization. The protein content in the solutions was determined by the method of Lowry et aI."
2.4 Preparation of chitosan membranes5~' The solution of chitosan (1 % w/v) in acetic acid (0.8 % w/v) at 60°C was prepared. Membranes supported with a glass fabric were cast on polyethylene plates (034 cm3 of chitosan solution per 1 cm2 surface area). The membranes were then dried at 65°C for 30 hours, neutralized with NaOH, washed with water and dried again. The thickness of the membranes obtained was 0.08-0.09mm (glass fabric included).
2.5 Immobilization of urease on chitosan membranes Urease was immobilized according to the following general procedure: the membranes were conditioned in water, then treated with glutaraldehyde at room temperature and washed until the washings were free of glutaraldehyde. The membranes were then reacted with urease solution for 1 h a t room temperature with occasional stirring, left overnight at 4°C and finally washed until the washings were free of urease. To optimise preparation of ureasexhitosan membranes, the effects of the following basic factors of the applied immobilization procedure on the activity yields of the ureasexhitosan system were studied: waterconditioning,
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B. Krajewska, M . Leszko, W . Zaborska
glutaraldehyde concentration, time of membrane-glutaraldehyde reaction, pH of glutaraldehyde solution, pH of urease solution and urease concentration. The study was performed in such a way that a chosen factor of the procedure under examination was changed while the remaining factors of the procedure were kept fixed. Unless otherwise stated, urease activity was determined as described in Section 2.2.
2.6 Determination of physicochemical properties of free and immobilized urease 2.6.1 Kinetic parameters KM,v,, Constant amounts of the free and immobilized urease were subjected to reaction of urea hydrolysis in solutions of increasing urea concentration. For each experiment the initial rate of the reaction was measured. The reactions of the free urease were stopped by inactivation of the enzyme with the chemicals of the phenolhypochlorite method. 2.6.2 Effect of pH on activity The effect of pH on activity of both the enzymes was studied in the phosphate buffers of pH ranging from 5.3 to 8.2. 2.6.3 Effect of temperature on activity The effect of temperature on activity of both the enzymes was studied between 10 and 95°C at the optimum pH of both the enzymes, i.e. 7.0. The samples of the ureases and of urea solutions were heated to a required temperature, then mixed and the reaction rate was measured at the same temperature. 2.6.4 Heat inactivation at 70°C The samples of both enzymes in the phosphate buffer pH 7.0 were incubated at 70°C for the desired times (between 0 and 250 min) and then their activities were assayed at 25°C. 2.6.5 Stability at 4°C To compare the storage stability of the examined enzymes at 4°C three types of samples were prepared: a solution of the free urease in the phosphate buffer pH 7.0 (0.1 mg ~ m - ~equal ) ; sized samples of the chitosan membrane with immobilized urease stored wet in the phosphate buffer pH 7.0; and equal sized samples of the chitosan membrane with immobilized urease stored dry (the membranes were dried for 3 h at room temperature after urease had been immobilized on them). All the samples were stored at 4°C. The activities of the consecutive fresh samples taken from the store were assayed periodically at 25°C. 2.6.6 Stability at 2YC The same types of urease samples as used for the storage stability assay at 4°C were prepared. All the samples were stored at 25°C. Activities of the samples newly taken from the store were assayed periodically.
Urease immobilized on chitosan membrane
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2.6.7 Reusability Two types of reusability assay were performed: one sample of the chitosan membrane-immobilizedurease was stored in the phosphate buffer pH 7.0, the other one dry, both at 4°C.The activities of the samples were measured every other day at 25°C. After each activity determination the samples were washed with the buffer pH 7.0and left stored till the next assay, the first sample in the buffer, the other one dried after the assay. 2.6.8 Operational stability The reaction of urea hydrolysis catalyzed by the chitosan membrane-immobilized urease was carried out continuously for 120 h in static conditions. The sample was kept in the urea solution (10 g dm- 3), which was exchanged every 1.5 h, and the activity of the membrane was assayed periodically.
3 RESULTS A N D DISCUSSION 3.1 immobilization of urease onto chitosan membranes
The proposed method of immobilizationof urease on chitosan using glutaraldehyde involves the following reactions: Ch-NH,
-
+ q\C-(CH,)3-Cp H ’
Chitosan Ch-N=CH-(CH,),--C
8)
Ch-N=CH-(CH,),-C
\H
H ‘
Glutaraldehyde
8)+
HZN-E
aCh-N=CH-(CH,),xH=N-E
(3)
H‘
Glutaraldehyde-pretreated chitosan Enzyme
Reaction (2) should introduce aldehyde groups onto the chitosan membranes to facilitate the reaction with the enzyme. It was found, however, that maximum activity was obtained with membranes on which urease was immobilized by simple adsorption. This suggests that reaction (3) alters the enzyme structure so as to diminish its activity. Membranes with adsorbed urease, however, lacked stability and lost enzymatic activity because of easy desorption of the enzyme. On the other hand, glutaraldehyde concentrations greater than 0.03 % (w/v) and reaction times greater than 1.5 h render the membranes brittle as a result of cross-linking of chitosan. As a compromise, a glutaraldehyde concentration of 0-01% (w/v) and a reaction time of 1.5 h were considered optimal. Reaction (2) was not pH dependent and so aqueous glutaraldehyde solution was chosen for further studies. The study of reaction (3) showed that maximum immobilized enzyme activity was achieved with the lower pH urease solution. This may result from the fact that an acidic pH favours reaction between amino and aldehyde groups, and also from the fact that in solutions of pH 6 chitosan exists in
-=
B. Krajewska, M . Leszko, W.Zaborska
342
TABLE 1 Activities of Selected Urease-Carrier Systems Activity of urease-carrier system
Carrier
(pmol NH, min- cm carrier)
(jimol NH, min-' g - ' carrier)
1.56
1100 1200 215
'
Chitosan membrane Chitin powder Chitosan powder Gelatin membrane Collagen-poly (glycidyl methacrylate) graft copolymer
3.0-6.0
AQ-nylon membrane
ImmunodyneTMnylon membrane
Activity of free urease used (jimol NH, min-' mg-' protein)
32 80 0.487
32 165
1.2
116
0.36
0.088
Ref.
32
6 7 12 13 14 15
a gel form which probably helps more urease molecules to bind the membrane via adsorption or gel inclusion. Consequently a urease solution at pH 5.3 was chosen for the procedure. Finally it was found that in order to achieve maximum bound urease activity a urease concentration of at least 0-5 mg cm-3 for reaction (3) at pH 5.3 should be used. Prior to chemical treatment chitosan membranes were waterconditioned, the determined optimum time of waterconditioning ranges from 8 to 14 days.
3.2 Activity of immobilized urease The membrane-immobilized urease had the following activity: 1.56 pmol NH, min- cm-' membrane, i.e. 1100 pmol NH, min- ' g-' chitosan membrane, which corresponds to 00490mg protein immobilized on 1 cm2 of the membrane. For comparison, activities of selected ureasecarrier systems are listed in Table 1. The protein content ofthe membrane was 0.0507 mg cm-'; which means that the immobilized urease retained 94% of the activity of the free urease. This represents an improvement over values reported by other investigators (51 % , I 6 2 ( r 3 O X 6 and for the retained activity of immobilised urease.
'
3.3 Physicochemical properties of immobilized urease 3.3.1 Kinetic parameters KM, vmax The kinetic results obtained for the free and immobilized urease are presented in Fig. 1 as Lineweaver-Burk plots, l/u versus l/[S]. The linear nature of the Lineweaver-Burk plots proves that in the ranges of urea concentration examined both the enzymes follow the Michaelis-Menten kinetics represented by the
Urease immobilized on chitosan membrane
343
vma. * 0.337 mmolsa UREA/s q PROTEIN
3 10
K, = 5.01 mmol/dm5 vmqX * 0.236 mmoles UREA/s g PROTEIN
I
I
I
I
200
400
600
c
f/p] (moles UREA/dm,)-'
Fig. 1. Lineweaver-Burk plots, l/u versus l/[S], for free and chitosan membrane-immobilized urease. Reactions were carried out in phosphate buffer pH 7.0 (22 mmol dm-', 1 mmol dm-', EDTA, I (307)at 25°C.
following equation:
where v and v,,, are actual and maximum reactions rates, respectively, KM is the Michaelis constant and [S] is substrate concentration. For low concentrations of substrate the reaction is of the first order, for high concentrations it changes into zero order when the reaction rate is independent of substrate concentration. This range of concentration was used for enzyme activity determinations. The calculated values of the kinetic parameters: the Michaelis constants and the maximum reaction rates are listed in Fig. 1. The chitosan-immobilized urease exhibited K , values about 5 times higher than that of the free urease. This increase may be a consequence of either structural changes in the enzyme introduced by the applied immobilization procedure, or lower accessibility of the substrate to the active site of the immobilized enzyme. The latter may result either from diffusional resistances of stagnant solvent layers produced around the immobilized molecules, or from the confinement of enzyme molecules within the skin of the membrane during the process of immobilization (gelation of chitosan at pH 6.5). Consequently the maximum rate of reaction catalyzed by the immobilized urease was lower than that of the free urease. Increased K , of urease after immobilization has similarly been reported by various authors.' 7-20
-=
391
3.3.2 Effect of p H on activity Figure 2 shows that the immobilized urease was less sensitive to pH changes at
B. Krajewska, M . Leszko, W . Zaborska
344
I 0 IMMOBILIZED
1 5.0
1
I
I
6.0
zo
8.0
c
PH
Fig. 2. Effect of pH on activity of free and chitosan membrane-immobilized urease. Reactions were performed at 25°C in phosphate buffers (22 mmol dm-3, 1 mmol dm-3 EDTA, I 0.07) of various pH values.
pH c 7 and slightly more sensitive at pH > 7 than the free urease. Both the enzymes had optimum pH values of 7, but the optimum of the immobilized urease was broader at lower pH values. This effect is in agreement with a general observation that positively charged supports displace pH-activity curves of enzymes attached to them towards Lower pH values.21 Different pH-activity profiles of immobilized urease have been obtained depending on a support chosen and on an immobilization method a ~ p l i e d . ~3 .* 1' 6.1 7 * 9 , 2 2 * 2 3
'
3.3.3 Effect of temperature on activity Figure 3(a) shows the temperature optima curves for the free and immobilized urease preparations. The free urease had an optimum temperature of about 65T, whereas the temperature optimum of immobilized urease was shifted to 75°C. The results for the temperature range from 10°C to the optimum temperatures are also presented in the form of Arrhenius plots (Fig. 3(b)). The plots for both the enzymes were linear and the calculated values of activation energy were equal to 5.71 and 7.37 kcal mol-' for the free and chitosan-immobilized urease, respectively. The higher value of activation energy obtained for the immobilized urease indicates that the applied immobilization procedure introduced changes in the structure of urease molecules which impeded the enzymecatalyzed reaction. Similar results were reported previously.' 7.1 In contrast, cases where urease activation energy decrcased after immobilization have also been reported.' 9 , 2 2 3.3.4 Heat inactivation at 70°C As shown in Fig. 4 the immobilization of urease on chitosan protected the enzyme
Urease immobilized on chitosan membrane
345
4
0 TREE 0 IMMOBILIZED
ioo
5
4.0-
20
1
1.0
I
I
I
1
1
10
20
30
40
50
I
1
I
60
4
I
70 00 90 TEMPERATURE
PC]
100
-
a5 -
60
50
I
I
1
i0
I
&I
40
30
10
10
I
I
I
1
I
I
s2
53
3.9
15
0 I I
c
3.6 T-’ 10’ [K-‘]
(b) Fig. 3. Effect of temperature on activity of free and chitosan membrane-immobilized urease: (a) temperature optima; (b) Arrhenius plots, log (activity) versus 1/T. Reactions were performed in phosphate buffer pH 7.0 (22 mmol dm-), 1 mmol d m - 3 EDTA, I 0-07).
against heat inactivation. The half-times for the decay of activity were about 120 and 250 min for the free and immobilized enzyme, respectively. 3.3.5 Stability at 4°C As shown in Fig. 5 the free urease retained activity for 8 days, but thereafter activity quickly decreased, the half-time of activity decay was 13 days. The chitosanimmobilized urease, whether stored wet or dry, remained very active over a long
B. Krajewska, M . Leszko, W . Zaborska
346
t
IMMOBILIZED
\
I
1
50
100
'
I
A
150 200 250 PREINCUBATION TIME [min]
Fig. 4. Heat inactivation at 70°C of free and chitosan membrane-immobilized urease. Enzyme activity was determined at 25°C in phosphate buffer pH 7.0 (22 mmol 1 mmol dm-3 EDTA, I 007).
4°C
0 TREE 0 I M M O b I L I Z E D (MEMBRANE5 5TORED DRY) 0 IMMOBILIZED (MEMBRANE5 STORED WET)
I
I
I
1
I
1
8
16
LO
32
40
48
1
4
56 64 STORAGE T I M E [DAY51
Fig. 5. Storage stability (4°C) of free and chitosan membrane-immobilized urease (wet and dry). Enzyme activity was determined at 25°C in phosphate buffer pH 7.0 (22 mmol dm-', 1 mmol dm-3 EDTA, I 0.07).
Urease immobilized on chitosan membrane
347
U
0
0
0 TREE 0 I M M O I I L I Z E D (MEMBRANES STORED DRY) 0 IMMOBILIZED (MEMBRANES JTORED WET)
I
I
I
I
I
I
I
I*
8
i6
24
3.2
40
48
56
64
STORAGE T I M E [DAYS]
Fig. 6. Storage stability (25°C)of free and chitosan membrane-immobilizedurease (wet and dry). Enzyme activity was determined at 25°C in phosphate buffer pH 7.0 (22 mmol dm-3, 1 mmol dm-3 EDTA, I 0.07).
period of time: the membranes stored wet lost about 10%of their original activity over the period of 60 days, whereas membranes stored dry lost about 20% of their original activity over the same period of time. 3.3.6 Stability at 25°C As shown in Fig. 6 the free urease lost activity within 12 days, the half-time of activity decay was 3 days. The membranes stored wet lost 30% of their original activity within 10days and then remained active; the membranes stored dry slowly lost 20% of their original activity over the period of 60 days similarly to those stored at 4°C. Both these sets of data show that the chitosan-immobilized urease was considerably more stable than the free enzyme, and could be stored for extended periods, in both wet and dry forms, before use. Various other reports confirm that the storage stability of immobilized urease varies depending on the immobilization method applid.6.13.17.19.22-24 3.3.7 Reusability Figure 7 shows that the immobilized urease lost activity after 9 cycles of reuse. Drying of the membrane after each use considerably speeded up the process of activity decay. 3.3.8 Operational stability Figure 8 shows that the membrane lost about 30% of its activity within 5 h of the continuous process and then slowly decreased its activity to about 40% within 120 h.
B. Krajewska, M . Leszko, W.Zaborska
348
OMEMBRANES STOREbWET 0 MEMBRANE5 STORED DRY
1
4
3
2
I
I
5
6
I
I
1 -
8 9 NUMBER O f REU5E5
1
Fig. 7. Reusability of chitosan membrane-immobilized urease. Enzyme activity was determined at 25°C in phosphate buffer pH 7.0 (22 mmol dm-3, 1 mmol dm-3 EDTA, I @07).
40
t
t
1
I
I
1
I
10
20
30
40
I
I
If
1
I
*
50 60" 110 110 TIME OF OPERATION [HOURS]
Fig. 8. Operational stability ofchitosan membrane-immobilized urease. Enzyme activity was determined at 25°C in phosphate buffer pH 7.0 (22 mmol dm-3, 1 mmol dm-3 EDRA, 1 007).
Urease immobilized on chitosan membrane
349
4 CONCLUSIONS This study shows that urease can be successfully immobilized on glutaraldehydepretreated chitosan membranes. The ureasexhitosan membrane system offers various advantages: (1) chitosan is a relatively inexpensive material, it is inert and easy to cast in the form of membrane; (2) the proposed immobilization procedure is simple to carry out; and (3)the properties of the immobilized enzyme offer potential for use in production processes. The obtained material has a comparatively high enzymatic activity. The activity retention of urease after immobilization of 94 % is higher than others previously reported. The physicochemical properties of urease were changed after immobilization, but whilst the kinetic parameters are slightly less favourable than those of free urease the excellent storage stability, thermal stability, reusability and operational stability of the immobilized enzyme demonstrates the superior potential of the immobilized enzyme for practical application. ACKNOWLEDGEMENT This work was supported by the State Programme of Fundamental Research on Membranes and Membrane Processes (CPBP 02.11).
REFERENCES 1. Chang, T. M.S. In Immobilized Enzymes, Antigens, Antibodies, and Peptides, ed. H. H. Weetall. Marcel Dekker, Inc., New York, 1975, pp. 245-92. 2. Chang, T. M. S., Artificial cells: the use of hybrid systems. Artificial Organs, 4 (1980) 264-71. 3. Drukker, W., Parsons, F. M. & Gordon, A. In Replacement of Renal Function by Dialysis, ed. W. Drukker, F. M. Parsons & J. F. Maher. Martinus Nijhoff, The Hague, 1979, pp. 24458. 4. Krajewska, B. & Zaborska, W., Dialysate regeneration system of artificial kidney. Biocyb. Biomed. Eng., 9 (1989) 113-29. 5. Muzzarelli, R. A. A., Chitin. Pergamon, New York, 1977. 6. Iyengar, L. & Rao, A. V. S. P., Urease bound to chitin with glutaraldehyde. Biotechnol. Bioeng., 21 (1979) 1333-43. 7. Kasumi, T., Tsuji, M., Hayashi, K. & Tsumura, N., Preparation and some properties of chitosan bound enzymes. Agr. Biol. Chem., 41 (1977) 1865-72. 8. Hirano, S. & Miura, O., Alkaline phosphatase and pepsin immobilized in gels. Biotechnol. Bioeng., 21 (1979) 71 1-14. 9. Weatherburn, M. W., Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem., 39 (1967) 9 7 1 4 . 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J., Protein measurement with the Fohn phenol reagent. J . Biol. Chem., 193 (1951) 265-75. 1 1. Brzeski, M., Technologia Otrzymywania Chityny i Chitozanu z Pancerzy Kryla Antarktycznego oraz Zastosowanie Tych Polimerow d o Formowania Blon. PhD thesis, Szczecin Polytechnic, Szczecin, 1984.
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12. Tanny, G. B., Kedem, 0.& Bohak, Z., Coupling of transport and enzymatic reaction in a membrane composed of an anion exchanger and immobilized urease. 3. Membrane Sci., 4 (1979) 363-77. 13. Raghunath, K., Rao, K. P. &Joseph, K. T., Preparation and characterization of urease immobilized on to collagen-poly(glycidy1 methacrylate) graft copolymer. Biotechnol. Bioeng., 26 (1984) 1049. 14. Miyama, H., Kobayashi, T. & Nosaka, Y., Immobilization of enzyme on nylon containing pendant quaternized amine groups. Biotechnol. Bioeng., 26 (1984) 139C2. 15. Krajewska, B., Leszko, M. & Zaborska, W., Membrane-immobilized urease for possible use in dialysate regeneration system. Env. Prot. Eng., in press. 16. Karube, I. & Suzuki, S., Electrochemical preparation of urease-collagen membrane. Biochem. Biophys. Res. Commun., 47 (1972) 5 1 4 . 17. Madeira, V. M. C., Incorporation of urease into liposomes. Biochim. Biophys. Acta, 499 (1977) 202-11. 18. Onyezili, F. N. & Onitri, A. C., The effect of substrate flow-rate on immobilized urease assays. Biochim. Biophys. Acta, 659 (1981) 244-8. 19. Ramachandran, K. B. & Perlmutter, D. D., Effects of immobilization on the kinetics of enzymecatalyzed reactions. 11. Urease in a packedcolumn differential reactor system. Biotechnol. Bioeng., 18 (1976) 685-99. 20. Miyama, H., Kawata, M. & Nosaka, Y., Immobilization of enzyme on dimethylaminated nylon gels. Biotechnol. Bioeng., 27 (1985) 1403-10. 21. Zaborsky, O., Immobilized Enzymes. CRC Press, Cleveland, 1973. 22. Sundaram, P. V. & Hornby, W. E., Preparation and properties of urease chemically attached to nylon tube. FEES Letters, 10 (1970) 325-7. 23. Sundaram, P. V . &Crook, E. M., Preparation and properties ofsolid-supported urease. Can. J. Biochem., 49 (1971) 1388-94. 24. Riesel, E. & Katchalski, E., Preparation and properties of water-insoluble derivatives of urease. J . Biol. Chem., 239 (1964) 1521-4.
