Chapter 5

129

OPTIMIZATION OF LACCASE IMMOBILIZATION BY

POLYACRYLAMIDE GEL USING TAGUCHI DOE METHODOLOGY

ABSTRACT

In this chapter immobilization of laccase (LCC2) by polyacrylamide gel

(PAG) was optimized using Taguchi DOE methodology. This methodology optimizes

critical factors involved in the immobilization of enzyme like initiator and stimulator

to entrap the enzyme without appreciable loss of enzyme activity.

In the immobilization process, four factors viz, purified laccase LCC2,

acrylamide/bisacrylamide monomer (ABM) solution, ammonium persulphate (APS)

and N, N, N’, N’- tetramethyl-ethylenediamine (TEMED) in three levels with an OA

layout of L 9 (34) were selected for the experimental design. In total, nine experiments

were conducted with different combinations of four factors and the obtained

percentage of immobilization were analyzed by ANOVA available in the Qualitek-4

software at “bigger is better” as quality character. The optimized conditions shared an

enhanced immobilization percentage of 12.4% in the immobilized state. Individual

levels of various factors in the optimized condition are laccase (LCC2) 2.0 U, ABM

solution 0.85 ml, APS 80 mM and TEMED 10 mM. The contributions of various

factors involved in the optimized immobilized laccase in PAG are as follows, purified

laccase (LCC2) 41.34%, TEMED 39.16%, ABM solution 16.81% and APS 2.64%.

The optimized immobilized laccase was finally checked for its solvent and

thermostablility.

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130

5.1. INTRODUCTION

Enzymes exhibit a number of features and applications that make their use

advantageous as compared to conventional chemical catalysts. The idea of performing

an enzymatic reaction in non-aqueous media and high temperature seemed almost

heretical, but during the last few years, applications of enzymes for catalyzing various

chemical processes in water/organic media has become a widely used approach to

obtain biologically active compounds. However, despite numerous examples of

successful use of biocatalysts in non-aqueous media, removal of vicious effects of

organic solvents on enzymes still remains a problem. As is well known, direct contact

of enzyme molecules with an organic solvent affects the catalytically active

conformation of the enzyme. Immobilization of enzymes is one of the most hopeful

techniques to prevent enzymes from inactivation in organic solvents [Markvicheva et

al., 2005].

There are several reasons for using an enzyme in an immobilized form. Free

enzymes have limitation in industrial applications such as their high cost of isolation

and purification, their non-reusability, the instability of their structures, contamination

of products, thermal instability and their sensitivity to harsh process conditions

[Guisan et al., 1981; Taylor, 1991]. Moreover, enzyme immobilization has been

revealed in the last times as a very powerful tool to enhancing almost all enzyme

properties, if properly designed: e.g., stability, activity, specificity and selectivity,

reduction of inhibition [Blanco et al., 2007]. The broad substrate specificity of

laccases holds promise to use them for biotechnological purposes such as

biomechanical pulping, bleaching of pulp, degradation of dye and transformation and

detoxification of xenobiotic and other aromatic compounds [Nyanhongo et al., 2002;

Patel et al., 2008]. Moreover, immobilized laccase showed a broadening of the

Chapter 5

131

temperature range of enzyme activity [Al-Adhami et al., 2002; Dodor et al., 2004].

Consequently, many supports have been proposed for immobilization of laccase

[Duran et al., 2002]. Recently various immobilization methods of laccase have been

reported, including macroporous exchange resins [Zhang et al., 2010] and

diatomaceous earth support celite [Hubert et al., 2009]. Among the various

immobilization processes, entrapment is one of the simplest methods of

immobilization under milder conditions, and therefore, results in minimum

denaturation of the biocatalyst during the process.

The aim of the present study was to optimize the immobilization of laccase

(LCC2) in polyacrylamide gel using Taguchi DOE methodology and to analyze the

competence of solvent and thermostability of immobilized laccase.

5.2. MATERIALS AND METHODS

5.2.1. Chemicals

Acrylamide, ammonium per sulphate, N, N, N, N’- methylene bisacrylamide

(BlS), and TEMED were purchased from Himedia (Mumbai, India). Unless otherwise

stated all chemicals were purchased from s d fine-chem Limited, India and all other

chemicals were of analytical grade.

5.2.2. Different methods of laccase immobilization

Immobilization of laccase* by alginate gel was carried out according to the

method of Lu et al. [2007b]. 2% sodium alginate and 2% calcium chloride was used

to prepare the alginate immobilized laccase.

Immobilization of laccase* by gelatin was carried out according to the method

of Crecchio et al. [1995]. 2.5% gelatin was used to immobilize the laccase.

Chapter 5

132

Immobilization of laccase* (LCC2) by polyacrylamide gel was carried out

according to the method of Trevan and Grover [1979]. Buffered Monomer Solution

(2.0 g of acrylamide and 0.1 g of bisacrylamide in 10 ml of 100 mM sodium

phosphate buffer [pH 6.0]) was used to immobilize the laccase.

* Laccase represents purified laccase isoenzyme (LCC2).

5.2.3. Determination of immobilization percentage

To validate the effectiveness of immobilization in the matrix, immobilization

percentage was determined according to the following equation.

Immobilization (%) =

Aload = total loaded activity into the mixture of polymer solution assayed using

guaiacol as substrate.

Awash = laccase activity detected in curing solution and two washing solution

assayed using the same substrate.

5.2.4. Taguchi DOE methodology

The Taguchi method involves the establishment of a large number of

experimental situations described as orthogonal arrays (OA) to reduce experimental

errors and to enhance the efficiency and reproducibility of laboratory experiments.

Kindly refer the second chapter for details (2.2.2).

5.2.5. Experimental design

In the immobilization process priority was given to immobilize the laccase in

polyacrylamide gel with minimum loss of activity and having more durability. Based

Aload –Awash

Aload

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133

on the obtained experimental data from our initial studies, four factors were selected

for the optimization of laccase immobilization in PAG. The selected three levels of

four factors were as follows; laccase (LCC2), ABM solution, initiator; ammonium

persulphate and stimulator; TEMED. All the factors were assigned with three levels,

with a layout of L9 (34) are shown in table 5.2. The details of the individual

combinations of the 9 experimental trials and their obtained results for the

immobilization percentage are shown in table 5.3. The obtained results were further

processed with Qualitek-4 software at bigger is better as quality character.

5.2.6. Immobilization of laccase

The concentration of the ABM solution used for the immobilization of purified

laccase LCC2 was adopted from the protocol of Trevan and Grover [1979] with minor

modification. The total volume of the reaction mixture was made up to 3.5 ml

uniformly and it was poured into a specific block to cast the gel. Usually casting of

the gel takes 25 minutes. After polymerization, the gel was washed twice with 100

mM sodium phosphate buffer pH 6.0 to remove unpolymerized gel and

unimmobilized enzyme. Then the gel was cut uniformly to get uniform size for further

analysis.

5.2.7. Laccase assay

Laccase activity was determined using guaiacol as the substrate according to the

method of Sandhu and Arora [1985]. Kindly refer the first chapter for details (1.2.6).

5.2.8. Qualitek-4 software

The Qualitek-4 software (Nutek Inc .MI) allows designing experiments using

any of the L-4 to L-81, L-16 and L-18 (modified) arrays. Kindly refer the second

chapter for details (2.2.7).

Chapter 5

134

5.2.9. Thermal stability

Thermal stability of the optimized immobilized laccase (LCC2) was

determined after 30 min incubation at different temperature ranges from 50 to 80 °C.

Residual activity was determined with guaiacol as a substrate in 100 mM sodium

phosphate buffer pH 6.0.

5.2.10. Solvent stability

The effect of organic solvent such as ethanol, methanol and isobutyl alcohol

on immobilized laccase (LCC2) were determined. The optimized immobilized laccase

(LCC2) was incubated with respective organic solvents (final concentration 10% v/v)

for 30 min at room temperature and the percentage of residual activity was measured.

Free enzyme served as control.

5.3. RESULTS

Various methods of immobilization of the purified laccase were tried to retain

their maximum activity. The efficiency of the immobilization was determined on the

basis of immobilization percentage (Table 5.1). Among the different materials studied

polyacrylamide gel entrapment provides excellent protection to laccase (LCC2). To

improve the immobilization percentage of the laccase (LCC2) in PAG, the

immobilization parameters were further optimized. Table 5.2 shows the selected

factors and their assigned levels for laccase immobilization in PAG. Table 5.3 shown

the variation in immobilization percentage according to the experiments conducted

based on the Taguchi DOE method. The average effect of the factors, along with

interaction at the assigned levels on the immobilization percentage are shown in table

5.4, in which initiator APS shows highest in level 1, but in level 2 and level 3 laccase

and TEMED showed highest effect respectively. It is well known that larger the

Chapter 5

135

difference, the stronger is the influence. The difference between level 2 and level 1

implies the influence of the factors in immobilization of enzyme. Among the factors

and their levels studied on the immobilization, laccase and TEMED showed stronger

influence (L2-L1), when compared to other factors studied viz. ABM solution and

APS. It can be seen from table 5.5, highest two severity index (48.5% and 40.7%)

were observed might be due to the interaction of laccase with APS and ABM solution

respectively.

Figure 5.1 shows the variation of immobilization percentage at chosen levels.

ANOVA with the percentage of contribution of each factor with interactions are

shown in table 5.6. It can be observed from the table laccase (LCC2) and ABM

solution are the most significant factor in the immobilization process.

Figure 5.2 shows the contribution of individual factors for achieving the optimum

immobilization percentage. Optimum condition and their performance in terms of

contribution for achieving effective immobilization are shown in table 5.7. The

maximum contribution was given by purified laccase (LCC2). Figure 5.3 shows the

activity of optimized immobilized laccase in PAG.

Figure 5.4 shows the thermal stability of the free laccase; unoptimized

immobilized laccase and optimized immobilized laccase were studied in the range

from 50 to 80 °C. At 60 °C when compared with free laccase unoptimized

immobilized laccase lose its 45.8% of residual activity, but the loss of residual

activity in optimized immobilized laccase was 25%. Moreover, at 70 °C residual

activities of the optimized and unoptimized laccase were higher than free laccase.

Optimized immobilized laccase shows 13.3% increased residual activity than

unoptimized immobilized laccase at 70 °C.

Chapter 5

136

In order to determine the stability of immobilized laccase in organic solvents,

three solvents viz ethanol, methanol and isobutyl alcohol were selected as shown in

the table 5.8. In all the solvents optimized immobilized laccase retains maximum

activity when compared with free laccase. In the case of methanol both optimized and

unoptimized immobilized laccase retains 52±0.16% and 57±0.61% residual activity,

while free laccase have only 35±0.12% of activity.

5.4. DISCUSSION

When compared with alginate gel and gelatin gel the immobilization

percentage was good in polyacrylamide gel. Among the above three methods,

immobilization of laccase in polyacrylamide gel showed 75% of immobilization in

the matrix (Table 5.1). The advantage of immobilization of laccase (LCC2) by

polyacrylamide entrapment is quickness of the process, minimum bonding with

enzyme and high durability. The key factors and their levels (Table 5.2) selected for

the present study were identified based on the earlier data obtained from our

laboratory experiments and previous references in connection to this study [Fournier

et al., 1996; Ge et al., 1998; Demirel et al. 2006]. As per the Taguchi methodological

design, 9 experimental trials with combination of four factors at three levels were

carried out and the results obtained from each set defined the immobilization

percentage (Table 5.3).

According to the table 5.4 increasing the unit of laccase (LCC2) from 1.5 U to

2.0 U leads to 16.8% increase in the activity of the immobilized laccase. Further

increase of laccase (LCC2) unit did not show any significance in immobilization

percentage. This may be due to the overcrowding of enzyme in the polymer matrix

which affects the activity of the enzyme. The above statement was correlated with the

statement of Hotchkis et al. [2010]. Loss of the enzyme activity with increased

Chapter 5

137

loading has been observed on other supports and has been attributed to crowding of

the enzyme at the surface which leads to spatial restrictions, limited active site

accessibility, or denaturing of the protein [Hotchkis et al., 2010].

When the volume of ABM solution in the immobilization process was

increased from 0.75 ml to 0.85 ml it leads to increase in the activity of the enzyme by

10.4%. Subsequent increase in the volume reduces the immobilization percentage to

1.5%, this may be due to excess amount of polymer in the matrix which interferes

with the activity of the enzyme during polymerization. According to the table 5.5

second highest severity index was obtained when laccase (LCC2) interact with ABM

solution shows its significance in the immobilization process. As per the table 5.6

laccase (LCC2) and ABM solution are the most important two significant factors to

achieve optimum immobilization percentage.

Increasing the concentration of APS above the optimum level leads to

decrease the immobilization percentage this may due to the excess free radicals

produced by the initiator during the polymerization process. According to Delbem et

al. [2002] enzyme immobilization can be also difficult because enzymatic activity

may decrease as a consequence of enzyme/free radical reactions. Figure 5.1, depicts

relative influence of factors in the activity of immobilized laccase at chosen levels.

It is evident from the figure 5.2 that upon considering the optimum levels of

factors from the experiment designed the immobilization percentage can be increased

to 19.6%. To validate the proposed experimental methodology, immobilization

experiments were carried out according to the table 5.7, showed an increased

immobilization percentage of 87.4 from 75 (12.4% improved in immobilization

percentage). Figure 5.3 shows the laccase (LCC2) entrapped in optimized PAG

matrix.

Chapter 5

138

Finally the optimized immobilized laccase was subjected to temperature and

organic solvent stability. In the case of thermostability, optimized immobilized

laccase retains high activity at 70 °C and 80 °C, when compared to free laccase

(Figure 5.4). In all cases, the immobilized laccase showed higher stability at

temperatures higher than 60 °C than the free one. From the experimental data it was

proved that optimization of immobilization may increase the thermostability of

immobilized laccase in PAG. In general, the immobilization support has a protecting

effect at high temperatures when enzyme deactivation occurs. The conformational

flexibility of the enzyme is affected by immobilization. The immobilization step

causes an increase in enzyme rigidity, commonly reflected by an increase in stability

towards denaturation by raising the temperature [Abdel-Naby, 1993].

In the case of free laccase, the stability in these organic solvents is low when

compared with immobilized laccase (table 5.8). This may due to their higher dielectric

constant of these solvents towards free enzyme. The immobilized laccase may

maintain its activity in organic solvents due to the entrapment of laccase in PAG

which reduce the unfolding of the three dimensional structure of protein by the

organic solvents. The above results are in good agreement with the results of Roy and

Abraham [2006].

5.5. CONCLUSION

Based on above optimization of immobilization of laccase by polyacrylamide

gel, the performance of the immobilized biocatalysts described in this work was

satisfactory. A satisfactory prediction for the immobilization of enzyme was derived

and demonstrated. This immobilization procedure did not affect the enzyme

specificity. This model could be used to predict the effect of individual factors and

their effects in the immobilization process. The ratio between polymer matrix and

Chapter 5

139

concentration of laccase plays crucial role in the immobilization process. This process

is easy immobilization procedure, required very minimum time; desired shape of the

immobilized enzyme can be fabricated and maintains good stability while use. To our

knowledge, this is the first report on immobilization of laccase on polyacrylamide gel.

Moreover after the optimization the immobilized shows both temperature and organic

solvent stability which is a preferable quality for industrial applications. The simple

immobilization method may be a viable alternative to using more expensive

commercial beads or the more brittle alumina and silica beads. Our immobilization

method has following advantages over various matrices reported in literature (1)

inexpensive starting materials, (2) a rapid simple method and (3) minimum loss of

activity.

Chapter 5

140

10

20

30

40

50

60

70

80

90

100

Aver

age

Lac

case

(LC

C2)

TE

ME

D

AB

M

AP

S

Immobilization factors

Imm

ob

iliz

atio

n (

%)

Average Laccase(LCC2) ABM APS TEMED

Figure.5.1. Relative influence of factors in the laccase immobilization.

Figure 5.2. Optimum performance with the major factors contribution of

laccase immobilization.

TEMED APS ABM Laccase(LCC2) Error

Chapter 5

141

Figure 5.3. Immobilized laccase (LCC2) in polyacrylamide matrix and their

activity.

Figure 5.4. Temperature profiles of the free, unoptimized immobilized PAG

laccase and optimized immobilized laccase. Data were obtained from

three replicates, standard deviation values were less than 3%.

0

10

20

30

40

50

60

70

80

90

100

20 30 40 50 60 70 80

Temperature

Resi

du

al

acti

vit

y (

%)

Free laccase (LCC2)

unoptimized immobilized laccase (LCC2)

optimized immobilized laccase (LCC2)

(ºC)

Chapter 5

142

Table 5.1. Immobilization percentage of laccase by different methods

Immobilization matrix Concentration Immobilization percentage

Alginate 2% (w/v) 65%

Gelatin 2.5% (w/v) 50%

Polyacrylamide (20:1) 21% 75%

Table 5.2. Selected condition factors and assigned levels

Factor Level 1 Level 2 Level 3

Laccase (LCC2) (U) 1.5 2.0 2.5

ABM solution (ml) 0.75 0.85 0.95

APS (mM) 50 80 100

TEMED (mM) 8 10 12

Chapter 5

143

Table 5.3. L9 (34) orthogonal array of designed experiments

Experiment

No

Column Immobilization

percentage* 1 2 3 4

1 1 1 1 1 55.52 0.4

2 1 2 2 2 80.65 0.5

3 1 3 3 3 72.24 0.5

4 2 1 2 3 81.68 0.4

5 2 2 3 1 78.70 0.7

6 2 3 1 2 90.05 0.6

7 3 1 3 2 76.5 0.4

8 3 2 1 3 79.14 0.4

9 3 3 2 1 72.65 0.6

*Mean SD, n =3

Table 5.4. Main effects of selected factors for laccase immobilization

Factors L1 L2 L3 L2-L1

Laccase (LCC2) (U) 69.473 83.482 76.098 14.009

ABM solution (ml) 71.236 79.503 78.314 8.266

APS (mM) 74.907 78.331 75.815 3.424

TEMED (mM) 68.959 82.403 77.691 13.444

Chapter 5

144

Table 5.5. Estimated interaction of severity index for different factors

Factors Columns SI (%) Reserved

column Levels

Laccase (LCC2) x APS 1 × 3 48.5 2 [2,1]

Laccase (LCC2) x ABM solution 1 × 2 40.7 3 [2,3]

APS x TEMED 3 × 4 38.4 7 [1,2]

ABM solution x APS 2 × 3 35.69 1 [3,1]

ABM solution x TEMED 2 × 4 27.55 6 [3,2]

Laccase (LCC2) x TEMED 1 × 4 19.96 5 [2,2]

Table 5.6. Analysis of variance (ANOVA)

Factors DOF Sum of

squares Variance F Ratio Pure sum Percentage

Laccase (LCC2) 2 883.984 441.992 10712.525 883.901 41.335

ABM solution 2 359.539 179.769 4357.068 359.457 16.809

APS 2 56.626 28.313 686.22 56.543 2.644

TEMED 2 837.473 418.736 10148.881 837.39 39.16

Other error 18 0.742 0.041 0.052

Total 26 2138.366 100

Chapter 5

145

Table 5.7. Optimum conditions and their contribution in laccase immobilization

Factors Values Level Contribution

Laccase (LCC2) (U) 2.0 2 7.13

ABM solution (ml) 0.85 2 3.151

APS (mM) 80 2 1.979

TEMED (mM) 10 2 6.051

Total contribution from all factors 18.31

Current grand average of performance 76.351

Expected result at optimum condition 94.662

Table 5.8. Comparative organic stability of Pleurotus ostreatus IMI 395545 free

purified laccase (LCC2), unoptimized PAG immobilized purified

laccase (LCC2) and optimized PAG immobilized purified laccase

(LCC2)

Solvent

Final

Concentration

(%)

Residual activity percentage (%)

Free laccase

(LCC2)

Unoptimized

immobilized

laccase (LCC2)

Optimized

immobilized

laccase (LCC2)

Ethanol 10 27±0.31 38±0.42 45±0.23

Methanol 10 35±0.22 52±0.16 57±0.61

Iso butyl

alcohol

10 18±0.24 29±0.50 36±0.41