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Stabilization of Papain by Modification with Chitosan

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Papain (EC 3.4.22.2) was immobilized on chitosan by adsorption and subsequent cross-linking with glutaraldehyde. The immobilized papain displayed a lower specific activity than did the native enzyme. The thermal stability of the immobilized papain, relative to that of the free enzyme, was markedly increased. The storage stability of the conjugated enzyme was enhanced such that more than 85% of the initial activity remained after a month storage at 45?C. The optimum pH of immobilized papain was shifted to the acidic region and enzyme stability was increased at pH levels below 3,5.
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Turk J Chem
26 (2002) , 311 – 316.
c T ¨
UB?ITAK
Stabilization of Papain by Modi?cation with Chitosan
Ali KILINC
¸ , Se¸
cil ¨
ONAL and Azmi TELEFONCU
Department of Biochemistry, Faculty of Science, Ege University,
35100 Bornova, ?Izmir-TURKEY
e-mail:akilinc@sci.ege.edu.tr
Received 08.05.2001
Papain (EC 3.4.22.2) was immobilized on chitosan by adsorption and subsequent cross-linking with
glutaraldehyde. The immobilized papain displayed a lower speci?c activity than did the native enzyme.
The thermal stability of the immobilized papain, relative to that of the free enzyme, was markedly
increased. The storage stability of the conjugated enzyme was enhanced such that more than 85% of
the initial activity remained after a month storage at 45?C. The optimum pH of immobilized papain was
shifted to the acidic region and enzyme stability was increased at pH levels below 3.5.
Key Words: Enzyme stabilization, immobilized papain, chitosan
Introduction
The chemical modi?cation of enzymes with macromolecules constitutes a useful strategy for improving the
stability of these biocatalysts. In this context, the use of several natural or synthetic polymers such as
dextran, polyethylene glycol and carboxymethylcellulose has been reported1?4.
Amino acid residues of enzymes contain reactive groups such as amino (Lys, N-terminus), thiol (Cys),
carboxyl (Asp, Glu, C-terminus), aromatic hydroxyl (Tyr) and aliphatic hydroxyl (Ser and Thr). Chemical,
ionic or chelation reactions with such groups enable us to attach the amino acids and hence proteins to soluble
or insoluble inert supports. Immobilization is one of the best ways of stabilizing enzymes. Immobilization
imparts stability to proteins by restricting the movement of the protein molecule by attachment to an inert
body via chemical bonds. The various domains are therefore held in the correct orientation to retain activity
at least over an extended period of time when compared with enzymes in free solution.
The addition of polyhydroxyl compounds to enzyme solutions has been shown to increase the stability
of enzymes5?8. This is thought to be due to the interaction of the polyhydroxyl compound with water
in the system.
This e?ectively reduces the protein water interactions as the polyhydroxyl compounds
become preferentially hydrated and thus the hydrophobic interactions of the protein structure are e?ectively
strengthened. This leads to an increased resistance to thermal denaturation of the protein structure and
an increase in the stability of the enzyme. Polysaccharide amines were e?ective in reducing hydrophobic
interactions when they were used as spacers, and they prolonged enzyme life in use when compared to alkane
spacer molecules9,10. Chitosan is in polycationic form at acidic and neutral pH regions. The association of
the polyelectrolyte with the enzyme probably results in the formation of a cage around the enzyme molecule.
311

Stabilization of Papain by Modi?cation with Chitosan, A. KILINC
¸ , et al.,
Some proteases, including papain, can hydrolyze the peptide bonds of collagen and keratin in the
stratum corneum of the skin. The controlled skin damage can trigger skin repair and bring to the surface
a layer of smoother, softer skin11. Papain is one of the potential active ingredients in cosmetic products.
The aim of this work is the stabilization of papain by coupling with chitosan, which is a cationic, bioactive,
biodegradable, biocompatible polysaccharide suitable for many applications in cosmetics.
Experimental
Materials
Papain (EC 3.4.22.2) and N-benzoyl-L-arginine ethyl ester (BAEE) were obtained from Sigma Chem. Co.
(St. Louis, USA). Glutaraldehyde (25%) was purchased from BDH (Dorset, UK). Chitosan was obtained
from Fluka Chemie AG (Buch, Switzerland). All other chemicals were of analytical grade.
Preparation of Chitosan-Papain Conjugate
Chitosan was dissolved in 2% (v/v) acetic acid, precipitated by adding 1 M NaOH up to pH 10.0 and
then extensively washed with 30 mM citrate/phosphate bu?er pH 6.0 (Bu?er-I). Then 100 mg chitosan was
suspended in 10 ml of citrate/phosphate bu?er and 50 mg papain was added. The suspension was kept at
4?C overnight under stirring and subsequently centrifuged at 10,000xg for 15 min. The resulting pellet was
resuspended in Bu?er-I containing 2% glutaraldehyde. The suspension was stirred at room temperature for
30 min under vacuum and then centrifugated again at 10,000xg for 15 min. The chitosan-papain conjugate
was washed with small aliquots of Bu?er-I containing 2M KCl and Bu?er-I alone until no protein was
detected in the washings.
Determination of Immobilized Protein
The protein content of the chitosan-papain conjugate was calculated by substracting the amount of protein
determined in the centrifugate and washings following immobilization from the amount of protein used for
immobilization. The protein content in the solutions was determined by the Bradford method12.
Enzyme Assay
Enzyme activity in native and modi?ed papain was determined titrimetrically, using N-benzoyl-L-arginine
ethyl ester (BAEE) as substrate. The reaction mixtures contained 125 mmole of substrate, 25 mmole of
cysteine, 10 mmole of EDTA, and the appropriate amount of enzyme in a total volume of 5 ml. The
enzymatically liberated N-benzoyl-L-arginine was titrated with 0.01 N NaOH using a pH-stat13. One unit
enzyme was taken to hydrolyze 1.0 mmole of BAEE per min at pH 6.2 and 25?C.
Stability Tests
In order to evaluate the e?ect of chemical modi?cation on papain thermostability, two di?erent types of
experiment were performed. Firstly, native and modi?ed papain were incubated at di?erent temperatures in
50 mM phosphate bu?er, pH 7.0. Aliquots were removed after 10 min incubation, chilled quickly and assayed
312

Stabilization of Papain by Modi?cation with Chitosan, A. KILINC
¸ , et al.,
for enzymatic activity. Secondly, native and modi?ed papain were incubated at 75?C in 50 mM phosphate
bu?er (pH 7.0). Aliquots were removed at scheduled times, chilled quickly and assayed for enzymatic activity.
For the estimation of pH stability, native and modi?ed papain were incubated at 25?C in 50 mM
citrate/phosphate bu?er, pH 2.2-6.2 and 50 mM phosphate bu?er, pH 6.2-8.6. Aliquots were removed after
30 min incubation and assayed for enzymatic acitivity.
The storage stability of the enzyme preparations was also studied.
Native and modi?ed papain
preparations were stored at 25?C and 45?C, and enzymatic activity was measured at scheduled times. It
is generally accepted that a month’s stability of an enzyme at 45?C is roughly equal to that of one year at
room temperature8.
Results and Discussion
Chitosan was selected as a carrier for papain immobilization on account of the following properties:
(i) polycationic, (ii) hydrophilic, (iii) biocompatible and biodegradable, (iv) bioactive. Papain was immo-
bilized by cross-linking the enzyme and chitosan by means of glutaraldehyde. The optimal cross-linking
incubation time and glutaraldehyde concentration were found to be about 30 min and 2%, respectively.
Above this incubation time the immobilization yield appeared to increase slightly but the speci?c activity of
the immobilized enzyme decreased. The chitosan-papain conjugate contained about 18 mg protein/100mg
chitosan (Table).
Table Some properties of native and chitosan-linked papain (cross-linking process time: 30 min; glutaraldehyde
concentration in the coupling medium: 2%).
Property
native papain
chitosan-papain conjugate
Protein content (%)
81.7
15.3 (18 mg/100 mg chitosan)
Speci?c activity
12.4
10.2
(mmole/min.mg protein)
pH optimum
6.5
6.0
Temperature optimum (?C)
65
75
Storage stability (% activity retained
16
85
after one month at 45?C)
The recovery yield of papain activity was about 82% right after immobilization. The speci?c activity
reduction in immobilized enzymes with respect to their native forms has been widely reported14,15. Structural
changes by covalent attachment and di?usional limitations could be the reasons for these activity losses16.
The stability of papain was dramatically improved by conjugation with chitosan-compared with that
of native enzyme. In order to evaluate the e?ect of the modi?cation on enzyme thermostability, two di?erent
types of experiments were performed. Figure 1 shows the thermal stability pro?le of chitosan-bound papain
after 10 min of incubation at di?erent temperatures.
The thermal stability of immobilized papain was
markedly increased relative to that of the native enzyme. After conjugation, T50 (the temperature at which
50% of initial activity was retained) was increased from 75?C to 83?C. As shown in Figure 2, the heat
stability of chitosan-linked papain at 75?C was improved dramatically.
313

Stabilization of Papain by Modi?cation with Chitosan, A. KILINC
¸ , et al.,
100
80
(%)
60
40
Relative Activity 20
0
25
35
45
55
65
75
85
95
Temperature ( °C)
Figure 1. Thermal stability pro?le of native (o) and immobilized papain (•). Native and immobilized papain
preparations were incubated at preset temperatures in 50 mM phosphate bu?er (pH 7.0) for 10 min and then assayed
under standard test conditions.
100
80
60
Activity (%)
40
20
log Relative
0
0
50
100
150
200
250
Time (min)
Figure 2. Heat stability of native (o) and immobilized papain (•) at 75?C. The enzyme preparations were incubated
at 75?C in 50 mM phosphate bu?er (pH 7.0) for scheduled times and then assayed at standard test conditions.
The storage stability of papain was also dramatically improved by conjugation with chitosan compared
with that of native enzyme. About 85% of initial activity remained after a month of storage at 45?C
(Fig. 3).
100
80
(%)
60
40
Relative Activity 20
0 0
5
10
15
20
25
30
Time (day)
Figure 3. Stability of native (o) and chitosan modi?ed papain (•) when stored at 25?C (—) and 45?C (- - -).
314

Stabilization of Papain by Modi?cation with Chitosan, A. KILINC
¸ , et al.,
As shown in Figures 1-3, the thermal stability and storage stability of chitosan-conjugated papain
were improved. The conformational stabilization of papain molecules due to intramolecular cross-linking is
the most important factor underlying this e?ect17. Protein aggregation plays an important role in thermal
denaturation of enzymes and the electrostatic repulsion between the enzyme molecules linked to chitosan
(a cationic polymer) could be another factor for the improved thermostability of the modi?ed enzyme.
The activities of native and immobilized papains were determined at di?erent pH values. As can
be seen from Figure 4, the pH optimum of the chitosan-papain conjugate was shifted to the acidic region
relative to native papain. The immobilization of enzymes to charged supports often leads to displacements
in the pH activity pro?le, ascribable to unequal partitioning of H+ and OH? between the microenvironment
of the immobilized enzyme and the bulk phase due the electrostatic interactions with the matrix16,18.
100
(%)
80
60
40
Relative Activity
20
02
4
6
8
10
pH
Figure 4. pH-activity curves for native (o) and immobilized papain (•).
100
(%) 80
60
40
Relative Activity 20
0
2
4
6
8
10
pH
Figure 5. E?ect of pH on the stability of native (o) and immobilized papain (•). The test solution (1 ml) in the
appropriate bu?er and containing papain or immobilized papain was incubated at 25?C for 30 min. The esterase
activity was determined under standard test conditions.
The pH stability of free and immobilized papain was determined in the pH range 2.2-8.6. Figure 5
shows that pH stability was increased for chitosan-papain conjugate in the range between 2.2 and 6.2. Since
chitosan is positively charged at acidic or neutral pH values, this polysaccharide is able to form intramolecular
salt bridges with anionic groups of the protein. This electrostatic interaction could improve the stability
of papain, because intramolecular salt bridges are, in fact, one of the principal forces contributing to the
maintenance of the active conformation of enzymes19.
315

Stabilization of Papain by Modi?cation with Chitosan, A. KILINC
¸ , et al.,
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3. Y. Inada et al. Meth. Enzymol. 242, 65-90 (1994).
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5. Y. Fujita, Y. Iwada, and Y. Noda, Bull. Chem. Soc. Jpn. 55, 793-800 (1982).
6. T. Arakawa, and S. N. Marhe?, Biochemistry 21, 6536-6544 (1982).
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10. T. D. Gibson, PhD Thesis, Leeds University, Leeds UK (1991).
11. J. P. Forrestier, Int. J. Cosmetic Sci. 14, 47-55 (1992).
12. M. M. Bradford, Anal. Biochem. 72, 248-253 (1976).
13. R. Arnon, “Papain” in Methods in Enzymology, Vol. 19, eds. G. E. Perlman and L. Lorand, pp. 226-244,
Pergamon Press.
14. R. Ayen, and S. Ernback, Eur. J. Biochem. 18, 351-356 (1971).
15. V. Ivanova and E. Dobreva, Process Biochem. 29, 607-612 (1994).
16. C. R. Carrera and A. C. Rubiolo, Process Biochem. 31, 243-248 (1996).
17. A. M. Klibanov, Adv. Appl. Microbiol. 29, 1-28 (1983).
18. A. Telefoncu, Biotechnol. Bioeng. 25, 713-724 (1983).
19. M. L. Perutz, Science 201, 1187-1191 (1978).
316

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