Immobilization and stabilization of papain on chelating sepharose : a metal chelate regenerable carrier

EJB Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.4 No.3, Issue of December 15, 2001
© 2001 by Universidad Católica de Valparaíso -- Chile Received April 9, 2001 / Accepted July 27, 2001
RESEARCH ARTICLE
Immobilization and stabilization of papain on chelating sepharose: a metal
chelate regenerable carrier
Sarah Afaq
Research Scholar in Biochemistry
Department of Biochemistry, Faculty of Life Sciences
Aligarh Muslim University, Aligarh-202002 (U.P.), India
Tel: 091 571-700741
Jawaid Iqbal*
Senior Lecturer
Department of Biochemistry, Faculty of Life Sciences
Aligarh Muslim University, Aligarh-202002 (U.P.), India
Tel: 091 571-700741
E-mail: j.iqbal@angelfire.com
Financial support: Council of Scientific and Industrial Research (C.S.I.R.).
Keywords: papain, immobilized metal ion (IMI) carrier, immobilization, thermal stability, regeneration of matrix.
A method for immobilization of papain has been
lymphocytes in transplantation immunology (Hausmann et
selected based on the interaction between its histidine,
al. 1993) and in the treatment of red blood cells with
cysteine and tryptophan residues with the immobilized
immobilized papain prior to use in antibody-dependent cell-
metal ion (IMI) carrier for maximum binding on a
mediated cytotoxicity (ADCC) assays with lymphocytes
small volume of the carrier. The immobilized papain
(Kumpel and Bakacs, 1992).
retained high activity has improved thermal stability
and the carrier could be recovered from the spent
Various attempts have been made to stabilize papain for a
bound enzyme, to be reused. Reimmobilization of
more efficient use. Papain and other proteolytic enzymes
papain on the regenerated matrix was equally effective
have been immobilized by radiation polymerization of
with the retention of maximum enzyme activity.
various monomers (Kumakura and Kaetsu, 1984) and
insolubilized by using polyclonal antibodies (Khan and
Iqbal, 2000). Covalent coupling of papain has also been
Numerous approaches have been explored for the
shown in different studies performed by several workers
preparation of immobilized enzymes because they have
(Huckel et al. 1996; Zhuang and Butterfield, 1992).
considerable advantages over enzymes in bulk solution
However, the biomatrices with entrapped enzymes tend to
(Surinenaite et al. 1996; Gomara et al. 2000; Guo and
leak proteins with time. This resulted in the activity losses
Yang, 2000; Wadu-Mesthrige et al. 2000). Papain, a thiol
as well as contamination of the product with the enzymes,
protease, is well characterized kinetically and structurally
which is not acceptable for pharmaceutical applications.
(Liu and Hanzlik, 1993; Mellor et al. 1993; Vernet et al.
The covalent coupling of enzyme can produce a
1995) being a suitable model to compare the efficiency of
considerable loss of activity due to the influence of the
various immobilization procedures. The need for the
coupling conditions and to conformational changes in
immobilization of papain has been due to its great industrial
enzyme structure (Goldman et al. 1968). However,
and medicinal potential. For example, papain is used as a
irreversible binding of enzyme to the carrier during
chill proofing agent during beer finishing operations in the
covalent coupling does not allow the recovery of the carrier
brewing process (Wiseman, 1993). This enzyme also
from the carrier-enzyme complex (Kise and Hayakawa,
facilitates the tenderization of meat in the meat industry
1991; Huckel et al. 1996; Huang et al. 1997). A method is,
(Swanson et al. 1992). The potential uses of papain include
therefore, needed in which the carrier should be easily
its frequent use as a biocatalyst for amino acid ester and
regenerated and reused without reducing the
peptide synthesis (Lozano et al. 1993), as well as the
immobilization yield. Attempts have been made in this
treatment of acute destructive lactation mastitis (Storozhuk
direction and a metal chelate regenerable carrier has been
et al. 1985). The biopharmaceutical potential of
used to immobilize the papain. This immobilization is
immobilized papain can be well illustrated by the
based on the ability of protein side chains of cysteine,
interaction of papain digested HLA class-I molecules with
histidine and tryptophan to substitute weakly bonded
alloreactive cluster of differentiation and cytotoxic-T-
ligands in the metal complexes. This method has a big
*Corresponding author
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Afaq, S. and Iqbal, J.
Table 1. Immobilization and reimmobilization of papain on chelating Sepharose activated by Cu2+ ions. Results
represent the mean and standard deviation of three different immobilized preparations. Activity and immobilization yields
were calculated from mean values.
No. of cycles
Enzyme activity (Units / mL gel)
Activity Yield
Immobilization
(%)
Yield (%)
Added
Unbound
Immobilized papain
C/A x 100
(A-B)/A x 100
(A)
(B)
(C)
Ist
65197 ± 2015
2313 ± 341
50867 ± 1210
78
96
IInd
69460 ± 1235
1494 ± 141
51841 ± 1339
75
98
IIIrd
69324 ± 508
2910 ± 176
49852 ± 692
72
96
IVth
67636 ± 2181
3266 ± 102
50116 ± 996
74
95
potential and may be more versatile since it allows a
and 0.5M NaCl) for 1 hour at room temperature. It was then
selection among many chelating metal ions.
centrifuged, supernatant was collected and matrix was
washed extensively with 10mM phosphate buffer, pH 8.2.
Materials and Methods
The colourless matrix was resuspended in 0.5 mL of
washing buffer and enzyme activity was determined in the
Papain (lyophilized powder) was obtained from Sigma
collected supernatant, eluant and colourless matrix.
Chemical Co. USA. Chelating Sepharose was the product
of Pharmacia Biotech. Uppsala, Sweden. Casein, cysteine
The whole matrix was reactivated with Cu2+ ions as
and other chemicals were from Sisco Research Lab.,
described earlier to perform a second cycle of papain
Mumbai, India.
immobilization by using the same enzyme concentration.
The above procedure was repeated for a total of four cycles.
Immobilization of papain on metal chelating
Sepharose activated by Cu2+ ions
Reimmobilization of papain on regenerated
Sepharose activated by Co2+ ions
0.5 mL gel of chelating Sepharose was mixed with 1 mL
distilled water and centrifuged for 30 sec. This procedure
0.2mL of regenerated (colorless) matrix was taken and
was repeated four times. The thoroughly washed matrix
washed with 1mL distilled water (3 times). Supernatant was
was loaded with metal ion (Cu2+) by the procedure
discarded and matrix was mixed with equal volume
described in the Instruction bulletin of Pharmacia Biotech,
(0.2mL) of 0.4M CoCl2. The solution was stirred at room
Uppsala, Sweden (1997). Equal volume of matrix (0.5 mL)
temperature for 30 min., centrifuged, and supernatant was
was mixed with equal volume of 0.4 M CuCl2 (0.5 mL),
discarded. The matrix was washed twice with 1 mL
stirred at room temperature for 30 minutes and centrifuged
distilled water and 10 mM phosphate buffer (pH 8.2).
briefly to discard supernatant. Washing of the matrix was
Finally, the whole matrix was suspended in 1.0 mL papain
done with 1 mL distilled water four times and finally with 1
solution (8.9 mg/mL). The same procedure was followed as
mL, 10 mM sodium phosphate buffer pH 8.2. This matrix
outlined in the section "Papain immobilization on Cu2+
was mixed with 1.5 mL of papain solution (8.9 mg/mL),
activated chelating Sepharose matrix". The matrix was
stirred at room temperature for 3 hours, centrifuged for 30
resuspended in 0.8mL of same washing buffer. The assay
sec and supernatant was collected. The matrix was finally
for enzyme activity was done in supernatant, washings,
washed repeatedly with 1 mL, 10 mM phosphate buffer pH
matrix and stock enzyme solution as described below.
8.2, until no detectable activity was obtained in the
washings. Finally the matrix was suspended in the same
Enzyme assay
buffer. The maximum bound units were calculated by
subtracting the activity remaining in supernatant and
The activity of soluble papain was determined by the
washings from the units added to chelating Sepharose
matrix. The actual units of bound papain were obtained by
method of Kunitz as described by others (Keay and Wildi,
measuring the activity in the immobilized enzyme
1970) using casein as substrate at 37ºC and pH 8.2. The
suspension.
enzyme activity of immobilized papain was determined in a
similar manner except that the reaction mixture was
Regeneration of matrix from immobilized papain
continuously stirred during the reaction. One unit of
enzyme activity is the amount of enzyme, which produces
0.5 mL blue coloured enzyme bound matrix was
TCA soluble peptides or amino acids giving a blue colour
regenerated by suspending in 2 mL eluant (0.05 M EDTA
equivalent to that of 0.5µg tyrosine per minute at 37ºC.
2

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Immobilization and stabilization of papain on chelating sepharose: a metal chelate regenerable carrier
Table 2. Reimmobilization of papain on regenerated chelating Sepharose activated by Co2+ ions.
Enzyme activity (Units / mL gel)
Activity Yield (%)
Immobilization Yield (%)
C/A x 100
(A-B)/A x 100
Added (A)
Unbound (B)
Immobilized papain (C)
109685
10425
78750
72
90
The activity yield remaining after immobilization was
defined as Activity yield (%) = C/Ax 100 and
Immobilization yield (%) = A- B/Ax 100 where A is the
total activity of enzyme added in the initial immobilization
solution; B, the activity of the residual enzyme in the
immobilization and washing solutions after the
immobilization procedure; and C, the activity of the
immobilized enzyme.
Results and Discussion
The results of the immobilization of papain on chelating
Sepharose activated with Cu (II) ions are summarized in
Table 1. As evident from the table, the activity and
immobilization yields were 78% and 98% respectively,
which were significantly higher when compared to the
covalently coupled papain (Zhuang and Butterfield, 1992).
The activity and immobilization yields of covalently
coupled papain were only 6% and 10.5% respectively in a
study performed by Zhuang and Butterfield, 1992.
Figure 1. Thermal inactivation of soluble and
Goldstein et al. 1970, have shown that different water
immobilized papain. Thermal stability of soluble and
insoluble derivatives of papain prepared by selecting
immobilized papain was determined by incubating at 65ºC
polyfunctional diazotizable resins exhibited only 25 to 50%
for various durations. Papain activity was determined at the
activity yields. In our study, the high activity yield of
end of incubation under standard condition. (o) Soluble
immobilized papain (Table 1) suggests for an easy
papain, (•) Immobilized papain.
accessibility of enzyme for its substrate. Glycoproteins
have also been immobilized on IMI carrier by using Cu (II)
The potential of substituting the metal ion and higher
ions but this inhibits the biological activity of certain
enzyme loading was also investigated on the recovered
proteins and only a limited number of proteins could be
carrier. In a different set of experiments Co (II) was used
used on these carriers (Chaga, 1994). There was no
for the reactivation of the regenerated matrix obtained from
considerable loss of biological activity of immobilized
Cu(II) activated papain bound IMI carrier. The results are
papain due to Cu(II) ions as evident by high activity yield
shown in Table 2. As evident from the table, the activity
(Table 1). Therefore, this carrier and metal ion are
yield was comparable with the values of Table 1, where
considered suitable for papain immobilization.
fresh as well as regenerated carrier was activated with Cu
(II) ions, suggesting that metal replacement is feasible and
effective for enzyme immobilization on IMI carrier. Such
A remarkable advantage of these carriers is its regeneration
replacement of metal ion is essential in certain cases, where
and recovery from spent immobilized enzyme. After the
enzymes are inactivated by binding to specific types of
elution of papain from Cu (II) activated IMI carrier, the
metal ion bound to IMI carrier (Chaga, 1994). The similar
regeneration and reactivation with same metal ion was
activity yield at two different enzyme loadings (Table 1 and
highly effective as evident by the activity yield of
Table 2) suggests for equal accessibility of enzyme
immobilized papain during subsequent cycles of
molecules to the substrate indicating that the bound enzyme
reimmobilization (Table 1). The activity yields for 2nd, 3rd
being held in a favourable orientation or location.
and 4th cycles were 75, 72 and 74% respectively. This
Wasserman et al. (1981) have demonstrated that even with
clearly indicates that the matrix can be efficiently
a nonporous matrix, the lower activity yield was achieved
regenerated, which is not possible in the case of covalently
at high enzyme binding levels. The immobilization yield
and cross-linked preparations (Iqbal and Saleemuddin,
shown in Table 2 was 90% which was lower than the values
1983a; Iqbal and Saleemuddin, 1983b).
obtained in Table 1 and such decrease in the immobilization
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Afaq, S. and Iqbal, J.
yield (Table 2) could be due to higher enzyme units loaded
Gomara, M.J.; Ercilla, G.; Alsima, M.A. and Haro, I.
on matrix for immobilization.
(2000). Assessment of synthetic peotides for hepatitis A
diagnosis using biosensor technology. Journal of
The thermal stability of soluble and immobilized papain
Immunological Methods 246:13-24.
was examined by heating at 65ºC for longer duration. As
shown in Figure 1, the immobilized papain exhibited a
Guo, L.H. and Yang, S.K. (2000). Study on the gallic acid
marked increase in thermostability and retained 87% of its
preparation by using immobilized tannase from Aspergillus
original activity after 1 hour incubation at 65ºC, while its
niger. Shen Wu Gong Chang Xue Bao 16:614-617.
soluble counterpart lost almost 75% of its original activity.
The higher stability of the IMI bound papain could be due
Hausmann, R.; Zavazava, N.; Steinmann, J. and Muller-
to the diminished autolysis of the enzyme fixed to the
Ruchholtz, W. (1993). Interaction of papain-digested HCA
support. The second possible explanation is related to the
class I molecules with human alloreactive cytotoxic T
rigidity of the conformation of the enzyme molecules
lymphocytes (CTL). Clinical and Experimental
resulting from binding to the matrix. Most proteolytic
Immunology 91:183-188.
enzymes studied in their soluble form are highly susceptible
to autolysis and get inactivated at elevated temperatures
Huang, X.L.; Catignani, G.L. and Swaisgood, H.E. (1997).
(Zhuang and Butterfield, 1992). The enhancement in
Comparison of the properties of trypsin immobilized on 2
thermal stability of membrane bound papain and covalently
celite derivatives. Journal of Biotechnology 53:21-27.
coupled chymotrypsin has also been reported in many other
enzyme immobilization studies (Butterfield et al. 1994;
Huckel, M.; Wirth, H.J. and Hearn, M.T. (1996) Porous
Schnapp and Shalitin, 1976).
Zirconia: a new support material for enzyme
immobilization. Journal of Biochemical and Biophysical
The cost of the carriers used for industrial applications is
Methods 31:165-179.
very important. The regenerability of IMI carrier is,
therefore, relevant. The mild conditions used for papain
Instruction bulletin of Pharmacia Biotech (1997). Chelating
immobilization, the high recovery of immobilized
Sepharose fast flow instructions. TK1, Uppsala, Sweden.
preparations and the regenerability of the matrix are the
pp. 1-20.
main features of the method reported here.
Iqbal, J. and Saleemuddin, M. (1983a). Preparation and
Acknowledgments
properties of Con A cellulose immobilized glucose oxidase.
Indian Journal of Biochemistry and Biophysics 20:33-38.
The financial support from Council of Scientific and
Industrial Research (C.S.I.R.) in the form of grant is highly
Iqbal, J. and Saleemuddin, M. (1983b). Activity and
acknowledged.
stability of glucose oxidase and invertase immobilized on
concanavalin A Sepharose: Influence of lectin
References
concentration. Biotechnology and Bioengineering 25:3191-
3195.
Butterfield, D.A.; Lee, J.; Ganapathi, S. and Bhattacharyya,
D. (1994). Bifunctional membrane Part IV. Active site
Keay, A.J. and Wildi, B.S. (1970). Proteases of the genus
structure and stability of an immobilized enzyme, papain,
Bacillus. I Neutral proteases. Biotechnology and
on modified polysulfone membranes studied by electron
Bioengineering 12:179-212.
paramagnetic resonance and kinetics. Journal of Membrane
Science 91:47-64.
Khan, S.A. and Iqbal, J. (2000).Polyclonal antibody-
mediated insolubilization and stabilization of papain.
Chaga, G. (1994). A general method for immobilization of
Biotechnology and Applied Biochemistry 32:89-94.
glycoproteins on regenerable immobilized metal ion
carriers: Application to glucose oxidase from pencillium
Kise, H. and Hayakawa, A. (1991). Immobilization of
chrysogenum and horseradish peroxidase. Biotechnology
and Applied Biochemistry 20:43-53.
proteases to porous chitosan beads and their catalysis for
ester and peptide synthesis in organic solvents. Enzyme and
Goldman, R.; Kadam, O.; Silman, I.H.; Caplan, S.R. and
Microbial Technology 13:584-588.
Katchalski, E. (1968). Papain-colloidion membrane. I
preparations and properties. Biochemistry 7:486-500.
Kumakura, M. and Kaetsu, I. (1984). Behaviour of enzyme
activity in immobilized proteases. International Journal of
Goldstein, L.; Pecht, M.; Blumberg, D.A. and Levin, Y.
Biochemistry. 16:1159-1161.
(1970). Water-insoluble enzymes. Synthesis of a new
carrier and its utilization for preparation of insoluble
Kumpel, B.M. and Bakacs, T. (1992). Comparison of lysis
derivatives of papain, trypsin and subtilopeptidase A.
of bromelin and papain treated red cells in ADCC assays.
Biochemistry 9:2322-2333.
Immunology Letters 31:237-240.
4

---

Immobilization and stabilization of papain on chelating sepharose: a metal chelate regenerable carrier
Liu, S. and Hanzlik, R.P. (1993). The contribution on
Zhuang, P. and Butterfield, D.A. (1992). Spin labeling and
intermolecular hydrogen bonding to the kinetic specificity
kinetic studies of a membrane immobilized proteolytic
of papain. Biochemica et Biophysica Acta 1158:264-272.
enzyme. Biotechnology Progress 8:204-210.
Lozano, P.; Cano, J.; Iborra, J.L. and Manjon, A. (1993).
Influence of polyhydroxylic cosolvents on papain
thermostability. Enzyme and Microbial Technology
15:868-873.
Mellor, G.W.; Thomas, E.W.; Topham, C.M. and
Brocklehurst, K. (1993). Ionization characterstics of the
cys-25/His-159 and of the modulatory group of papain:
resolution of ambiguity by electronic perturbation of the
quasi-2 mercaptopyridine leaving group in a new pyrimidyl
disulfide reactivity probe. Biochemical Journal 290:289-
296.
Schnapp, J. and Shalitin, Y. (1976). Immobilization of
enzymes by covalent binding to amine supports via
cyanogens bromide activation. Biochemical and
Biophysical Research Communications 70:8-14.
Storozhuk, V.T.; Shamolina, I.I. and Lobova, A.B. (1985).
Immobilized papain in the treatment of acute destructive
lactation mastitis. Vestn. Khir Im II Grek 135:42-46.
Surinenaite, B.R.; Bendikene, V.G. and Iuodka, B.A.
(1996). Immobilization of enzymes on carriers with
magnetic properties: the search for optimum conditions for
immobilization of alkaline phosphatase from the chicken
graft. Prikl. Biokhim. Mikrobiol. 32:609-614.
Swanson, M.C.; Boiano, J.M.; Galson, S.K.; Grauvogel,
L.W. and Reed, C.E. (1992). Immunochemical
quantification and particle size distribution of airborn
papain in a meat portioning facility. American Industrial
Hygiene Association Journal 53:1-5.
Vernet, T.; Tessier, D.C.; Chatellier, J.; Plouffe, C.; Lee,
T.S.; Thomas, D.Y.; Storer, A.C. and Menard, R. (1995).
Processing of the papain precursor. The ionization state of a
conserved amino acid motif within the proregion
participates in the regulation of intramolecular processing.
Journal of Biological Chemistry 27:16645-16652.
Wadu-Mesthrige, K.; Amro, N.A. and Liu, G.Y. (2000).
Immobilization of proteins on self-assembled monolayers.
Scanning 22:380-388.
Wasserman, B.P.; Jacobson, B.S. and Hultin, H.O. (1981).
Explanation of anamolous binding kinetics with a high
yield immobilized enzyme system. Biochemica et
Biophysica Acta 657:52-57.
Wiseman, A. (1993). Designer enzyme and cell
applications in industry and in environmental monitoring.
Journal of Chemical Technology and Biotechnology 56:3-
13.
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