S.A. Ansari, Q. Husain / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 68–74
73
Table 4
inhibition mediated by galactose as compared to the native enzyme
Fig. 5). Some earlier investigators have also reported the competi-
Lactose hydrolysis in the columns filled with CCG at different flow rates.
(
Number of days
Lactose hydrolysis (%)
tive inhibition of  galactosidase by its reaction product, galactose
−1
Flow rate (mL h
)
[27–29].
FTIR spectra reveal the presence of amino group at 1643.64 cm 1
which was due to free –NH2 groups of Con A [30]. The presence
of C O group in  galactosidase immobilized on Con A-cellulose
−
10
20
30
Control
5
100
95.0
100
91.45
100
87.3
−
1
1
1
2
2
3
3
4
4
5
5
6
0
5
0
5
0
5
0
5
0
5
0
95.0
94.3
94.3
94.3
92.0
92.0
92.0
91.8
90.0
90.0
90.0
91.45
90.0
90.0
89.8
89.8
89.8
88.4
87.0
87.0
87.0
86.9
87.0
87.0
87.0
84.9
84.9
83.0
83.0
81.7
81.7
80.0
80.0
has been confirmed by its peak value at 1053.28 cm . Moreover,
the peak at 1343.37 and 1440.07 cm further confirmed the pres-
ence of –C–N groups in this complex (Fig. 7). We further state that
−
1
−
1
major shift of peak occurs within the region of 1000–2000 cm
due to binding of Con A-cellulose with  galactosidase. It was also
observed visually by change in colour from white to pale yellow.
This interaction evidenced the formation of strong poly-electrolyte
complex [31].
The results for the continuous hydrolysis of lactose in batch pro-
cess exhibited that the rate of hydrolysis was more in case of soluble
Hydrolysis of lactose in the continuous reactors filled with CCG (2000 U) was per-
formed as described in text (Section 2.13). Each value represents the mean for three
independent experiments performed in duplicates, with average standard devia-
tions, <5%.

galactosidase for the first few hours as compared to crosslinked
Con A-cellulose adsorbed  galactosidase (Table 3). This was due to
the fact that the soluble enzyme was more accessible for the hydrol-
ysis of lactose for first few hours but after prolonged incubation,
the rate of lactose hydrolysis decreased because of more inhibition
of soluble enzyme by its own product. This phenomenon of inhi-
bition of soluble  galactosidase by its products has already been
explained earlier [32]. Immobilized  galactosidase was found to
be more resistant to inhibition by product thus it hydrolyzed more
lactose on prolong incubation. Pessela et al. [33] have also reported
more hydrolysis of lactose by immobilized enzyme as compared to
soluble  galactosidase.
and this bioaffinity support was exploited for the surface immo-
bilization of  galactosidase. Several investigators have earlier
reported the use of glutaraldehyde as crosslinking agent in differ-
ent ways [20–22]. Here, Con A-cellulose adsorbed  galactosidase
was crosslinked by glutaraldehyde in order to prevent the dissoci-
ation of enzyme from the support. Crosslinking of enzyme resulted
in a marginal loss of enzyme activity (Table 1). Several workers
have already reported that crosslinking of adsorbed enzymes by
glutaraldehyde prevented the dissociation of enzymes from Con
A supports in the presence of glucose, mannose and N-acetyl glu-
cosamines and it also significantly enhanced stability of enzymes
against various physical and chemical denaturants [18,19,23].
There was a shift in the temperature-optimum for the immobi-
Lactose hydrolysis was also analyzed at different flow rates by
the crosslinked Con A-cellulose bound  galactosidase. The greater
extent of lactose was hydrolyzed by the enzyme present in the col-
−
1
umn at the flow rate of 10 mL h (Table 4). It was due to more
residence time of lactose inside the column. A similar finding for the
continuous hydrolysis of lactose in a continuous column by calcium
alginate entrapped  galactosidase has been reported by Haider
and Husain [14]. The greater stability of this inexpensive immobi-
lized  galactosidase preparation at higher temperatures for longer
duration might bring about its use in the continuous hydrolysis of
lactose at large scale.
◦
lized  galactosidase from 50 to 60 C (Fig. 2). El-Masry et al. [24]
previously showed that  galactosidase from A. oryzae immobilized
via diazotization or condensation on nylon membranes grafted
with glycidyl methacrylate had a higher temperature-optimum
than its soluble counterpart. This increased activity at higher tem-
peratures showed significant stabilization against heat induced
inactivation. Crosslinked Con A-cellulose adsorbed  galactosidase
has shown remarkably very high stabilization against heat induced
denaturation (Fig. 3). Tanriseven and Dogan [25] also reported sim-
ilar findings when the enzyme was immobilized in fibers composed
of alginate and gelatin hardened by glutaraldehyde. Moreover,
crosslinking of enzyme with glutaraldehyde, a bi-functional agent
enhanced thermal stability of the enzyme due to the formation of
several linkages between enzyme and support [10,23].
Milk is a common health drink consumed by people of all age
groups. Calcium is one of the major minerals found in milk that is
required in large quantities for bone growth, development of soft
tissues and to maintain the casein micelle structure. The deficiency
of calcium can thus lead to several disorders in an individual [8,26].
Therefore, we have investigated the effect of CaCl2 on the activity
of soluble and immobilized  galactosidase. Immobilized enzyme
exhibited significantly higher stabilization against exposure to Ca2+
ions (Fig. 4). Haider and Husain [19] earlier reported that the sol-
5. Conclusion
This study deals with a simple and inexpensive procedure for
the immobilization of  galactosidase by using inexpensive Con A
and cellulose. Immobilized  galactosidase exhibited significantly
higher stability against various types of denaturants and on storage
than the free enzyme. The reusability experiment demonstrated
that the enzyme does not dissociate from the matrix apprecia-
bly upon repeated uses and thus this immobilized  galactosidase
preparation could be successfully employed for the hydrolysis of
lactose from milk and whey in a batch process. In view of the stabil-
ity and utility of immobilized enzyme to hydrolyze lactose in batch
and continuous processes, such preparation could be exploited for
the continuous hydrolysis of lactose from milk and whey continu-
ously for longer durations in reactors.
◦
uble enzyme exposed to 5% CaCl2 for 1 h at 37 C resulted in a loss
of nearly half of the original activity while the calcium alginate
Acknowledgment
entrapped crosslinked Con A- galactosidase retained more than
The authors are thankful to the Council of Science and Tech-
nology, Lucknow, Uttar Pradesh for funding the project entitled
8
0% of the original activity.
Galactose is one of the products of  galactosidase catalyzed
“Immobilization of plant and fungal  galactosidases by using
hydrolysis of lactose. Our findings indicated that the crosslinked
adsorbed Con A-cellulose  galactosidase and adsorbed Con A-
cellulose  galactosidase were significantly more resistant to the
bioaffinity supports—Its application in the hydrolysis of lactose in
whey and milk.”