Immobilization of α-amylase and amyloglucosidase onto ion-exchange resin beads and hydrolysis of natural starch at high concentration

ORIGINAL PAPER

Immobilization of a-amylase and amyloglucosidase ontoion-exchange resin beads and hydrolysis of natural starchat high concentration

Kapish Gupta • Asim Kumar Jana •

Sandeep Kumar • Mithu Maiti

Received: 29 January 2013 / Accepted: 24 March 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract a-Amylase was immobilized on Dowex MAC-

3 with 88 % yield and amyloglucosidase on Amberlite

IRA-400 ion-exchange resin beads with 54 % yield by

adsorption process. Immobilized enzymes were character-

ized to measure the kinetic parameters and optimal oper-

ational parameters. Optimum substrate concentration and

temperature were higher for immobilized enzymes. The

thermal stability of the enzymes enhanced after the

immobilization. Immobilized enzymes were used in the

hydrolysis of the natural starch at high concentration (35 %

w/v). The time required for liquefaction of starch to 10

dextrose equivalent (DE) and saccharification of liquefied

starch to 96 DE increased. Immobilized enzymes showed

the potential for use in starch hydrolysis as done in

industry.

Keywords a-Amylase � Amyloglucosidase �Ion-exchange resin beads � Immobilizations �Starch hydrolysis

Introduction

a-Amylase (AAM) used in the liquefaction of starch-pro-

ducing soluble dextrins and glucoamylase (amyloglucosi-

dase, AMG) used for further hydrolysis of the dextrins to

glucose in the saccharification step are of great significance

in present day biotechnology with applications ranging

from food, baking, brewing, detergent applications and

textile desizing, paper industries to analysis in medicinal

and clinical chemistry [1, 2]. Industrial applications of

these enzymes are often hampered by a lack of long-term

operational stability and difficulty in recovery and re-use of

the enzyme. It has also been encountered that the enzyme

used, require extra downstream processing steps for its

removal. To overcome such hurdles, immobilization tech-

niques are generally employed. The immobilization matrix

or support allows exchange with, but remains separated

from the bulk phase in which substrate is present. Various

immobilization methods can be classified into three major

types, i.e. binding to a carrier [3, 4], encapsulation in an

inorganic or organic polymeric gel, or by cross-linking of

protein molecules. There are many earlier reports on

immobilization of amylases on carrier poly(glycidyl

methacrylate-Co-ethylene dimethacrylate) [5], polysac-

charide matrices [6], PS/PNaSS microspheres [7], poly

(o-toluidine) [8], polyaniline polymer [9], and magnetic

carrier [10, 11] and in reverse micelle [12].

For commercial success of the immobilized enzyme

technology, cost of the immobilized enzymes and opera-

tional parameters are very important. Inexpensive immobi-

lization techniques require simple procedure with minimum

steps and high activity retention. Amongst the immobiliza-

tion methods, physical adsorption onto a solid support is a

simplest and least expensive procedure. Because the

adsorption involves weak interactions, the effects of immo-

bilization on the activity of the enzymes are less severe.

However, owing to weak nature of the binding forces,

retention of the enzymes on the carriers becomes a serious

problem. This problem can be reduced using ionic binding

which is much stronger than mere adsorption [13]. The use of

ion-exchange resins as carriers for immobilization is often

K. Gupta � A. K. Jana (&) � S. Kumar

Department of Biotechnology, National Institute of Technology,

G T Road Bye Pass, P. O. REC, Jalandhar 144011, Punjab, India

e-mail: [email protected]

M. Maiti

Department of Chemistry, Lovely Professional University,

Phagwara, Punjab, India

123

Bioprocess Biosyst Eng

DOI 10.1007/s00449-013-0946-y

used in the food industry, involving ionic and electrostatic

interactions between ions of proteins and ions of opposite

charge from the resin.

Most of the studies involving immobilized AMM/AMG

dealt with hydrolysis of soluble starch at very low con-

centration (1–5 %) [14, 15], but natural starch at high

concentration is being used for hydrolysis in the industries

due to economic reasons. The enzyme activities may be

inhibited and non-ideal conditions are generated because of

high viscosities at high starch concentrations. The studies

done using low starch/substrate concentration cannot be

replicated faithfully at industrial scale. Thus, it is preferred

to do the studies on the hydrolysis of starch by immobilized

enzyme near to the conditions as encountered in industrial

scale production. Currently, there are no reports on

hydrolysis of natural starch at higher concentration using

sequential treatment with immobilized enzymes.

In the present study, AMM and AMG were immobilized

on ion-exchange resin beads by simple adsorption process

and the kinetic parameters, operational parameters and

stability of the immobilized preparations were elucidated.

Immobilized enzymes were initially characterized by

hydrolysis of soluble starch. The operational parameters of

the immobilized enzymes were analysed on the basis of

hydrolysis of natural starch at very higher concentration

([30 % w/v) to replicate actual industrial conditions.

Materials and methods

Chemicals

a-Amylase (E.C. 3.2.1.1 from Bacillus licheniformis) and

glucoamylase (AMG) (E.C. 3.2.1.3 from Aspergillus niger)

were obtained from NOVO industry, Bagsvaerd, Denmark.

The protein content in AAM and AMG was 1.2 and

1.08 mg ml-1, respectively. Corn starch was supplied by

Sukhjit Starch Industries Ltd., Phagwara, India. Dinitro-

salicylic acid (DNS), sodium sulfite, sodium acetate and

soluble starch were purchased from Central Drug House

(P) Ltd. (CDH), New Delhi, India. Folin Ciocalteu’s phenol

reagent (2N) and bovine serum albumin (BSA) were pur-

chased from Ranbaxy Laboratories Ltd., New Delhi, India.

Ion-exchange resins Amberlite IR 120 (H? form), Am-

berlite IRA 400 (OH- form), Amberlite IRA 67 free base,

Dowex MAC-3 (H? form), all were made by Rohm and

Haas, Philadelphia. All chemicals were of analytical grade.

Characterization of free enzymes

Enzymes were characterized by hydrolysis of soluble

starch. The starch concentration was optimized followed by

optimization of temperature and pH. Free AAM was

characterized by studying the effect on the enzyme activ-

ities by varying the final starch (soluble) concentration

0.83, 1.67, 2.5, 3.33, 4.16, 5.0 and 5.83 % w/v (70 �C, pH

7.0), temperature 60, 65, 70, 73, 75 and 80 �C (starch

4.16 % w/v, pH 7.0); pH 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0

(starch 4.16 % w/v, 73 �C). The optimized parameters

were used in the optimization of the next parameters. AMG

was characterized by studying the effect on the enzyme

activities by varying the starch (soluble) concentrations

0.31, 0.63, 0.94, 1.25, 1.56 and 1.88 % w/v (60 �C, pH

4.0), temperature 40, 45, 50, 55, 60 and 65 �C (starch

0.63 % w/v, pH 4.0); pH 4.0, 4.1, 4.2, 4.3 and 4.4 (starch

0.63 % w/v, 50 �C). Kinetic parameters Vmax and KM for

enzymes were determined using their Lineweaver–Burk

plot.

Enzyme activity assay

The activities of amylases were assayed on the basis of

starch hydrolysis. The liberated reducing sugar was esti-

mated by DNS method [16]. For determination of activity

of AAM under optimized conditions, 3 ml of reaction

mixture consisted of 0.25 ml enzyme solution (diluted

459), 0.25 ml of phosphate buffer (0.2 M, pH 7) and

2.50 ml of gelatinized starch suspension (5 % w/v). The

reaction was carried out for 15 min at 73 �C. A unit of

activity (U) of AAM was defined as the amount of enzyme

required to produce 1 lmol of glucose per minute. For

determination of activity of AMG under optimized condi-

tion, 2 ml of reaction mixture of 0.1 ml enzyme solution,

0.65 ml of sodium acetate buffer (50 mM, pH 4.1) and

1.25 ml of gelatinized starch suspension (1.0 % w/v). The


activity (U) of AMG was defined as the amount of enzyme

required to produce 1 lmol of glucose per minute. Specific

activity was measured in terms of U mg -1 protein. Protein

content of enzyme was determined by Lowry’s method

taking BSA as standard protein [17].

Studies on sequential hydrolysis of natural corn starch

by free enzymes

Enzyme characterizations were done by the hydrolysis of

natural corn starch. Starch was hydrolyzed sequentially as

done in the industries, at first liquefaction by AAM fol-

lowed by saccharification using AMG [18]. The product

was analyzed in terms of dextrose equivalent (DE) [19].

Liquefaction step generally yield 10 DE and saccharifica-

tion of liquefied starch yield 96 DE. Liquefaction was car-

ried out taking 5 ml gelatinised starch solution and 0.4 ml


123

AAM (diluted 459, amount of enzyme required was also

optimized, but data not shown). Optimization was done on

the basis of minimum time required to achieve desired DE.

Optimal conditions for liquefaction was established by

varying starch concentrations 26, 28, 30, 32 and 34 % w/v

(73 �C, pH 7.0); temperature 70, 73, 80, 83, 85, 87, 89 and

95 �C (starch 32 % w/v, pH 7.0); pH 5.0, 5.4, 5.8, 6.2, 6.4,

6.8, 7.0 and 7.1 (starch 32 % w/v, 87 �C). The optimized

parameters were used in the optimization of the next

parameters.The product of liquefaction under optimized

conditions was saccharified by AMG (0.5 ml, amount of

enzyme required was also optimized, but data not shown).

Operational parameters were optimized for this step by

varying temperature 50, 55, 60, 65, 70 �C (pH 4.1) and pH

3.5, 4, 4.1, 4.5, 5.0, 5.5 (60 �C).

Immobilization of enzymes

The AAM enzyme was immobilized by adsorption on weak

cation exchange resin, Dowex MAC-3 (H? form) after

screening of different ion-exchange resins. For finding the

optimum weight of resin beads for the immobilization of

AAM, various bead weights were immobilized with same

enzyme solution. 0.2–0.4 mg Dowex MAC-3 (H? form) of

the carrier ion-exchange resin matrix was soaked in

0.25 ml of 459 diluted AAM, 6,127 U mg-1 protein in

0.2 M sodium phosphate buffer pH 7.0 at temperature

30 �C for the 24 h. The immobilized beads were washed

with buffer. The beads were air dried at temperature 30 �C

for 72 h. The amount of resin beads were optimized to get

maximum immobilization yield.

AMG was immobilized by adsorption on strong anion

exchange resin beads, Amberlite IRA 400 (OH- form)

after the screening of different ion-exchange resins. For

finding the optimum resin beads weight for the immobili-

zation of glucoamylase, various beads weights were

immobilized with same enzyme solution. 0.02–0.10 mg of

the carrier ion-exchange resin matrix was soaked with

0.1 ml AMG enzyme (1.31 U mg-1 proteins) in 0.05 M

sodium acetate buffer pH 4.0 at 30 �C for 28 h. The

immobilized beads were washed with 1.5 ml distilled water

such that no activity of enzyme or protein content was

detected in the washings. The beads were air-dried for

72 h. The amount of resin beads were optimized for the

maximum immobilization yield.

Efficiency of immobilization was determined as immo-

bilization yield using following equation [20]

Immobilization yield %ð Þ ¼ I � 100

ðA� BÞ

where A = added enzyme (U g carrier-1), B = unbound

enzyme (U g carrier-1); I = immobilized enzyme (U g

carrier-1).

Characterization of immobilized enzyme

Immobilized enzymes were also characterized by hydro-

lysis of soluble starch. Immobilized AAM was character-

ized by studying the effect on the enzyme activities by

varying final substrate concentration 0.83, 1.67, 2.5, 3.33,

4.167, 5.0 and 5.83 % w/v (73 �C, pH 7.0); temperature 70,

73, 76, 79, 80, 82, 85, 90 and 80 �C (starch 5 % w/v, pH

7.0) and pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 (starch 5 %

w/v, 82 �C). The optimized parameters were used in the

optimization of the next parameters. Similarly, immobi-

lized AMG was characterized by studying the effect on the

enzyme activities by varying starch concentration 0.5, 0.83,

1.0, 1.32, 1.45, 1.6, 1.9 and 2.1 % w/v (50 �C, pH 4.1),

temperature 50, 55, 60, 62, 65 and 70 �C (starch 1.6 %

w/v, pH 4.1) and pH 3.8, 3.9, 4.0, 4.1, 4.2, 4.3 and 4.4

(starch 1.6 % w/v, 62 �C). Kinetic parameters Vmax and KM

for enzymes were determined using the Lineweaver–Burk

plot.

Assay for activities of immobilized enzymes

The activities of amylases were assayed on the basis of

starch hydrolysis. The liberated reducing sugar was esti-

mated by DNS [16]. For determination of activity of

immobilized AAM under optimized conditions, 3 ml

reaction mixture consisted of 0.25 ml enzyme solution

(diluted 459) immobilized on optimized support resin

matrix, 0.5 ml of phosphate buffer (0.2 M, pH 7) and

2.50 ml of gelatinized starch solution (6 % w/v). The


activity (U) was defined as amount of enzyme required to

produce 1 lmol of glucose per minute. For determination

of immobilized AMG activity, 2 ml reaction mixture

consisted of 0.1 ml AMG immobilized on optimized resin

matrix, 0.75 ml of sodium acetate buffer (50 mM, pH 4.1)

and 1.25 ml of gelatinized starch suspension (1.6 % w/v).

The reaction was carried out for 15 min at 62 �C. A unit of

activity (U) was defined as amount of enzyme required to

produce 1 lmol of glucose per minute.

Studies on the sequential hydrolysis of natural corn

starch by immobilized enzymes

Enzyme characterizations were also done by the hydrolysis

of natural starch. Initial physical conditions used were

similar to those for free enzymes for hydrolysis of natural

starch. First, starch was liquefied by adding 0.4 ml AAM

(diluted 459) immobilized over 0.53 mg Dowex MAC-3

beads to 5 ml starch solution. Optimization was done on

the basis of minimum time required to achieve desired DE.

Optimization of liquefaction was done using various starch

concentrations 26, 28, 30, 33, 35 and 37 % w/v (87 �C, pH


123

5.8); temperature 83, 85, 87, 90, 92 and 95 �C (starch

35.0 % w/v, pH 5.8); pH 5.0, 5.4, 5.8, 6.2, 6.5, 6.8, 7.0 and

7.5 (starch 35.0 % w/v, 92 �C). The optimized parameters

were used in the optimization of the next parameters. The

product of liquefaction under optimized condition was

saccharified by AMG (0.5 ml AMG immobilized over

225 mg Amberlite IRA 400 beads). Operational conditions

for saccharification were optimized by varying temperature

50, 55, 60, 65, 68, 70 �C (pH 4) and pH 3.0, 3.5, 4, 4.5, 5.0,

5.5 (68 �C).

Stability of immobilized enzymes

The thermal stability of free and immobilized AAM was

tested by observing the retention of % maximal activity

during the course of hydrolysis. The reaction conditions

were substrate concentration 30 % w/v (free), 35 % w/v

(immobilized), pH 5.8 (free) and 7.0 (immobilized). The

temperature was maintained at 87 �C for free and at 92 �C

for immobilized enzyme.

The thermal stability of free and immobilized AMG

were tested by the retention of maximal activity (%) during

the course of hydrolysis of liquefied starch (10 DE), pH 4.0

(free) and 3.5 (immobilized). The temperature was main-

tained at 60 �C for free and 68 �C for immobilized.

Results and discussion

Characterization of free enzymes

Enzymes were characterized for optimum operational

parameters using soluble starch as substrate. In case of AAM,

it was found that starch concentration 4.16 % w/v, temper-

ature 73 �C and pH 7.0 was optimal for maximum enzyme

activity (Fig. 1a). Kinetic parameters were measured using

Lineweaver–Burk plot. Vmax was found to be 6,219 U mg-1

and KM 0.07 % w/v. In case of AMG it was found that starch

concentration 0.62 % w/v, temperature 50 �C and pH 4.1

was optimal for maximum enzyme activity (Fig. 1b). Kinetic

parameters Vmax was 1.46 U mg-1 and KM 0.09 % w/v were

determined by Lineweaver–Burk plot.

It was observed that the reaction velocity increased with

increase in initial substrate concentration and reached to

saturation. This leveling off of the reaction rate at high

substrate concentrations was owing to the saturation of the

binding sites of enzymes for substrate molecules. At low

temperature, the dominant influence on enzyme catalyzed

reaction was increase in reaction rate with increase in

temperature. Above the optimum temperature, thermal

agitations at the molecular levels tend to disrupt tertiary

structure of the proteins causing denaturation and decrease

in reaction rate. Deviation from optimum pH changed the

ionic character of amino acids disturbing the electrostatic

balance that maintained the biologically active three-

dimensional structure of the enzyme. KM values showed

the affinity between enzyme and substrate and alteration to

lower/higher KM would indicate a higher/lower affinity of

the enzyme for the substrate due to immobilizations.

Immobilization of enzymes and their characterizations

Preliminary screening trials were done to observe the

potential of the different carriers for immobilization of the

Act

ivity

(U m

l-1 )

0

1000

2000

3000

4000

5000

6000

7000

Starch concentrationTemperaturepH

pH

Starch concentration (% w/v)

Temperature (oC)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

pH

0 1 2 3 4 5 6 7

55 60 65 70 75 80 85

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

35 40 45 50 55 60 65 70

3.9 4.0 4.1 4.2 4.3 4.4 4.5


Act

ivity

(U m

l-1 )


Temperature (oC)

a

b

Fig. 1 a Effect of starch concentration (70 �C, pH 7.0), temperature

(starch 4.16 % w/v, pH 7.0) and pH (starch 4.16 % w/v, 73 �C) on

activity of a-amylase (AAM). b Effect of starch concentration (60 �C,

pH 4.0), temperature (starch 0.62 % w/v, pH 4.0) and pH (starch

0.62 % w/v, 50 �C) on activity of glucoamylase (AMG)


123

enzymes. Different resins considered were (a) strong cation

exchange resin, Amberlite IR 120 (H? form), particle size

16–50 mesh; (b) strong anion exchange resin, Amberlite

IRA 400 (OH- form), particle size 16–50 mesh; (c) weak

cation exchange resin, Dowex MAC-3 (H? form), particle

size 16–50 mesh; (d) weak anion exchange resin, Amber-

lite IRA 67 free base, particle size 16–50 mesh. The

immobilizations were carried out by taking 22 mg support

with 0.25 ml AAM (diluted 459) and 0.1 ml AMG for 2 h

(37 �C, pH 7.0 for AAM and pH 4.0 for AMG) and the

beads were air-dried for 2 days. The results of the pre-

liminary screening of the ion-exchange resins are shown in

Table 1. Out of the different ion-exchange resins screened,

the retention of considerable activity was observed only in

case of weak cationic resin Dowex MAC-3 for AAM and in

case of strong cation exchange resin Amberlite IRA 400 for

AMG.

In the subsequent experiment, optimizations of amount

of carrier material to get the maximum immobilization of

the enzymes were carried out. It was found that 0.33 mg

beads (Dowex MAC-3) with 0.25 ml 459 diluted AAM

and 45 mg beads (Amberlite IRA 400) with 0.1 ml AMG

gave the maximum immobilization yield (Fig. 2). The

percentage yields obtained under optimized bead loadings

are summarized in Table 2. Abdel-Naby et al. [20] reported

immobilization yield of 24.6 % in case of physical

absorption of AAM onto AS-alumina and 77.6 % in case of

covalent binding of AAM with chitin. They proposed that

the decrease in the activity upon immobilization may be

attributed to diffusion limitation with macromolecule like

starch as substrate. Ahmed et al. [21] in a study similar to

the present study involving immobilization of AAM on to

cationic resin reported an immobilization yield of 86.4 %.

Immobilization yield obtained in present study are also

much better than recent studies involving immobilization

of amylases using other methods of immobilization [14,

22].

Immobilized enzymes were characterized for optimum

operational conditions. In case of immobilized AAM, it

was found that starch concentration 5 % w/v, temperature

82 �C and pH 7.0 was optimal for maximum enzyme

activity (Fig. 3a). Vmax was 4,373.11 U mg-1 and KM was

0.14 % w/v determined by Lineweaver–Burk plot. In case

of immobilized AMG, starch concentration was 1.0 % w/v,

temperature 62 �C and pH 3.9 was optimal for maximum

enzyme activity (Fig. 3b). Vmax was found to be

0.56 U mg-1 and KM 0.21 % w/v. The optimal operational

Table 1 Activities immobilized a-amylase (AAM) and glucoamylase (AMG) during preliminary screening of ion-exchange resins

Carrier resin Functional group Specific activity of a-amylase

(AAM) (U g carrier-1)

Specific activity of glucoamylase

(AMG) (U g carrier-1)

Amberlite IR 120 (H? form) SO3- 0.024 ± 0.00014 0.001 ± 0.0

Amberlite IRA 400 (OH- form) N? R3 2.154 ± 0.014 0.08 ± 0.000087

Dowex MAC-3 (H? form) COO- 23.0 ± 0.017 0.00 ± 0.0

Amberlite IRA 67 N? R3, N? R2 1.649 ± 0.0098 0.00 ± 0.0

0

20

40

60

80

100

120

140

160

180

200

220

0.020 0.022 0.024 0.026 0.028 0.030 0.032 0.034 0.036 0.038

0.00 0.02 0.04 0.06 0.08 0.10 0.120.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

AMGAAM

Dowex MAC-3 (H+ form) added (mg)

Act

ivity

of

imm

obili

zed

AM

G (

U g

car

rier

-1)

Amberlite IRA 400 (OH- form) added (mg) Act

ivity

of

imm

obili

zed

AM

G (

U g

car

rier

-1)

Fig. 2 Variation of activities of immobilized enzymes with the

amount of carriers added to the enzyme solutions (0.25 ml 459

diluted AAM and 0.1 ml AMG)

Table 2 Optimized immobilization yield of a-amylase (AAM) and glucoamylase (AMG) on ion-exchange resins

Carrier Enzyme added

(U g carrier-1)

Enzyme in

washing

(U g carrier-1)

Enzyme

immobilized

(U g carrier-1)

Immobilization

yield (%)

Weak cation exchange resin Dowex MAC-3

(H? form)

1,237 ± 11.9 (33 mg carrier added

to 0.25 ml 459 diluted AAM)

976.32 ± 9.39 230.08 ± 2.01 88

Strong anion exchange resin Amberlite IRA 400

(OH- form)

3.14 ± 0.11 (45 mg carrier

added to 0.1 ml AMG)

2.71 ± 0.1 0.23 ± 0.008 54


123

parameters of the free and immobilized enzymes are shown

in Table 3. Optimum substrate concentration was higher

for immobilized amylases as compared to that of the free

enzyme due to porous ion-exchange resin immobilization.

The results showed that the temperature optima of immo-

bilized AAM shifted toward higher temperature from 73 to

82 �C and for immobilized AMG shifted from 50 to 62 �C.

A similar increase in temperature optima had been found in

immobilized amylases [23, 24]. El-Batal et al. [25] also

reported increase in optimum temperature of AAM from 40

to 50 �C. In case of binding on the supports, diffusion

effects protects enzyme against heat denaturation. El-Batal

et al. [25] hypothesised that the actual temperature in the

environment of the carrier is lower than in bulk solution,

Hence, the optimum temperatures of the immobilized

enzymes become higher than that of free enzyme. Immo-

bilized enzymes exhibited KM values higher than the free

enzyme due to the lower accessibility of the substrate to the

active site of the immobilized enzyme [25, 26] or due to

variation in microenvironment of immobilized enzyme [27,

28]. Variation in optimum pH as observed may be attrib-

uted to partition effects that cause different concentrations

of charged species (e.g., substrates, products, hydrogen

ions, hydroxyl ions, etc) in the microenvironment of the

immobilized enzyme and in the bulk solution, due to

electrostatic interactions with charges on the support.

Sequential hydrolysis of natural starch

As shown in Table 5, most of the studies related to

immobilization of AAM and AMG deal with their appli-

cation with soluble starch at concentration around 1–2 %.

Secondly most of the studies dealt with efficiency of

individual enzyme for starch hydrolysis. Thus, there always

remain many hurdles in implementing the immobilization

method at industrial scale as in industries very high starch

concentrations are employed and starch hydrolysis take

place in two sequential steps (liquefaction and saccharifi-

cation). The main focus of current study was to employ an

immobilization technique and prove its practical applica-

tion in industrial scenario. Thus, starch was hydrolysed as

0

1000

2000

3000

4000

5000

pH


Act

ivity

(U m

l-1 )


Temperature (oC)

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

pH

0 1 2 3 4 5 6 7

65 70 75 80 85 90 95 100

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

0.2 0.4 0.6 0.8 1.0 1.2 1.4

45 50 55 60 65 70 75

3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5


Act

ivity

(U m

l-1 )


Temperature (oC)

a

b


(starch 5 % w/v, pH 7) and pH (starch 5 % w/v, 82 �C) on activity of

immobilized a-amylase (AAM). b Effect of starch concentration

(50 �C, pH 4.1), temperature (starch 1.0 % w/v, pH 4.1) and pH (starch

1.0 % w/v, 62 �C) on activity of immobilized glucoamylase (AMG)

Table 3 Optimal operational parameters and kinetic properties of free and immobilized enzymes during hydrolysis of soluble starch

Properties a-Amylase (AAM) Glucoamylase (AMG)

Free Immobilized Free Immobilized

Optimum starch concentration (% w/v) 4.16 5.0 0.62 1.0

Optimum temperature (�C) 73.0 82.0 50.0 62.0

Optimum pH 7.0 7.0 4.10 3.9

Vmax (U mg-1 protein) 6,219.68 4,373.11 1.46 0.56

KM (% w/v) 0.07 0.14 0.09 0.21


123

in the industries using the free and immobilized enzymes.

At first, starch was liquefied to 10 DE by AAM and sub-

sequently saccharified to 96 DE by AMG. Parameters for

hydrolysis of natural starch by free and immobilized

enzymes were studied. Starch concentration, temperature

and pH were optimized for minimum time requirement for

liquefaction of starch to reach 10 DE by free and immo-

bilized AAM (Fig. 4a, b). In case of free AAM enzyme

with starch concentration 30.0 % w/v, temperature 87 �C

and pH 5.8, the minimum time required to 10 DE was

65 min, whereas, in case of immobilized AAM, with starch

concentration 35 % w/v, temperature 92 �C and pH 7.0, the

minimum time required to reach 10 DE was 85 min. In

case of saccharification of liquefied starch by free enzyme

AMG, minimum time required was 35 h (Fig. 5a) and 65 h

in case of immobilized enzyme (Fig. 5b). Optimum tem-

perature and pH conditions are tabulated in Table 4. The

time required to reach optimum DE was higher in case of

immobilized enzymes. The results showed that the immo-

bilized enzymes can be used for production of glucose

from natural starch as in industry. The increased time may

be attributed to the decrease in activity of enzyme upon

immobilization. The amount of free enzyme and the


Tim

e R

equi

red

for

10 D

E (

min

utes

)

50

60

70

80

90

100

110

120

Temperature (oC)

pH


80

85

90

95

100

105

110

115

24 26 28 30 32 34 36

65 70 75 80 85 90 95 100

4.5 5.0 5.5 6.0 6.5 7.0 7.5

24 26 28 30 32 34 36 38

82 84 86 88 90 92 94 96

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

Starch concentration TemperaturepHT

ime

Req

uire

d fo

r 10

DE

(m

inut

es)

pH

Temperature (oC)


a

b



time required for liquefaction of natural starch to 10 DE by a-amylase

(AAM). b Effect of starch concentration (87 �C, pH 5.8), temperature


time required for liquefaction of natural starch to 10 DE by

immobilized a-amylase (AAM)

34

36

38

40

42

44

pH

TemperaturepHT

ime

Req

uire

d fo

r 96

DE

(ho

urs)

Temperature (oC)

60

65

70

75

80

85

90

95

45 50 55 60 65 70 75

3.0 3.5 4.0 4.5 5.0 5.5 6.0

45 50 55 60 65 70 75

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

TemperaturepH

Tim

e R

equi

red

for

96 D

E (

hour

s)

pH

Temperature (oC)

a

b

Fig. 5 a Effect of temperature (pH 4.1) and pH (60 �C) on time

required for saccharification of liquefied starch (10 DE) to 96 DE by

glucoamylase (AMG). b Effect of temperature (pH 4.0) and pH

(68 �C) on time required for saccharification of liquefied starch (10

DE) to 96 DE by immobilized glucoamylase (AMG)


123

amount of immobilized enzyme obtained using same

amount of free enzyme were used for starch processing.

Because the amount of enzyme immobilized on carriers

was less as compared to the enzyme used in immobilization

(there was also a fraction of enzyme in washings which

remain unimmobilized as shown in Table 2), the increased

time requirement may be attributed to the actually less

quantity of enzyme in case of immobilized enzyme than

the free enzyme.

It was observed that there were some alterations in

optimum conditions in case of sequential hydrolysis of

natural starch by the free and immobilized enzymes

(Table 4). Optimum temperature for AAM increased from

87 �C in case of free enzyme to 92 �C in case of immo-

bilized enzyme, optimum pH for AAM increased from 5.8

in case of free enzyme to 7.0 in case of immobilized

enzyme. Similarly, optimum temperature and pH for AMG

have also altered. The same reasons as attributed to chan-

ges in optimum parameters in case of soluble starch

hydrolysis may be applicable in this case also.

Two sets of optimization were carried out for immobi-

lized enzymes (mentioned in ‘‘Characterization of immo-

bilized enzyme’’ and ‘‘Studies on the sequential hydrolysis

of natural corn starch by immobilized enzymes’’ in

‘‘Materials and methods’’). As detailed in ‘‘Characteriza-

tion of immobilized enzyme’’ (results shown in Fig. 3a, b),

the purposes of first optimization was (1) compare the

characteristic parameters of immobilized enzymes with the

free enzymes where low concentration soluble starches

were used during characterizations, (2) both the amylases

were characterized independently. This was similar to the

different immobilization studies reported earlier in

literature.

Most of the earlier studies extrapolated the results

obtained using low concentration soluble starch to the sit-

uation in industrial practice directly. However, these con-

ditions are never encountered in industries. To make the

process economical and fast, high concentrations of natural

starch instead of soluble starch are used. Hence, opera-

tional parameters of immobilized enzymes were again

optimized in the similar conditions [i.e. high concentration

(*30 %) of natural starch] as used in the industry. The

results varied from earlier optimizations because (1) the

enzymes followed different kinetic behaviour as the source

and concentration of starch changed, (2) factors, such as

substrate inhibition, steric interference, etc might have

affected the kinetics of the reaction. Another reason for

optimizing again was the inclusion of effect of liquefaction

on saccharification. This was done to mimic the industrial

scenario. In the result shown in Fig. 5b, the substrate used

was the product obtained after liquefaction under opti-

mized conditions (shown in Fig. 4b) and further used for

optimization of saccharification. This is in contrast to the

result shown in Fig. 3b, where stand alone optimization of

AMG was carried out using soluble starch as substrate (not

hydrolysed product by AAM).

Table 4 Optimal operational parameters of free and immobilized enzymes during sequential hydrolysis of natural starch

Properties a-Amylase (AAM) (liquification) Glucoamylase (AMG) (saccharification)

Free Immobilized Free Immobilized

Optimum starch concentration (% w/v) 30.0 35.0 – –

Optimum temperature (�C) 87 92 60 68

Optimum pH 5.8 7.0 4.0 3.5

Res

idua

l act

ivity

(%

)

65

70

75

80

85

90

95

100

105

Immobilized AAMFree AAM

Time (hours)

0 20 40 60 80 100 120 140 160 180

0 20 40 60 80 100

Res

idua

l act

ivity

(%

)

70

75

80

85

90

95

100

105

Immobilized AMGFree AMG

a

b

Fig. 6 a Temperature stability of free and immobilized a-amylase

(AAM). b Temperature stability of free and immobilized glucoam-

ylase (AMG)


123

Thus, Fig. 3a and b showed general characterization and

was used just as starting point for optimization of hydro-

lysis of natural starch (Fig. 4a, b) and also for determining

immobilization efficiency. The actual problem that we tried

to assess was industrial applicability and thus additional

study of Fig. 4a and b was necessary.

Stability of immobilized enzymes

One of the most important considerations in the large scale

application of immobilized enzyme is its stability. As

evident from Fig. 6a and b, the stability of immobilized

enzymes improved upon immobilizations. Immobilized

AAM and AMG retained 95 % of maximum activity after

3 h incubation at 92 �C and 85 % of maximum activity

after 60 h incubation at 68 �C, respectively. Silva et al. [9]

reported 70 % activity retention after 3 h incubation at

50 �C in case of glucoamylase, whereas Tardioli et al. [31]

reported 50 % activity retention after 40 h incubation at

55 �C. In a study similar to ours involving cationic resins

for immobilization of AAM, Ahmed et al. [21] showed that

immobilized enzyme retained 50 % activity after 1 h

incubation at 60 �C. As shown in Table 5, the thermal

stability achieved in current study is far better than many

earlier reports. In case of AAM although the immobiliza-

tion efficiency was not as much as reported in other reports,

but its thermal stability (in same temperature regime) was

far better than shown in other reports. Thus, increased

thermal stabilization may complement the results and

overall efficacy of the immobilization technique is com-

parable (and even better) than those reported earlier. There

does not seem to be a general pattern of response to tem-

perature changes and it is difficult to judge whether dif-

ferences in thermal stability arise from the type of carrier,

the immobilization procedure or the nature of the enzyme

itself. It may be possible that the increase stability resulted

from the prevention of conformational inactivation of the

enzyme and the steric shielding that minimizes attack by

reactive solutes. The nature of the solid phase can also

profoundly influence the stability of the attached enzyme.

It may also be possible that electrostatic interactions

between carrier and enzyme increased the enzyme stability.

The real cause of improvement of stability may be difficult

to determine, but the important factor is that the thermal

stability has improved. Such result may have important

impact on industries using these enzymes.

At the end, one important point is needed to mention

that the enzymes belong to low value; high volume appli-

cation in industry and the immobilization matrices used in

the study are expensive. The practical use of the immobi-

lized enzymes would depend on (1) the effective separation

of the immobilized enzymes from the solution and it is

Table 5 Comparison of present study with earlier studies

Method of immobilization Enzyme Immobilization

efficiency (%)

Starch

used

Stability References

Ionic binding on DEAE cellulose AAM (Bacillusamyloliquifaciens)

92.12 Soluble

starch

(2 %)

50 % activity retention

after 86.5 h

incubation at 60 �C

[13]

Immobilization on Amberlite MB-150 beads AAM (soya bean) 70.4 Starch

(1 %)


after 100 days


[22]

Adsorption on polymer Eudragit S-100 AAM (porcine

pancreas)

59 Soluble

starch

Complete activity lost

within 30 min


[29]

Immobilization on poly aniline grafted magnetic

hydrogel via adsorption/crosslinking

AMG (A. niger) 43 Potato

starch

(0.5 %)


after 2 h incubation at

65 �C

[30]

Immobilization by adsorption and covalent

binding onto poly (o) tolidine

AMG (Rhizopusspp.)

91.5 – 48 % activity retention


55 �C

[8]

Adsorption on ion-exchange resin beads,

a-amylase on Dowex MAC-3 and

amyloglucosidase on Amberlite IRA-400

AAM (Bacilluslicheniformis)

88 Soluble

starch

(4.16 %)



92 �C

Present

study

AMG (A. niger) 54 Soluble

starch

(0.62 %)

85 % activity after 60 h


Raw

starch

(35 %)


123

reuse for number of cycles requiring lesser enzymes, (2)

the regeneration of the matrix for it’s reuse number of

times and reduction in the cost of matrix per unit enzyme,

(3) the enhanced stability of enzymes to be used for longer

duration so that catalytic efficiency would be higher

requiring lesser enzymes, (4) reduction in cost of purifi-

cation of product because of decreased load of protein

contamination due to immobilization of the enzymes.

Conclusion

a-Amylase was immobilized on Dowex MAC-3 and AMG

on Amberlite IRA-400 ion-exchange resin beads by simple

economical adsorption process. The immobilized enzymes

were characterized by kinetic parameters, optimal opera-

tional parameters and thermal stabilities. Optimum sub-

strate concentration and temperature were higher for

immobilized enzymes. Thermal stability of the enzymes

enhanced after the immobilization. Immobilized enzymes

showed the potential for use in production of glucose

through sequential liquefaction and saccharification of

natural starch at high concentration (35 % w/v) as done in

industry.

References

1. Souza PMD, Magalhaes PDOE (2010) Braz J Microbiol

41:850–861

2. Vihinen M, Mantsala P (1990) Biotechnol Appl Biochem

12:427–435

3. Richetti A, Munaretto CB, Lerin LA, Batistella L, Oliveira VJ,

Dallago RM, Astolfi V, Luccio MD, Mazutti MA, Oliveira DD,

Treichel H (2012) Bioprocess Biosyst Eng 35:383–388

4. Bayramoglu G, Yilmaz M, Yakup Arica M (2010) Bioprocess

Biosyst Eng 35:439–448

5. Svec F, Kalal J, Menyailova II, Nakhapetyan LA (1978) Bio-

technol Bioeng 20:1319–1328

6. Lai J-T, Wu S-C, Liu H-S (1998) Bioprocess Eng 18:155–161

7. Oh JT, Kim JH (2000) Enzyme Microb Technol 27:356–361

8. Ashly PC, Mohanan PV (2010) Process Biochem 45:1422–1426

9. Silva RN, Asquieri ER, Fernandes KF (2005) Process Biochem

40:1155–1159

10. Khan M, Husain Q, Azam A (2012) Biotechnol Bioprocess Eng

17:377–384

11. Wang J, Zhao G, Li Y, Liu X, Hou P (2013) Appl Microbiol

Biotechnol 97:681–692

12. Shah C, Sellappan S, Madamwar D (2000) Process Biochem

35:971–975

13. Kikani BA, Pandey S, Singh SP (2012) Bioprocess Biosyst Eng

1–11 (Epub ahead of print)

14. Jaiswal N, Prakash O, Talat M, Hasan SH, Pandey RK (2012) J

Genet Eng Biotechnol 10:161–167

15. Tanriseven A, Olcer Z (2008) Biochem Eng J 39:430–434

16. Miller GL (1959) Anal Chem 31:426–428

17. Lowry BH, Rosebrough NJ, Farr AL, Randall RJ (1951) J Biol

Chem 193:265–275

18. Crabb WD, Shetty JK (1999) Curr Opin Microbiol 2:252–256

19. Lane JH, Eynon L (1923) J Soc Chem Ind 42:32T–37T

20. Abdel-Naby MA, Hashem AM, Esawy MA, Abdel-Fattah AF

(1999) Microbiol Res 153:319–325

21. Ahmed SA, Mahdy ESME, Hassan O-K (2008) Aust J Basic Appl

Sci 2:466–474

22. Kumari A, Kayastha AM (2011) J Mol Catal B Enzyme 69:8–14

23. Yagar H, Ertan F, Balkan B (2007) Prep Biochem Biotechnol

38:13–23

24. Pandya PH, Jasra RV, Newalkar BL, Bhatt PN (2005) Micro-

porous Mesoporous Mater 77:67–77

25. El-Batal AI, Atia KS, Eid M (2005) Radiat Phys Chem

74:96–101

26. Kara A, Osman B, Yavuz H, Besirli N, Denizli A (2005) React

Funct Polym 62:61–68

27. Liu H, Liu J, Tan B, Zhou F, Qin Y, Yang R (2012) Bioprocess

Biosyst Eng 35:1287–1295

28. Dhingra S, Khanna M, Pundir CS (2006) Indian Chem Tech

13:119–121

29. Sardar M, Agarwal R, Kumar A, Gupta MN (1997) Enzyme

Microb Technol 20:361–367

30. Bayramoglu G, Altintas B, Arica MY (2013) Appl Microbiol

Biotechnol 97:1149–1159

31. Tardioli PW, Vieira MF, Vieira AMS, Zanin GM, Betancor L,

Mateo C, Fernandez-Lorente G, Guisan JM (2011) Process Bio-

chem 46:409–412


123

Documents

Immobilization of α-amylase and amyloglucosidase onto ion-exchange resin beads and hydrolysis of natural starch at high concentration