Comparative Study of the Immobilization of Pancreatin and Papain on Activated Carbon and Alumina, Using Whey as Protein Substrate

World Applied Sciences Journal 2 (3): 175-183, 2007
ISSN 1818-4952
© IDOSI Publications, 2007
Comparative Study of the Immobilization of Pancreatin and Papain on
Activated Carbon and Alumina, Using Whey as Protein Substrate
Viviane D.M. Silva, Letícia M. De Marco, Wendel de O. Afonso,
Daniella C.F. Lopes and Marialice P.C. Silvestre
Departamento de Alimentos, Faculdade de Farmácia,
Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Abstract: Aiming the preparation of low cost dietary supplements, using whey as hydrolysed protein source,
this work involved the immobilization of papain and pancreatin on activated carbon (AC) and alumina (AL) and
the use of these immobilized enzymes to hydrolyze whey proteins. The extent of immobilization (EI) was
determined by the quantification of the non-adsorbed enzyme. The type of support affected the EI of both
enzymes, which were much higher on activated carbon than on alumina. In most cases, the use of 30 min
at 25°C, gave the highest values for EI. Considering the operational stability, AL showed better results than
AC for papain, since the enzyme activity remained unchanged after 15 and 5 times, respectively. The reusability
of pancreatin was 5 and 2 cycles of use on AC and AL, respectively.
Key words: Enzymatic immobilization % immobilization supports % enzymes % proteases
INTRODUCTION
Several approaches have been explored for the
preparation of immobilized enzymes because they have
Proteolytic enzymes have been used in food
shown several advantages over enzymes in bulk
industries [1, 2], for several purposes such as preparation
solution [16]. Thus, immobilization leads to ease of
of protein hydrolysates with dietary applications [3-10].
recoverability and reusability of the enzymes [17]. In
Enzymatic hydrolysates rich in oligopeptides,
fact, for large-scale production, the process would be
especially di- and tripeptides, represents a way of
more economical if the enzyme could be reused, for
improving protein utilization [11]. These preparations
example, by immobilization [18, 19]. Moreover, these
have been used in some countries in the manufacture
systems may increase the stability of enzymes to thermal
of special food for several groups, such as those
treatments and extremes of pH [20].
with protein intolerance or allergy, phenylketonuria,
Other positive aspects of immobilization of enzymes
cystic fibrosis, Crohn´s desease, etc. [3, 5]. Moreover,
on solid supports have been mentioned by Moreno and
these hydrolysates may be useful in the dietetic
Sinisterra [21], such as high concentration and even
supplementation for old people, athletes as well as for
distribution of the enzyme, enhancing its operational
weight control diets [12].
stability. According to Kilinç et al. [22], immobilization
Casein, the main milk protein, has been the first
imparts stability to proteins by restricting the movement
choice as protein source for preparing protein
of the protein molecule by attachment to an inert body
hydrolysates [13, 14]. However, in underdeveloped
via chemical bonds. The various domains are therefore
countries, this protein needs to be imported which
held in the correct orientation to retain activity at least
represents an important increase in production costs.
over an extended period of time when compared with
Thus, the use of alternative sources must be investigated,
enzymes in free solution.
such as whey, whose proteins are ready assimilated by
Immobilization of enzymes has been carried out by
the organism, showing a high protein efficiency ratio [15].
entrapment, ionic interaction, complex formation with
Also, the utilization of whey may contribute to reduce
metal, covalent attachment, encapsulation and adsorption
environmental pollution.
to hydrophobic or hydrophilic surfaces [23]. Among these
Corresponding Author: Marialice P.C. Silvestre, Departamento de Alimentos, Faculdade de Farmácia, UFMG. Avenida Antônio
Carlos, 6627, sala 3070-B3, CEP. 31270-901 - Belo Horizonte, MG, Brasil
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World Appl. Sci J., 2 (3): 175-183, 2007
methods, adsorption has been considered as a simple
MATERIALS AND METHODS
and an economical mechanism for immobilization [24].
Moreover, enzyme immobilization by adsorption has the
L-phenylalanine, L-tyrosine, L-tryptophan, pancreatin
benefit of wide applicability and may provide relatively
(P-1500, from porcine pancreas, activity at least equivalent
small perturbation of the enzymes native structure and
to USP specifications), papain (P-3375, from Carica
function, which contributes to the maintenance of enzyme
papaya, 1.5 - 3.5 units / mg solid), alumina (A-5 type) and
activity [25].
activated carbon (20-60 mesh) were purchased from
Various techniques have been used to evaluate
Sigma (St. Louis, MO, USA). Whey (powder) was kindly
the amount of the adsorbed enzyme [26]. Some are based
furnished by a food producer (Minas Gerais, Brazil).
on measuring the decrease in protein concentration in the
The stirrer was from Fisatom (São Paulo, Brazil) and
solution after the solid surface has come in contact with
the spectrophotometer was CECIL (CE2041, Buck
the solution. For this purpose, some authors [19, 27-29]
Scientific, England).
used the Lowy method [30] and others [18, 23, 31, 32] the

Bradford method [33].
Immobilization process of papain and pancreatin: Papain
Other techniques employ the direct measurement
and pancreatin were immobilized by adsorption on two
of proteins adsorbed on the surface, using more
supports: activated carbon (AC) and alumina (AL).
sophisticated methods such as enzyme-linked
Volumes of 10 mL (papain) and 50 mL (pancreatin) of 0.1%
immunosorbent,
radiolabeling, quartz crystal
(w/v) enzymatic solutions in phosphate buffer 0.1 mol/L,
microbalance, ellipsometry, total internal reflection
pH 7.5 and 10 g of the support were placed in a beaker.
fluorescence and neutron reflection [26].
After 30 min, 60 min and 90 min of contact at 25°C as well
Our interest in immobilizing papain and pancreatin
as 12 h at 5°C, the mixtures were centrifuged at 11.000 x g
is associated with their reuse in the process of
for 20 min, at 25°C (Centrifuge Jouan, BR4i model, France).
hydrolysing proteins to produce dietary supplements
Blanks were prepared using only phosphate buffer.
rich in oligopeptides with various clinical applications.
The action of papain [7, 34] and pancreatin [35-37] in a
Determination of the extent of immobilization of the
bulk solution has already been tested in our laboratory
enzymes: The enzyme:support complexes were filtered
and found to be efficient in preparing protein
through quantitative paper under vacuum and the
hydrolysates with high oligopeptide content.
residues were washed three times with 50 mL of distilled
Second derivative spectrophotometry (SDS) was
water. The filtrates and the waste waters were collected
chosen for estimating the operational stability of
for the determination of the unabsorbed protein content,
these enzymes, because our group has been testing
by the Lowry method [30, 46], using bovine serum
this technique for several purposes for many years
albumin as a standard.
ago. Thus, we used successfully SDS for determining
The same method was used for protein quantification
the hydrolysis degree of protein hydrolysates [38],
in the 0.1% enzymatic solution. The amount of
for evaluating the encapsulation rate of protein
immobilized enzyme was obtained by the difference
hydrolysates [39], as well as for estimating the Phe
between the protein content of the enzymatic solution
removal from skim milk [8, 10] and whey hydrolysates [40].
and that found for the sum of the filtrates and waste
Although several supports have been tested for
waters. The extent of immobilization was calculated by
immobilizing papain [16, 17, 22, 41, 42], no work was found
the equation bellow:
in the literature about the use of activated carbon or
Amount of
alumina for this purpose. Concerning pancreatin, some
immobilized enzyme
reports describe the immobilization of some constituents
% of immobilized enzyme =
100
×
Amount of
of this enzyme [43-45], but no work with the whole enzyme
added enzyme
was found.
In the present work, we studied the immobilization of
Determination of the operational stability of immobilized
a papain and a pancreatin on these two supports, aiming
enzymes: The reusability of papain and pancreatin was
for preparation of low cost dietary supplements, using
evaluated by measuring the residual activity of the
whey as a hydrolysed protein source.
enzymes after 20 cycles o f use. The quantification of
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World Appl. Sci J., 2 (3): 175-183, 2007
0.16
50E-07
B
0.14
a
0.12
b
0
Absorbance
0.10
Absorbance
c
0.08
d
-50E-07
A
0.
0.04
255
260
265
270
275
255
260
265
270
275
Wavelength (nm)
Wavelength (nm)
0.7
40E-06

a
D
C
0.6
20E-06
b
0.5
0
c
Absorbance
Absorbance
d

0.4
-20E-06

-40E-
0.3
255
260
06
265
270
275
255
260
265
270
275
Wavelength (nm)
Wavelength (nm)
Fig. 1: Absorbance and second derivative spectra of Phe in a solution containing Tyr and Trp (A and B, respectively)
and of hydrolysate obtained after the second hydrolysis (C and D, respectively), in pH 6.0
phenylalanine (Phe) exposed by the enzymatic hydrolysis
absorbance of the samples was measured from 250 to
of whey proteins was used for measuring enzyme activity.
280 nm. Second derivative spectra were drawn (CECIL
As described before by our group [47], this corresponds
spectrophotometer, CE2041 model, Buck Scientific,
to the exposure rate (ER) of Phe (see bellow), which may
England) and the area of the third negative peak “c”
be measured by second derivative spectrophotometry
(Figure 1) was used to calculate the amount of Phe in the
(SDS).
samples, employing a standard curve. A software
First, the enzymes were immobilized, as described
GRAMS-UV (Galactic Industries Corporation, Salem, NH,
above, using the condition that produced the highest
EUA) was used to draw the second derivative spectra.
extent of immobilization. Then, appropriate volumes of
For a standard curve, stock solutions of Phe
a 10% (w/v) whey solution were added to the enzyme:
(6.05 x 10-4 mol/L), Tyr (5.52 x 10




-4 mol/L) and Trp
support complexes in order to obtain an enzyme: substrate
(4.90 x 10 mol/L) were prepared in 0.
-4
01 mol/L phosphate
ratio (E:S) of 1%. Then, the mixtures were stirred for 1 h,
buffer, pH 6.0. Then, 10 mL of each solution were
at 25°C, at a velocity sufficient to keep the complex in
mixed and successive dilutions of this mixture were
suspension. Then, they were centrifuged at 11.000 x g for
made so as to have Phe concentrations in a range from
20 min, at 25°C and filtered through quantitative paper
0.13 to 1.01 x 10 mol/L. Spectra of these diluted solut
-4
ions
under vacuum. The residues were washed three times
were recorded from 250 to 280 nm and the area of the
with 5.0 mL of distilled water and submitted to successive
third negative peak of Phe was used to draw the standard
hydrolysis giving rise to six hydrolysates, for each
curve as a function of its concentration.
enzyme, corresponding to the first, second, fifth, 10 , 15
th
th
and 20 utilizations of the immobilized enzymes. Th
th
e
Statistical analysis: All experiments were carried out in
hydrolysates were then, freeze-dryed (Labconco, 77500
triplicate. Differences between means of areas of negative
model, Kansas City, MI, USA) and submitted to the SDS,
peaks were evaluated by analysis of variance (ANOVA)
as described bellow.
and Duncan test [48]. Differences were considered to be
significant at p<0.05 throughout this study. The least
Determination of exposure rate of phenylalanine: The
square method was used to fit the standard curve and the
exposure of phenylalanine was measured by SDS, as
adequacy of the linear model (Y = aX + b) was tested at
previously described by our group [8, 47]. Briefly, the
p<0.05.
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World Appl. Sci J., 2 (3): 175-183, 2007
RESULTS AND DISCUSSION
Considering that no report about the immobilization
of papain on either activated carbon or alumina was
Extent of immobilization of papain and pancreatin: The
found in the literature, the data obtained in the present
data in Table 1 show that the type of support affected the
work were compared to those using other supports.
extent of immobilization of both enzymes, which were
Four studies were found relating to hydrophilic
much higher for activated carbon (AC) than for alumina
supports. Thus, working with silica and using different
(AL). This result could be explained by the fact that
conditions of pH, the extent of immobilization varied
proteins having high structural stability, called hard
from 13% to 16%, near to the values found here for
proteins, are barely adsorbed on hydrophilic supports,
alumina [50]. For other supports, the results were higher
such as alumina [26, 49]. Papain could be considered as
than those of the present work with alumina. This was
a hard protein, since it has low molecular mass, high
the case of zirconia, a kind of crystalline silica, for which
thermal stability and three disulfide cross linkages.
the extent of immobilization ranged from 30% to 80% [50].
Pancreatin could also fit this category, since one of its
Papain was immobilized on chitosan, a hydrophylecand
main components, chymotrypsin, is considered a hard
polycationic polysaccharide, at a pH near 7.0 and showed
protein [1, 26].
36% of immobilization [22]. The use of sepharose,
When AC was used as the support for papain, no
activated by cupric ions, for immobilizing papain reached
significant difference was observed with respect to extent
an extensively high immobilization level, i.e. 96%,
of binding after 30 min and 60 min at 25°C, as well as 12 h
according to Afaq and Iqbal [16].
at 5°C, which were all barely superior (97 to 99%) to 90 min
Only one report was found concerning hydrophobic
at 25°C (95%). The best results for binding with AL were
supports. Thus, Zhuang et al. [17], using a polyvinyl
obtained after 30 min at 25°C and 12 h at 5°C, which were
membrane, obtained an immobilization level varying
similar (13%). Thus, for both supports, 30 min, at 25°C,
from 4% to 13%, which is much lower that of the present
was chosen for evaluating hydrolysis efficiency since it
work with activated carbon.
employed the shortest time which is advantageous from
No report was found in the literature concerning the
an economical point of view.
immobilization of pancreatin on solid supports. Some
Also, the efficiency of pancreatin immobilization on
work has been performed with the enzymes which
AC was much greater than on AL, as shown by the values
compose the pancreatin, such as trypsin, chymotrypsin
of its extent of immobilization in Table 1. Around 100% of
and carboxypeptidade A. In one of these studies, trypsin
this enzyme was immobilized on AC, during 30 min, at
was immobilized by adsorption on a polymer (Eudragit
25°C and 96% was immobilized when using the two other
S-100, 2% and 4%) and 99% of the enzyme was linked to
conditions. With AL, the immobilization efficiency of
the support [43]. This result is close to that obtained in
this enzyme changed from 11% (60 min, 25°C) to 37%
this work for pancreatin,when using AC as the support.
(90 min, 25°C).
Vanková et al. [45] immobilized "-chymotrypsin from
Table 1: Immobilization levels of papain and pancreatin on activated carbon and alumina
Immobilization supports
-----------------------------------------------------------------------------------------------------------------------------------------------------
Activated Carbon
Alumina
-----------------------------------------------------------------------------------------------------------------------------------------------------
EI * (%)
EI * (%)
Immobilization
----------------------------------------------------------
----------------------------------------------------------
parameters
Papain
Pancreatin
Papain
Pancreatin
30 min / 25°C
97a,1,y
100a,1,x
13a,2,y
17b,2,x
60 min / 25°C
99a,1,x
96

b,1,x
10b,2,x
11c,2,x
90 min / 25°C
95b,1,x
96

b,1,x
4c,2,y
37a,2,x
12 h / 5 °C
98a,1,x
99a,1,x
13a,2,x
8d,2,y
*EI = extent of immobilization. Each value represents the mean of triple determination. For each enzyme, different letters (a,b) are significantly different
(p < 0.05) for different treatments and the same support. For each enzyme, different numbers are significantly different (p < 0.05) for different supports and
submitted to the same treatment. For different enzymes, different letters (x,y) are significantly different (p < 0.05) for different supports and submitted to the
same treatment
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World Appl. Sci J., 2 (3): 175-183, 2007
Table 2: Operational stability of immobilized papain and pancreatin on activated carbon and alumina
Immobilization supports
Activated carbon
Alumina
----------------------------------------------------------------------------
-------------------------------------------------------------------------
Enzyme activity
Variation of Enzyme
Enzyme activity
Variation of Enzyme
(Exposure rate of Phe)*
activity, (%)
(Exposure rate of Phe)*
activity, (%)
Number of times of
----------------------------------
-----------------------------------
----------------------------------
-------------------------------
utilization of the enzyme
Papain
Pancreatin
Papain
Pancreatin
Papain
Pancreatin
Papain
Pancreatin
1
75a,2,x
59 a,2,y
100a,1,x
100ª,1,x
124a,1,y
176 a,1,x
100a,1,x
100ª,1,x
2
72a,2,x
59 a,2,y
100a,1,x
100ª,1,x
122a,1,y
180 a,1,x
100a,1,x
100ª,1,x
5
72a,2,x
62 a,2,y
100a,1,x
100ª,1,x
120a,1,y
143 b,1,x
100a,1,x
82b,2,y
10
58b,2,x
50 b,2,y
82b,2,x
83b,1,x
118a,1,y
153 b,1,x
100ª,1,x
82b,1y
15
40c,2,x
36 d,2,y
55c,2,y
65c,2,x
116a,1,y
142 b,1,x
100ª,1,x
82b,1,y
20
38d,2,y
42 c,2,x
54c,2,y
65c,1,x
85b,1,y
119 c,1,x
74b,1,x
67c,1,y
Exposure rate of Phe, in mg of Phe/100g of hydrolysate. Variation of enzyme activity: the highest values represent 100% of the enzyme activity. Each value
represents the mean of triple determination. For each enzyme, different letters (a,b) are significantly different (p < 0.05) for different number of times of utilization
of the enzyme using the same support. For each enzyme, different numbers are significantly different (p < 0.05) for different supports and submitted to the same
number of times of utilization of the enzyme. For different enzymes, different letters (x,y) are significantly different (p < 0.05) for the same support and submitted
to different number of times of utilization of the enzyme
bovine pancreas on two supports containing saccharide
Thus, five peaks for this amino acid in pH 7.0 were
residues and the best immobilization level was 30% using
shown by Ichikawa and Terada [51], while Miclo et al. [52]
the "-galactosyl-polyacrilamida as support. This result
described the presence of six, in pH 1.9. These
was similar to just one value obtained in the present work
discrepancies could be associated to several factors
for pancreatin with AL (90 min, 25°C) and much less
such as forms of Phe (free or ester), type of equipment
inferior to those obtained here with AC. The extent of
(spectrophotometer and software used for measuring
immobilization of carboxypeptidade A on polyethylene
absorption and derivative spectra), properties of the
terephtalate was around 70%, i.e., lower than those
solvent and pH used [51, 53].
obtained here for pancreatin with CA [44].
Concerning the standard curve of Phe, the one
using the area of negative peak “c” presented the best
Second Derivative Spectrophotometry Spectra of
correlation coefficient (the highest and most significant).
phenylalanine and enzymatic hydrolysates: The
The curve equation and the correlation coefficient
absorbance and SDS spectra of Phe, in a mixture of
were y = 3.0077x + 0.7587 e R = 0.9576. This result is in
2
aromatic amino acids and of the hydrolysate obtained
agreement with others in the literature [51, 54] and
after the second hydrolysis, in pH 6.0, are shown in
also with previous studies carried out in our laboratory
Fig. 1. In case of Phe (Fig. 1b), we can see four negative
[8, 10, 47], since in all these studies a linearity for the
peaks, indicated by letters “a”, “b”, “c” and “d”, situated
standard curve of Phe, in the presence of Tyr and Trp in
within the range of 250 to 280 nm with maxima at 253, 258,
several concentrations, was shown.
263, 268 and 273 nm and minima at 257, 262, 267 and
272 nm.
Operational stability of immobilized enzymes: As shown
The SDS spectrum of the hydrolysate is close to that
in Table 2, the activities of papain and pancreatin were
of Phe, with negative peaks situated in almost the same
much higher on AL than AC. Also, one can see in this
wavelengths. The likeness among the spectra of standard
table that, using AC as a support, the activity of papain
amino acids and proteins has previously been described
was greater than pancreatin up to 15 times of enzyme use.
by Ichikawa and Terada [51], working with several native
Only at 20 cycles of use, did the activity of papain show
and denaturated proteins. The same result was previously
a lower value than pancreatin. The activities of these
achieved in our laboratory using different enzymes for
enzymes were constant at 100% up to five cycles of use
hydrolysing casein and skim milk [8, 10, 47].
(around 71 and 61 mg Phe/100g of hydrolysate, for papain
In some reports, the number of negative peaks for
and pancreatin, respectively). With 10 cycles of use, there
Phe was different from that found in the present work.
was a decrease which was smaller for pancreatin than for
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World Appl. Sci J., 2 (3): 175-183, 2007
papain. It is worth stating that even after using these
studies must be done in order to explain all the results
enzymes for 20 cycles, their activities were 54% and
obtained here. These results show clearly that the
65% of the initial values, respectively, which may be
immobilization of these enzymes on activated carbon
considered good, compared to other data in the literature
and alumina may be considered when the interest is
[41, 42].
associated to the use of such enzymes in large scale
In contrast, using AL as support, the activity of
industrial processes.
pancreatin was higher than that of papain up 20 times
enzyme use (Table 2). On the other hand, the activity of
CONCLUSIONS
papain remained stable at 100% until 15 cycles of use,
while for pancreatin this behavior persisted only through
A activated carbon was more efficient than alumina
two cycles of enzyme use. Similarly to AC, the final
for immobilizing pancreatin and papain, yielded a very
activities of these enzymes (after 20 cycles of enzyme
high level immobilization of 96-100% and 95-99%,
use) were still high, i. e., 74% and 67% of the initial values,
respectively. In the test for hydrolyzing whey, this
for papain and pancreatin, respectively.
support was also better than alumina for immobilizing
Although Whitaker [1] states that immobilized
pancreatin, since it could be used for up five times with
enzymes may be reused up to 50% of their initial activity,
no change in enzyme activity. However, with papain, the
in our case it would be important to keep their activity
alumina showed better results than activated carbon,
near 100%, because our interest is associated with
since enzyme activity remained unchanged after 15 cycles
hydrolyzing proteins in order to remove phenylalanine
of use.
(Phe)
and prepare dietetic supplements for
phenylketonurics, since the more the protein is
ACKNOWLEDGEMENTS
hydrolyzed the higher the Phe exposure and the easier
will be the expected removal of this amino acid.
The authors thank CNPq and FAPEMIG for their
No report concerning the operational stability of
financial support.
papain in either activated carbon or alumina was found
in the literature. The data using other supports were lower
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