Indian Journal of Fihrc & Tcxtilc Rcscarch
Vol. �l). MardI �OO). PI'. 7)-X I
Immobilisation of amylase by various techniques
S R Shukla" & Lalil Jajpura
Matunga. Mumbai 40() O I l)
Dcpartmcnt of rihrcs and Tcxtiic Proccssing Tcchnology. Institutc of Chcmical Tcchnology. Univcrsity of Mumhai.
/?ecein'd 2(j Septell/ber 20W: 1I('('epted 23 April 2()().J
alginatc gcl clltrapmcnt tcchniquc was uscd for thc cnzyme cntrapmcnl. Nylon 6 hcads and kniltcd fahric wcrc thc supports
Thc enzymc u-amylasc was subjccted to immobilisation by entrapment and covalent binding mcthods. Calcium
choscn for thc covalcnt bonding tcchniquc using glutaraldchydc with and without chitosan. Thc charaL·tcristics of thc
immobiliscd cnzyme havc hccn discusscd. Covaicnt bonding gavc bcltcr stability and rcusability of thc immohiliscd cnzymc
than thc calcium alginatc bead cntrapmenl.
Keywords: u-Amylasc. Calcium alginatc. Enzymc immobilisation. Glutaraldchyde. Nylon (,
fPC Code: Inl. CI. f)06B I WOO
7
1 Introduction
The textile wet processing industry is one of the
maj or contributors of pollution to the global
environment. particularly through effluent generation
of diverse characteristics. The cotton fabric pre­
treatment undergoes desizing to remove added starch.
scouring to remove naturally present impurities and
bleaching to make the fabric white. The conventional
chemicals used for these purposes contribute
substantially to the chemical and biological load of
the eftluent. It is. therefore. desirable to replace them
by the ecofriendly alternatives and in this respect. the
enzymes play a key role.
In recent years, the interest in the use of enzymes
in textile processing has increased many folds. The
mostly used enzymes 1·1 are et-amylase for desizing of
starch-sized cotton, catalase as a peroxide neutraliser
after bleaching. hemicellulase and pectinase for
relting of hast fibres. lipase for scouring. and cellulase
and protease for denim washing. biopolishing. etc.
Although the enzymcs are ccofriendly in nature, the
enzyme treatments are sometimes much costlier
compared to the conventional chemical treatments.
After completion of a desircd reaction, the enzymc is
discarded as is the case with any other chemicals used
for treatments. The activity of an enzyme, howcver, is
Phonc: 2-11-15616: Fax:
+ <)1-22-2-11-1561-1:
"To whom all tht: cOITt:spondcncc should bc addrcsscd.
E-mail: sanjccl'[email protected]()m
scarcely diminished during reactions carried out under
optimum conditions. Draining away the enzyme IS
thus a wasteful practice.
Reusing the enzyme several times is a possible
remedy to ovcrcome this problem. The increase in the
concentration of product, byproduct and other
impurities in the same treatment bath. however. cause
a drastic decrease in the enzyme activity.
An immohilised enzyme may simply be rccovcred
at the time of drainage of the reaction waste products
and be reused in fresh treatment bath in a batch-wise
manner. In a continuous flow rcactor containing
immohilised enzyme. the substrate may be fed
continuously,
the
product
being
collected
simultaneously. Enzyme may thus be recovered and
reused. greatly improving the economy of the
operation. Enzyme can be immobilised by various
techniques,
such
as
adsorption,
entrapment.
-l s
microencapsulation and covalent bonding · .
Before weaving a cotton fahric. the starch sill' is
applied to the warp threads to strengthen them to
sustain mechanical adversities. Removal of this starch
through desizing operation after weaving is essential
to impart absorbency for further wet processing. The
enzyme a-amylase catalyses the hydrolysis of 1.4
linked D-glucose units of starch size to produce
maltose and larger oligosaccharides. which are
.
<)·11
�
so I uble In water
.
Commonly used cntrapment mcdia for enzyme
immobilisation are polyacrylamides, calcium alginate
76
INDIAN J. FIBRE TEXT. RES., MARCH 200S
and gelatine. In all the protocols, enzymes are well
mixed with monomers/polymers and cross-linking
agents in a solution. The solution is then exposed to
polymerisation promoters to start the process of gel
formation and moulded into the desired shape and
size. Spherical beads, whose size can be controlled
conveniently, are generally prepared and then
4
hardened to enhance structural integrity I2-1 .
Enzymes have been covalently bound to insoluble
cellulose derivatives by various methods. The enzyme
must, of course, be linked at some distance from the
active site of the enzyme. Such i mmobilised enzymes
retain their activities, although their immobility may
reduce the reaction rate. The anionic or cationic
nature of the carrier may alter the pH optimum for the
reaction. The binding of the enzyme to carrier may
result in steric hindrance and impose restrictions on
its specificity. This is most apparent where the
substrate is a large molecule, such as protein, rather
IS.
than a small molecule like a peptide
Nylon is a particularly convenient immobilisation
matrix; it is inexpensive, inert, non-toxic, readily
available and can be obtained in a number of forms. The
earliest practical procedure involved partial acid
hydrolysis of the nylon surface to generate more number
of amino and carboxyl end groups, which could be
coupled to proteins with glutaraldehyde. The enzyme
can be immobilised onto nylon support with
glutaraldehyde crosslinking using chitosan as a spacerl619. It is an unbranched 13 ( 1 � 4)-linked polymer of
2-acetamido-2-deoxY-D-glucose
(N-acetyl-D-glucose­
amine), and chitosan is a collective name given to a
group of N-deacetylated chitin derivatives 20.21 .
In the present work, alginate salt was used for
entrapment of the enzyme a-amylase, and nylon in the
form of beads and knitted fabric was used for its
immobilisation by coupling with glutaraldehyde in
absence and presence of chitosan as spacer molecule.
The optimisation and stability studies for the entrapped
or immobilised amylase towards pH and temperature
were carried out. The activity was measured by
estimating the amount of reducing groups formed on
reaction with starch using DNSA reagent22.
2 Materials and Methods
2.1 Materials
Aquazyme Ultra 250 L (Novo Nordisk), an a­
amylase, supplied by Zytex Pvt. Ltd, Mumbai, was
used for immobilisation.
Chitin, used for preparation of chitosan, was
supplied by S.D. Fine Chemicals Ltd. Soluble starch,
supplied by Loba Chemie, was used as the substrate
for reaction with the enzyme.
2.2 Methods
2.2.1 Preparation of 3, 5-Dinitro Salicylic Acid (DNSA) Reagent
The DNSA reagent was prepared by dissolving
1 0 g DNSA, 1 0 g NaOH, 200 g rochelle salt, 0.5 g
sodium metabisulphite and 2 g phenol in 1 litre
distilled water.
2.2.2 Determination of Enzyme Activity by DNSA Method
After enzymatic reaction with starch, the glucos�
formed was measured, for which 2 m1 of the
incubated solution and 2m! DNSA reagent were taken
in a test tube, stoppered with rubber plug, kept for 1 0
min in a water bath at boil and then diluted to 2 4 ml
with distilled water. The -N02 group of DNSA was
reduced to -NH2 group by glucose, which was formed
by the enzymatic reaction with starch. The absorbance
of the red colour formed was measured at 540 nm on
UV-Visible spectrophotometer by Techcomp 8500.
The quantity of glucose formed due to enzyme
reaction was then estimated by comparing this
absorbance value with the known absorbance on the
already prepared standard glucose curve.
2.2.3 Enzyme Entrapment by Calcium Alginate Gel
The solutions of sodium alginate (40 gIL) and the
enzyme Aquazyme Ultra 250 L (0.5mllL) were
prepared in distilled water and mixed together in
equal volumes. The mixture was extruded drop wise
through a 25 ml pipette into a beaker containing 1 00
mM calcium chloride solution from a height of 7.5 cm
(Fig. 1 ).
Spherical calcium alginate gel beads were formed
having entrapped enzyme. These were left to harden
Mixture
of Enzyme
and
Sodium A lginat e Solution
•
• •
•
•
Calcium Chloride Solution
• • • &..���--
•••
•
Calcium Alginate
Beads with Entrapped
Enzyme
Fig. I - Preparation of enzyme entrapped in calcium alginate
bead
SHUKLA & JAJPURA: IMMOBILISATION OF AMYLASE BY VARIOUS TECHNIQUES
in calcium chloride solution for 3 h, washed twice
with the same solution and stored in a refrigerator.
These were subsequently used for the starch
hydrolysis reaction.
Nylon 6 knitted fabric and beads of 2 mrn diameter
were used as an enzyme support for immobilization
through covalent bonding.
2.2.41mmobilisation ofAmylase Enzyme on Nylon 6 Support
2.2.4. 1
Partial Hydrolysis of Nylon
Pieces measuring - 10mm xl 0 mm were cut from
the knitted nylon fabric. For hydrolysis of the amide
bonds, the nylon beads and fabric pieces were placed
in a flask fitted with a magnetic stirrer and then
. incubated in 2.9 M hydrochloric acid for 2 h at room
temperature (2S°C). The reaction was terminated by
thorough washing with water and then with 0. 1 M
sodium phosphate buffer of pH 8.0.
2.2.4.2 Activation
of Nylon Beads and Knitted Fabric
The amino groups of nylon support were activated
by placing the samples in 2.S% glutaraldehyde
solution in 0. 1 M sodium phosphate buffer of pH 8.0
with material-to-liquor ratio 1 : 10. The reaction was
carried out at 2SoC for IS min. Unreacted
glutaraldehyde was removed by washing with water
and then with buffer of pH 8.0.
2.2.4.3
Synthesis of Chitosan
Chitosan was prepared by refluxing 2S g chitin
powder with ISO g of SO% NaOH solution in 1 litre
three-necked flask fitted with a water condenser and a
mechanical stirrer. The mixture was refluxed for 4 h
at 120°C and the completion of reaction was detected
by complete solubility of the product in dilute acetic
acid. The reaction mixture was cooled to room
temperature and the product was filtered and washed
with water till it was neutral. Final washing was given
with ethanol to get crude chitosan.
Further purification was done by dissolving
chitosan in 400 ml of 2% HCl. The mixture was well
stirred and filtered. Filtrate was made slightly alkaline
by gradual addition of sodium hydroxide solution
with constant stirring until a precipitate was obtained.
The precipitate was isolated by filtration, washed and
dried at 40°C. It was further dried in a desiccator and
then powdered. The powder was passed through 80
mesh sieve.
2.2.4.4
Bonding of Spacer Molecule
The chitosan spacer molecules were attached to the
nylon beads and fabric by treatment with O.S % (w/v)
77
chitosan dissolved in 0. 1 M sodium phosphate buffer
for 3 h at 2SoC using material-to-Iiquor ratio of 1 : 10.
This was followed by thorough washing with water
and then with buffer of pH 8.0.
2.2.4.5
lmmobilisation after Activation of Nylon
The chitosan-treated nylon supports were
reactivated by 2.S % glutaraldehyde using the
procedure mentioned in section 2.2.4.2. These samples
were then incubated in S.O % enzyme solution at 4°C
for 24 h with material-to-Iiquor ratio of 1 : IS. The
enzyme in 0. 1 M sodium phosphate pH 8.0 buffer
should be as concentrated as conveniently possible
(ideally 1 .0 mg/ml or more) but with prolonged time
(24 h or more), even the solution as dilute as 0. 1
mg/ml may give satisfactory levels of binding .
Thereafter, the supports were thoroughly washed with
water followed by pH 8.0 buffer solution and then
with 1 M sodium chloride solution in 0. 1 M sodium
phosphate buffer. These nylon supports, immobilised
with enzyme, were stored at pH 8.0 and temperature
SoC in a refrigerator.
2.2.5 Determination ofActivity of Immobilised Enzyme
2.2.5.1 Activity Determination of Entrapped Enzyme
Five ml of reaction mixture containing 0:8 ml of
2 % starch solution along with different arnounts of
CaCh buffered at pH 4.2 was added to a test tube
containing accurately weighted (- O.S g) entrapped
enzyme beads. After an incubation period of 30 min
at SOOC, the reaction mixture was collected and the
aliquot was estimated for the reducing sugar produced
by DNSA method to determine the activity of the
immobilised enzyme.
The effect of calcium chloride on the activity of
calcium alginate entrapped enzyme was studied by
taking out 4.2 ml of different concentrations of
calcium chloride in S rnl of reaction mixture. The
beads were stored in varying concentrations of
calcium chloride for 2, 1 2 and 24 h and the stability of
entrapped enzyme was measured by the method
discussed earlier.
2.2.5.2
Activity Detennination of Covalently Immobilised Enzyme
on Nylon
The activity of the enzyme immobilised on nylon
support was determined by adding S ml of reaction
mixture containing 0.8 ml of 2 % starch solution and
4.2 ml of different buffer solutions in test tubes
containing accurately weighted (- 0.3 g) immobilised
enzyme nylon beads and fabric. After an incubation
period of 30 min at SOOC, the reaction mixture was
collected and the aliquots were estimated for the
78
INDIAN 1. FIBRE TEXT. RES., MARCH 2005
reducing sugar produced by DNSA method. The
effect of time on reaction of immobilised enzyme and
of starch concentration was also studied.
2.2.6 Determination of Optimum pH of Immobilised Enzyme
In the experimental work, acetate, phosphate and
glycine-sodium hydroxide buffer solutions were used
respectively for pH ranges 3.5 - 5.5, 6.0 - 8.0 and 9.0 1 0.0. Optimum pH for the activity of immobilised
enzyme was determined by adding 5 ml of reaction
mixture containing 0.8 ml of the starch solution and
4.2 ml of the appropriate buffer in a test tube
containing the immobilised enzyme. After an
incubation period of 30 min at 50oe, the reaction
mixture was collected and the aliquots were estimated
for the reducing sugars by DNSA method.
2.2.7 Determination ofpH Stability ofImmobilised Enzyme
The immobilised enzyme samples were incubated
at 250e for 24 h in 5 ml of 0.1 M buffer solutions at
the appropriate pH. Residual activity in the support
was then determined as described in section 2.2.5.2.
2.2.8 Determination of Optimum Temperature of Immobilised
Enzyme
Activity of the enzyme immobilised onto the nylon
support was estimated at various temperatures ranging
from 300e to 900e at lOoe interval. The assay was
carried out as described in section 2.2.5.2.
2.2.9 Determination ofThermostability ofImmobilised Enzyme
The temperature stability of the immobilised
enzyme was determined at sOe and over a temperature
range of 30 - 900e at lOoe interval. Enzyme
immobilised support was incubated in S ml of pH 8
buffer solution at the appropriate temperature for 2 h.
After the incubation period was over, the support was
removed and the residual activity of the immobilised
enzyme was determined as described earlier in
section 2.2.5.2.
2.2.10 Recycling EffICiency ofImmobilised Enzyme
Immobilised enzyme was added to S ml of mixture
containing 0.8 ml starch solution (2%) and 4.2 ml of
pH 8.0 phosphate buffer for 30 min at sooe. Reaction
mixture was then decanted and reducing sugars
estimated using DNSA reagent. After washing the
once used immobilised enzyme support with distilled
water and pH 8.0 buffer solution, it was reused in
fresh batches of substrate under similar conditions.
Amax of S40 nm. Correlation coefficient and standard
error were found to be 0.9987 and 0.0196
respectively. Activity of the enzyme was thus gauged
indirectly by measuring the change in absorbance of
DNSA solution.
The amylase enzyme was entrapped in calcium
alginate gel. It was observed that with the increase in
time and starch concentration, the absorbance
increased. After the completion of reaction, the
calcium alginate beads got swollen and after
prolonged reaction time the beads got cracked,
making them unsuitable for any further reaction
involving reuse of the enzyme entrapped in these
beads.
Since the alginate beads were found to be stable in
calcium chloride solution, the reaction with starch
was carried out in it. Fig. 2 shows the effect of
concentration of calcium chloride solution on the
activity of enzyme entrapped in the freshly prepared
beads. When the reaction was carried out in 0.25%
calcium chloride solution, the activity of entrapped
enzyme was found to be maximum, which decreased
with the further increase in calcium chloride
concentration in the reaction mixture. This may be
due to increased hardening of beads, which reduces
the accessibility of starch molecules to the entrapped
enzyme and lor reduces the leaching out of the
enzyme molecules to react with starch. The absence
of calcium chloride in the reaction mixture gave less
activity of the enzyme.
The activity of entrapped enzyme was then
checked by keeping the beads in calcium chloride
solution of different concentrations for 24 h (Fig. 3).
2.0
.-------�
1.6
1.2
0.6
0.4
.0
o
L-__�__�___�__�__�
0.5
3 Results and Discussion
Good linear correlation between the glucose
concentration and the absorbance was observed at
Fig. 2
-
CaCl,
1.0
(%)
1.5
cone.
2.0
2.5
Effect of CaCl2 concentration on entrapped enzyme
reaction
79
SHUKLA & JAJPURA: IMMOBILISATION OF AMYLASE BY VARIOUS TECHNIQUES
It may be observed that the maximum activity of the
entrapped enzyme was for 2 h storage in 0.25%
calcium chloride solution and it decreased with
prolonged duration. This may be attributed to the
leaching out of some of the enzyme from the beads
since they got swollen and became more open.
Fig. 4 indicates that for the first use, the maximum
activity was observed for the beads reacted with
starch in water alone. Here, the beads were used as
such without washing from the stock stored in
calcium chloride solution for a few days. As
compared to the beads reacted with starch in presence
of different concentrations of calcium chloride, the
ones in virtual absence of calcium chloride (a very
little calcium chloride comes from the beads used
without washing) allows most of the enzyme to leach
out and react. In second use, therefore, it may be
observed that very little activity, the lowest among all,
was available for reaction. In all other cases, with the
increasing amount of calcium chloride in solution, the
beads hardened to more extent, thereby causing
hindrance either to leaching out of the enzyme or to
the accessibility of starch molecules towards enzyme.
Thus, in 2% calcium chloride solution, lowest activity
was observed for first use and it showed least
decrease among all the cases, keeping the enzyme
well entrapped and inaccessible till the beads cracked
at the fifth reuse.
a-amylase was immobilised by covalent bonding
on nylon beads and fabric through glutaraldehyde
activation. Chitosan was used as a spacer. The results
1.0 r-------�
on pH optimisation of the enzyme reaction are shown
in Fig. 5. The maximum activity of the immobilised
enzyme was observed at pH 6.0 and by using
chitosan, it was found to be little higher. The enzyme
immobilised on nylon fabric by using chitosan (nylon
fabric-GA-Chi-GA-Enz) when reacted with starch in
pH 6.0 buffer showed activity about 1 8% more than
that observed without using chitosan (nylon fabric­
GA-Enz), whereas in the case of beads, nylon bead­
GA-Chi-GA-Enz gave activity more by about 70% as
1.4 �-------...
1.2
1.0
Q)
(,)
c:::
ro
.c
...
«
0
I/)
.c
0.6
O�-L---�----�___-L�
2
Reuse Cycle
�
0
4
0.9
0.8
f!
c:
�
0.5
B
A
6
B
1.0
1.5
CaCI2 cone. (%)
0.6
-e
0
., 0.4
�
0.4
O
3
Fig. 4 -Reusability of entrapped enzyme at different CaCI2
concentrations [ __ 0 %, -e 0.25 %, �
0.50 %, ......
1.00 %, -.-1 .50 %, and-- 2.00 %]
l\I
0.2
0 .4
0.7
-e
0
I/)
�
<{
0.6
0.2
0.8
Q)
()
c:
l\I
0.8
<{
t-
El
0.5
0.3
0.2
0.1
2.0
2.5
Fig. 3- Effect of CaCI2 storage time on entrapped enzyme
stability [ --e- after 2h, -e- after 1 2 h, and -A- after 24 h]
a
G3
4
�
5
6
pH
e
7
B
9
10
Fig. 5 - Effect of pH on nylon-immobilised enzyme activity
[ --- Nylon fabric-GA-Chi-GA-Enz,-e- Nylon fabric-GA-Enz,
-Nylon bead-GA-Chi-GA-Enz, and -e- Nylon bead -GA-Enz]
80
INDIAN J. FIBRE TEXT. RES., MARCH 2005
compared to nylon bead-GA-Enz. The fabric support
already has more open structure and available surface
area as compared to the beads, causing higher level of
bonding with the enzyme. The effect of enhancement
of enzyme activity on fabric by using chitosan was,
therefore, less pronounced as compared to that
observed for the beads.
The maximum stability of the enzyme immobilised
on nylon support was observed in the buffer solution
of pH 6-7 (Fig. 6). Towards acidic pH, the enzyme
stability decreased, although not as severely as in the
case of free enzyme. The fabric support also showed
significant decrease in the stability, since the enzyme
is more accessible than in the case of beads.
The results on effect of temperature (Fig. 7)
indicate that the maximum activity of the immobilised
enzyme was observed at 70°C and with the increase in
temperature it decreased, although slightly as
compared to that of the free enzyme.
Fig. 8 shows the comparative stability of the free
enzyme stored in distilled water and calcium chloride
solution and that of the enzyme immobilised on nylon
supports. The maximum stability was observed for
enzyme immobilised on nylon beads and minimum
for free enzyme stored without calcium chloride. The
order of temperature stability was found to be nylon
beads-GA-Chi-GA-Enz > nylon fabric-GA-Chi-GA­
Enz > free enzyme with calcium chloride > free
enzyme without calcium chloride.
1 20 �------...,
1 00
�
1 2 0 �------�
1 00
�
80
�
60
.�
>
40
�---�----�--�
60
40
20
Temperature tel
1 00
80
Fig. 7- Effect of temperature on nylon-immobilised enzyme
activity [ __Nylon fabric-GA-Chi-GA-Enz, -+- Nylon bead­
GA-Chi-GA-Enz, and -&- Free enzyme)
���----,
100
90
80
�
�
.2:
U
«
70
60
50
40
30
20
�----�----����
10
o
o
Temperature tc)
20
40
60
100
80
Fig. 8 - Effect of temperature on free and immobilised enzyme
stability [ -A- Free enzyme stored in distilled water, -k- Free
enzyme stored in 0.25 % CaCI2 solution, ___ Nylon fabric-GAChi-GA-Enz, and -- Nylon bead-GA-Chi-GA-Enz ]
1 .6 ,.------,
80
.�
�
'c
'(ij
0>
<=
�
60
8
c:
�
40
..
�
20
o
L_
_
��___�____�___�
2
4
pH
6
8
1 .2
0.8
0.4
10
Fig. 6 - Effect of pH on nylon-immobilised enzyme stability
[ -+- Nylon fabric-GA-Chi-GA-Enz, -- Nylon bead-GA-Chi­
GA-Enz, and -A- Free enzyme)
o
2
4
6
Reuse Cycle
8
10
12
14
16
Fig. 9 - Reusability of nylon-immobilised enzyme [ __ Nylon
fabric-GA-Chi-GA-Enz. and --Nylon bead-GA-Chi-GA-Enz]
SHUKLA & JAJPURA: IMMOBILISATION OF AMYLASE BY VARIOUS TECHNIQUES
The immobilised enzyme was taken in starch
reaction mixture and then reused number of times
after washing it first with distilled water and then with
pH 8.0 buffer solution. Fig. 9 shows the number of
such reaction cycles and the residual activity of the
enzyme. After 8-10 times reuse, the immobilised
enzyme retained around 50% of its original activity
and even after 1 5lh reuse, 32% residual activity was
retained by the enzyme immobilised on nylon fabric.
Thus, it is clear that different ways are possible to
practically immobilise enzymes and such enzymes are
quite capable of being reused for a number of times in
a given reaction. The immobilisation technique may
proved to be an efficient way of conserving the
precious enzymes. However, from application point
of view, the reuse of enzymes immobilised on solid
supports causes certain difficulties. It will be very
much useful for reactions conducted in fluid medium.
Thus, in the case of textile processing applications,
the use of catalase in residual peroxide bath and in
effluent treatment should be quite promising.
4 Conclusions
The stability of calcium alginate beads improved
when calcium chloride was used in the enzyme
reaction as well as during storage. The cracking of
beads occurred on reuse, the enzyme leached out and
thus the reusability was limited. On the other hand,
the covalently immobilised enzyme on nylon support
was much stable and could be reused number of
times. For the same weight, the nylon fabric gave
more yield than nylon beads. With chitosan as a
spacer during covalent binding with glutaraldehyde,
the yield increased. The enzyme immobilised on
nylon could be reused up to 8-10 times with 50%
residual activity.
Once the retained activity of immobilised enzyme
which decreases after every reuse is understood, it is
81
possible to add the required amount of fresh free Or
immobilised enzyme to carry out the reaction in a
particular reused cycle.
Mitra A, Saylee P & Rathi C L, Chern Weekly, 12 ( 1995)
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