International Journal of Biological Macromolecules 67 (2014) 439–445

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Carboxymethyl cellulose-gelatin-silica nanohybrid: An efficient

carrier matrix for alpha amylase
Vandana Singh ∗ , Shakeel Ahmad
Department of Chemistry, University of Allahabad, Allahabad 211002, India

a r t i c l e i n f o a b s t r a c t

Article history: Carboxymethyl cellulose (CMC)-gelatin (G) dual templated polymerization of tetramethoxysilane (TMOS)
Received 2 February 2014 furnished an efficient hybrid carrier support for alpha amylase. The material has been characterized
Received in revised form 19 March 2014 using FTIR, XRD SEM, TGA and BET studies. The amylase was immobilized at the presynthesized hybrid
Accepted 26 March 2014
support by adsorption and the immobilized enzyme was used to optimize the conditions for soluble
Available online 4 April 2014
starch hydrolysis. The immobilization did not change the optimum working pH (pH 5) and tempera-
ture (40 ◦ C) of the enzymatic reaction. The kinetic parameters of the immobilized (Km = 9.970 mg mL−1 ;
Keywords:
Vmax = 66.23 mg mL−1 min−1 ) and free amylase (KM = 4.0509 mg mL−1 , Vmax = 4.2909 mg mL−1 min−1 ) indi-
Carboxymethyl cellulose
Silica
cated that the immobilization has enhanced the catalytic function of diastase alpha amylase. The
Gelatin, Dual templation, Amylase immobilized enzyme showed higher shelf life as compared to the free enzyme in solution and it could be
Adsorption, Soluble starch hydrolysis reused for seven consecutive cycles where 85% of the initial activity was exhibited even in the last cycle.
The present material is as efficient as our previously reported material CMC-AgNps-Si.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction synthesize CMC-silica biocomposites from silica alkoxide precursor

sols.
Biomineralization processes in the living systems are jointly reg- Gelatin is a fibrous animal protein derived from collagen which
ulated by proteins and polysaccharides [1]. Sol gel silica of different is generated as waste during animal slaughtering and processing
morphologies and microstructures can be designed by templating [17]. This protein is known to possess poor mechanical and
silica ensuation from the silica precursor sols. The polysaccha- thermal stability [18]. Carboxymethyl group of CMC may have sec-
rides are known for templating the sol–gel silica polymerization ondary interactions with ␧-amino groups of lysine/hydroxylysine
[2], while proteins can activate [3–5], control and regulate the side groups of gelatin [19] and in principle the presence of
silica morphology [6–10] by self-assembly. It is known that sil- gelatin may affect the conformational state of CMC. The con-
ica nucleates around carbohydrate macromolecules to generate a comitant use of CMC and gelatin during silica templation may
surrounding silica shell [11]. Polysaccharide protein binary sys- lead to efficient enzyme carrier matrices. Many carrier substrates
tems show synergistic interactions e.g anionic polysaccharides are increase the ability and stability of enzymes [20–25] because of
known to form complex with polycationic proteins [12]. The inter- the specific interactions between enzyme and the carrier sup-
action of carboxymethyl cellulose and gelatin is well studied [13]. port. Enzymes are highly selective biocatalysts [25] and can be
Cellulose is an abundant natural polysaccharide which has either monomeric or multimeric in nature. Enzyme immobiliza-
been used for synthesizing silica biocomposites in various derived tion is a method of choice for wider application and stability [26]
forms such as e.g. hydroxyethyl cellulose [14] and carboxymethyl of enzymes.
cellulose [15]. Carboxymethyl cellulose or cellulose gum (CMC) In the present study silica hybrids have been prepared by using
is soluble form of cellulose which has carboxymethyl groups the dual templation concept where carboxymethyl cellulose and
(CH2 COOH) at glucopyranose monomer units at cellulose back- gelatin have been chosen as polysaccharide and protein com-
bone. CMC-silica hybrid gels synthesized from aqueous silicates ponents respectively. Dual templation effect of CMC and gelatin
[15] are reported, while excluding our previous study on CMC- on silica alkoxide polymerization is not previously studied. The
AgNps-silica composite [16], no attempt has been made to synthesized hybrid has been used as immobilization matrix for
diastase ␣-amylase and using the immobilized enzyme; soluble
starch hydrolysis has been optimized. To understand the effect of
∗ Corresponding author. Tel.: +91 9415310942 (m); +91 532 2461236 (o). gelatin, in the present study we did not incorporate AgNps as we
E-mail address: [email protected] (V. Singh). did in our previous study (CMC-AgNps-Si) [16].

http://dx.doi.org/10.1016/j.ijbiomac.2014.03.051
0141-8130/© 2014 Elsevier B.V. All rights reserved.
440 V. Singh, S. Ahmad / International Journal of Biological Macromolecules 67 (2014) 439–445

2. Experimental 2.4.4. Immobilization of amylase onto the nanohybrids

0.1 mL of diastase (0.4 mg mL−1 ) was adsorbed at Bb3c (100 mg)
2.1. Reagents and left at 40 ◦ C for 2 h. The immobilized enzyme (Bb3c -Am) thus
obtained was washed well with deionized distilled water and dried
Tetramethoxysilane (98%), Merck, Germany was used as sil- in vacuum at 40 ◦ C before use. Bb3c -Am was characterized in com-
ica precursor. Carboxymethylcellulose from Loba Chem was used parison with Bb3c and was used for the optimization of soluble
as supplied. Analytical grades of fungal diastase ␣-amylase, starch hydrolysis [16]. The enzyme immobilization was tried in the
Merck, Germany; and 3, 5-dinitrosalicylic acid; and NaOH, pH range of pH 5 to pH 8 (the enzyme pH was adjusted using phos-
Merck, India were used. Phosphate salts from Merck, India were phate buffers), where optimum loading was witnessed at pH 5.5, so
used for the buffer preparation. Soluble starch, Merck Germany this pH was used for all the immobilization instances in the present
(Mv = 4.451 kDa) was used as hydrolysis substrate to evaluate the study.
bioactivity of amylase. Dialysis of diastase alpha amylase (Merck,
Germany) was done using 10 kDa dialysis bags (1 width) (Banglo
2.4.5. Activity of immobilized enzyme
Genei, Banglore India).
The enzyme bioactivity was determined using 3,5-
dinitrosalicyclic acid (DNS) method [27,28]. The method involved
2.2. Characterization the monitoring of the released reducing sugars during starch
hydrolysis. The bioactivity measurements were performed at
X-ray diffraction (XRD) was done on XRD Pananalytical X- different substrate concentrations, temperatures and pH values
Pert Pro X-ray powder diffractometer. Fourier transform infrared using the predialyzed free enzyme and the immobilized enzyme.
(FTIR) spectra of the samples were recorded using JASCO FTIR The enzyme was dialyzed before use in order to eliminate the
(resolution 4 cm−1 ) in the range of 400–4000 cm−1 Perkin–Elmer effect of stabilizers that may be possibly present in the commercial
TGA-7 was used for the thermogravimetric analysis of the sam- enzyme sample. The reaction mixture contained 0.5 mL soluble
ples under nitrogen atmosphere at a heating rate of 10 ◦ C min−1 starch (1% (w/v) in 0.02 M phosphate buffer), 0.1 mL dialyzed
EDAX, FEI Quanta 200 machine was used for the SEM analysis. The diastase alpha amylase (0.4 mg mL−1 ) and 0.4 mL distilled H2 O.
hybrids were calcined in N2 atmosphere using an electric muffle The bioactivity of 1 mL of this reaction mixture was determined
furnace (Metrex Scientific Instruments (P) Ltd., New Delhi). WT at pH 5 at 40 ◦ C after 10 min incubation. The hydrolysis of soluble
Classic BET Surface Area Analyzer WAKO, India was used for nitro- starch was also monitored under the identical conditions using
gen adsorption–desorption isotherms at 26 ◦ C. The samples were Bb3c -Am (40 ␮g diastase adsorbed at 100 mg Bb3c ) as biocatalyst.
degassed for 4 h at 533 K before the gas was adsorbed. Bb3c -Am was added to a mixture containing 0.5 mL of H2 O, and
0.5 mL starch solution (1% (w/v) in 0.02 M phosphate buffer).
2.3. Dialysis of enzyme The reaction mixture was incubated for 10 minutes at 40 ◦ C as in
the case of free enzyme hydrolysis. The enzymatic reaction was
For the dialysis, dialysis bags containing 5 mL of enzyme solu- interrupted by the addition of 1 mL 3,5-dinitrosalicylic acid reagent
tion (40 ␮g mL−1 ) were hanged in 0.02 M phosphate buffer solution and the reaction mixture was heated in boiling water for 4 min
(200 mL) for 2 h and then in 0.0 2 M sugar solution for 1 h. and then cooled to the room temperature. The optical density of
brown reaction product was determined spectrophotometrically
(at max 540 nm) after dilution with 10 mL of distilled water.
2.4. Material synthesis A blank was simultaneously prepared in the same manner but
without enzyme. The enzyme activity was expressed in terms of
2.4.1. Carboxymethyl cellulose templated nanohybrid (Aa ) micromoles of the reducing sugar. Maltose standard curve was
Aa was synthesized by stirring 20 mL of CMC solution (2%, w/v) used to determine the released reducing sugar during the reaction.
with 1.5 mL TMOS and 1.5 mL of methanol at magnetic stirrer for The bioactivities of these enzyme samples were assessed using
45 min at 30 ◦ C. The hydrogel was washed well with deioinzed dis- 0.5 mL of 1% (w/v) starch solution. Similarly known weight of the
tilled water and dried under reduced pressure. The optimization of immobilized enzyme was incubated at different temperatures
this hybrid gel synthesis has been described elsewhere [16]. (from 25 to 50 ◦ C) for 10 min with 0.5 mL of 1% (w/v) starch and
0.5 mL of H2 O. Enzyme bioactivity was measured as described
2.4.2. Carboxymethyl cellulose-gelatin nanohybrid (B series above and expressed in terms of enzyme units. One unit of enzyme
hybrids) was considered equivalent to the amount of enzyme that could
B series hybrid gels [21] (Ba to Bf ) were crafted by adding a produce 1 ␮mol of reducing sugar from 1% starch solution in 1 min
known weight of gelatin during the polymerization of TMOS under at 40 ◦ C.
the synthetic conditions of Aa . The used immobilized enzyme was separated from the reac-
Among B series gels, Bb sample showed optimum result in a tion mixture by centrifugation and recycled after washing well with
preliminary investigation as described in section 2.5.4. Under the distilled water. The kinetic study was performed using 0.25 to 4%
synthetic conditions of Bb , the amount of CMC, TMOS and MeOH (w/v) starch solutions at pH 5 while keeping the other conditions
were varied one by one, while keeping other parameters fixed. The as described above. The shelf life of the immobilized enzyme was
hybrid hydrogels thus obtained were named from Bb1 to Bb4 , Bbu - monitored by storing it as dry solid for the desired time period at
Bbx and Bb␣ to Bb␧ , respectively. room temperature (∼40 ◦ C).

2.4.3. Calcination 3. Results and discussion

The optimum “B” series hybrid (Bb ) was calcined at known tem-
perature ranging from 200 to 800 ◦ C for 2 h at each temperature Among the “B” series hybrids (CMC-G-Si), Bb was most effi-
and left overnight to obtain hybrids Bb2c to Bb8c . These heat treated cient in terms of amylase immobilization. Bb could immobilize
gels were evaluated for amylase immobilization and the immobi- 95% of the loaded enzyme (results not shown) and the enzyme
lized enzymes were used to hydrolyze soluble starch as described loaded Bb (Bb-Am) showed maximum bioactivity among the immo-
in section 2.5.4. bilized enzymes derived from the “B” series hybrids and Bb was
 V. Singh, S. Ahmad / International Journal of Biological Macromolecules 67 (2014) 439–445 441

Table 1
Optimization of the hybrid synthesis (bioactivity measurement: 100 mg Bb3c -Enz, temperature = 40 ◦ C, incubation time = 10 min, RPM = 200, pH 5, at 540 nm as described in
Section 2.4.4).

S. no Hybrid CMC (mg) TMOS (mL) MeOH (mL) H2 O (mL) Gelatin (mg) Gelation Calcination Activity
time (min) tempera- (U mg−1 )
ture
(◦ C)

4 Aa 200 1.5 1.5 20 – 45 – 16.50

5 Ba 200 1.5 1.5 20 5 60 16.55
6 Bb 200 1.5 1.5 20 10 60 17.45
7 Bc 200 1.5 1.5 20 15 75 10.94
8 Bd 200 1.5 1.5 20 20 75 16.41
9 Be 200 1.5 1.5 20 25 75 15.21
10 Bf 200 1.5 1.5 20 30 75 12.56
11 Bb1 100 1.5 1.5 20 10 60 9.73
12 Bb2 300 1.5 1.5 20 10 40 17.07
13 Bb3 400 1.5 1.5 20 10 40 5.07
14 Bb4 500 1.5 1.5 20 10 30 1.93
15 Bbu 200 0.5 1.5 20 10 60 14.77
16 Bbv 200 1.0 1.5 20 10 50 17.29
17 Bbw 200 2.0 1.5 20 10 45 15.60
18 Bbx 200 2.5 1.5 20 10 30 14.19
19 Bb␣ 200 1.5 0.5 20 10 60 14.43
20 Bb␤ 200 1.5 1.0 20 10 60 14.26
21 Bb␥ 200 1.5 1.5 20 10 65 14.60
22 Bb␦ 200 1.5 2.0 20 10 65 13.88
23 Bb␧ 200 1.5 2.5 20 10 70 13.75
24 Bb2c 200 1.5 1.5 20 10 60 200 17.31
25 Bb3c 200 1.5 1.5 20 10 60 300 22.18
26 Bb4c 200 1.5 1.5 20 10 60 400 14.05
27 Bb5c 200 1.5 1.5 20 10 60 500 11.91
28 Bb6c 200 1.5 1.5 20 10 60 600 10.30
29 Bb7c 200 1.5 1.5 20 10 60 700 6.85
30 Bb8c 200 1.5 1.5 20 10 60 800 5.43

thus selected for the calcination study. The hybrid samples Bb2c to the synthesis of Bb involved one fifth weight of the polysaccha-
Bb8c (Table 1) were obtained by calcining Bb at known temperature ride (CMC) and gelled in lesser time (60 min) in comparison to gum
(ranging from 200 to 800 ◦ C) for 2 h at each temperature. Calcina- acacia-gelatin-silica (90 min).
tion of Bb at 300 ◦ C led to hybrid (Bb3c) which on enzyme loading
offered optimum activity (22.18 U mg−1 ) sample. Enzyme loaded 3.1. Characterization
Bb3c (Bb3c -Am) showed maximum bioactivity (22.18 U mg−1 activ-
ity) and therefore soluble starch hydrolysis was optimized using 3.1.1. FTIR
Bb3c -Am. To understand the effect of calcination on the hybrid Silica incorporation in the hybrids was confirmed by Si-O
structure, Bb3c has been characterized in comparison with Bb using stretching modes that are visible below 1250 cm−1 (Fig. 1). The
FTIR, XRD, SEM, TGA and BET studies. The bioactivity of the Bb3c -Am FTIR spectrum of optimum CMC-silica hybrid (Bb3c ) showed peaks
was nearly same as that of our previously reported material: gum at 1078 cm−1 (Si-O stretching) and 792 cm−1 (Si-OH stretching),
acacia-gelatin-silica nanohybrids [21], whose synthesis involved which revealed the presence of Si-O-Si bonds (Fig. 1A). A com-
1 g gum acacia, 10 mL H2 O, 1 mL TMOS, 10 mg gelatin. For the bined peak due to silica surface silanols and CMC hydroxyl groups
present material, the gel was not formed in absence of co-solvent is visible at 3441 cm−1 . Symmetrical stretching peak of –CH2 – (at
(MeOH) in contrast to the synthesis of gum acacia-gelatin-silica carboxymethyl groups of CMC) is visible at 2925 cm−1 while C O
nanohybrids, where the hydrogel could be formed in absence of the stretching is observed as a strong asymmetric stretching band
co-solvent [21]. This difference may be attributed to the different at 1610 cm−1 C-H deformation peak of carboxymethyl methylene
primary and secondary structures of CMC and gum acacia. However group is seen at 1418 cm−1 . The peaks associated with gelatin are

Fig. 1. (A) IR spectrum Bb3c ; (B) IR spectrum Bb3c -Am.

442 V. Singh, S. Ahmad / International Journal of Biological Macromolecules 67 (2014) 439–445

3.1.3. SEM
SEM images of Bb and Bb3c (Fig. 3) reveal their surface mor-
phologies. The hybrid (Bb ) showed layered structure having surface
adhered gelatin. The adhesion of gelatin takes place due to its sticky
nature. The layered structure of the hybrid becomes more evident
and clear upon calcination (Bb3c ), since most of the gelatin is lost
during the calcination and probably this layered structure offers
additional binding site in Bb3c as compared to Bb .

3.1.4. TGA
Thermo gravimetric analysis indicated that the hybrids are ther-
mally stable (Fig. 4). A two stage weight loss was observed for both
Bb and Bb3c. In case of Bb , first 28% weight loss took place (up
to 600 ◦ C) in two stages, first loss was due to bound and trapped
water, solvent and gelatin (cf SEM), while the other slow loss (that
Fig. 2. (A) XRD of Bb3c ; (B) XRD of Bb3c -Am. extended up to 600 ◦ C) can be attributed to the loss of the CMC and
densification of the hybrid gel. The calcined material (Bb3c ) showed
only 20% weight loss in the first stage which extended up to 150 ◦ C.
This weight loss can be assigned to the loss of moisture and residual
not separately observed and are seen merged with the CMC and solvent as most of the solvent and gelatin has been already removed
silica related peaks. during calcination at 300 ◦ C. After this, a very slow weight loss is
No new peak is seen in the immobilized enzyme (Bb3c -Am) seen that extended up to 600 ◦ C. Weight loss in Bb3c revealed that
(Fig. 1B), however shifting of the peaks is seen e.g. SiO-H peak some organics (gelatin and CMC) were still present in the calcined
(merged with CMC hydroxyl) shifted to 3422 cm−1 The shift indi- sample and weight loss during TGA can be assigned to them.
cated that the immobilized enzyme had an affect on the association
state of the hybrid’s surface silanols. Enzyme amide (I) and amide 3.1.5. BET
(II) band are not observed separately as these peaks might have The surface area the optimum CMC-gelatin-Silica (Bb ) and cal-
overlapped with the carbonyl peaks of the CMC. Other notice- cined CMC-gelatin-Silica (Bb3c ) were determined by BET method
able shift is seen for C-H deformation of carboxymethyl methylene using nitrogen adsorption–desorption isotherms. Bb3c had ∼7 times
(from 1418 to 1426 cm−1 ). Minor shifts in C-H stretching (from higher surface area (28.88 m2 g−1 ) as compared Bb (4.39 m2 g−1 )
2925 to 2929 cm−1 ) and Si-O stretching (from 1078 to 1082 cm−1 ) which indicated that Bb3c has higher active surface for the immo-
peaks are also visible. The IR studies have thus indicated that the bilization as compared to Bb .
surface silanols and hydroxyl and carboxymethyl groups of CMC
(residual which escape loss on calcination) take part in enzyme
3.2. Immobilization mechanism
immobilization.
A novel templating environment was created by the com-
bined use of CMC and gelatin. This happened possibly due to
3.1.2. XRD the secondary interactions between CMC (carboxymethyl groups
The XRD (Fig. 2) evidenced Bb3c to be an amorphous material and hydroxyls) and gelatin (amino and hydroxyl functionalities
having different angle centre 2 25◦ from that of control silica (2 in lysine and hydroxylysine). Silica precursor (TMOS) polymer-
22◦ ) (XRD not shown). CMC-gelatin-Silica before (Bb ) and after cal- ized around the composite template to result CMC-gelatin-silica
cination (Bb3c ) showed the same amorphous nature, however in hybrid material. TGA of Bb showed 28.67% weight loss up to 600 ◦ C
the calcined material, the amorphous hump has slightly sharp- while Bb3c showed a weight loss of 20.4% that indicated only ∼8%
ened and shifted to higher 2 value (indicating some change in weight was lost during the calcination of Bb (Fig. 4). It is evident
iner-atomic distances upon calcination which in turn explains the that the silica network cage some organics from loss (CMC and
difference in the bioactivities of Bb -Am (17.45 U mg−1 ) and Bb3c -Am Gelatin) as much higher weight loss in CMC is reported in litera-
(22.18 U mg−1 ). ture (∼74% up to 500 ◦ C) [29]. It can be assumed that loss of gelatin

Fig. 3. (A) SEM image of Bb ; (B) SEM image of Bb3c .

 V. Singh, S. Ahmad / International Journal of Biological Macromolecules 67 (2014) 439–445 443

Fig. 4. (A) TGA of Bb ; (B) TGA of Bb3c .

Fig. 5. (I) Bioactivity of immobilized enzyme (A) and free (B) enzyme at different pH values; (II) Bioactivity of immobilized enzyme (A) and free (B) enzyme at different
temperatures

is also not complete during calcination at 300 ◦ C because gelatin 3.3.2. Effect of temperature on soluble starch hydrolysis
goes away is at >300 ◦ C [30,31]. It is evident that Bb3c still has some The bioactivities of free and immobilized (Bb3c -Am) enzyme
intact templating molecules which play role in immobilizing the were found dependent upon the biocatalytic reaction tempera-
enzyme (Scheme 1). Partial loss of templates opens up binding sites ture (Fig. 5B). The immobilized enzyme had higher activity in the
at residual CMC and gelatin that were previously involved in the studied temperature range; however 40 ◦ C proved to be the opti-
secondary interactions. The residual templating molecules at cal- mum temperature for Bbc3 -Am as that of free enzyme in solution.
cined material now have exposed functionalities for the enzyme The optimum activity temperature (40 ◦ C) for the present hybrid
attachment. is lower than what we observed for amylase loaded gum acacia-
gelatin-silica hybrid (50 ◦ C) and this low temperature activity is
attractive from the industrial point of view. The bioactivity of the
enzyme (for free and immobilized state both) declined >50 ◦ C, indi-
3.3. Enzyme immobilization
cating enzyme denaturation takes place at these temperatures.

Diastase has been impregnated on CMC-gelatin-silica (Bb3c ) to

obtain Bb3c -Am. Bb3c -Am has been used to optimize soluble starch
0.25
hydrolysis. Kinetic parmeters, KM and Vmax for the free and the (A)
y = 0.7574x + 0.0818
immobilized enzymes were derived from the initial rates of the R2 = 0.9455
0.2 (B)
hydrolysis reaction of starch solutions of different concentrations
using Lineweaver–Burk equation [32].
0.15
y = 0.67x + 0.0672
V-1

R2 = 0.9404
0.1
3.3.1. Effect of pH on soluble starch hydrolysis
The optimum pH for the enzymatic reaction was determined by 0.05
varying the pH of the assay reaction mixture with the help of phos-
phate buffers. The immobilization at hybrid Bb3c did not change the 0
0 0.05 0.1 0.15 0.2
pH optimum (pH 5) of the enzyme bioactivity (Fig. 5A). The bioac- -1 -1
S (mg/mL)
tivity of the enzyme was significantly dropped when the reaction
pH was increased from pH 6 to pH 8. Similar trend was observed in Fig. 6. (A) Line-Weaver Burke plots for free enzyme; (B) Line-Weaver Burke plot for
our previous report on gum acacia-gelatin-silica hybrid [21]. the immobilized enzyme (at pH 5 and temperature 40 ◦ C).
444 V. Singh, S. Ahmad / International Journal of Biological Macromolecules 67 (2014) 439–445

Scheme 1. Schematic model for the hybrid synthesis and the amylase immobilization.

3.3.3. Hydrolysis and kinetic parameters long time. In general, the Km of an enzyme in the immobilized
Kinetic parameters, the Michaelis constant KM and the max- state is not same as that of the free enzyme because of the dif-
imum activity Vmax were derived for free and the immobilized fusion limitations [33]. Moreover the immobilized enzyme has
diastase (Bb3c -Am) hydrolysis of starch using Lineweaver–Burk different steric [34] and ionic [35] surroundings that also affects
plots (Fig. 6). Km corresponds to the substrate concentration that the KM value. Vmax corresponds to the highest velocity when all
is sufficient to reach a reaction velocity of 1/2 Vmax . Large Km indi- the active sites of the enzyme remain saturated with the substrate.
cates that substrate and enzyme do not prefer to be close for a This parameter depends upon the intrinsic nature of the immobi-
lized enzyme and is normally affected by diffusional constraints.
For the present study the kinetic parameters of free enzyme
21.5
(KM = 4.0509 mg mL−1 , Vmax = 4.2909 mg mL−1 min−1 ) and immo-
21
bilized (Km = 9.970 mg mL−1 ; Vmax = 66.23 mg mL−1 min−1 ) enzyme
Activity (U/mg)

20.5
indicated that the immobilization has decreased the enzyme sub-
20
strate affinity. It can also be attributed to interparticle diffusional
19.5
mass transfer restrictions or the immobilization hinders some of
19
the active sites of enzyme which are now not available for the
18.5
substrate. The increased value of Vmax indicates that enzyme-
18
17.5
substrate complex (for immobilized enzyme) once formed can
0 2 4 6 8 quickly decompose to furnish product. This indicated that the
immobilization has enhanced the catalytic function of diastase
Cycles
alpha amylase. It can be assumed that on immobilization the
Fig. 7. Recycling of the biocatalyst. enzyme has adopted a conformation that is more conducive for
 V. Singh, S. Ahmad / International Journal of Biological Macromolecules 67 (2014) 439–445 445

the breaking of enzyme-substrate complex. Similar results have to Department of National Centre of Experimental Mineralogy and
been reported for invertase immobilization by crystalline cellulose Petrology, University of Allahabad, India.
and Nylon 6 [36]. This result is different from over previous stud-
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are thankful to Dr. Sagar Pal of Indian Institute of Mines, Dhanbad, [36] M. deLos Angeles Calixto-Romo, J.A. Santiago-Hernadez, V. Vallego-Becerra, L.
India for FTIR facility and to Indian Institute of Technology, Karag- Amaya-Delgado, M. de Carmen Montes-Hocasitasm, M.E. Hidalgo-Lara, J. Indus.
pur, India for providing SEM facility. XRD facility is acknowledged Microbiol. Biotechnol. 35 (2008) 1455–1463.