548 Journal of Basic Microbiology 2010, 50, 548 – 556

Research Paper
Optimization of laccase production by Pleurotus ostreatus
IMI 395545 using the Taguchi DOE methodology

Rathinasamy Periasamy and Thayumanavan Palvannan

Department of Biochemistry, Periyar University, Salem, Tamil Nadu, India

Production of laccase using a submerged culture of Pleurotus orstreatus IMI 395545 was opti-
mized by the Taguchi orthogonal array (OA) design of experiments (DOE) methodology. This
approach facilitates the study of the interactions of a large number of variables spanned by
factors and their settings, with a small number of experiments, leading to considerable savings
in time and cost for process optimization. This methodology optimizes the number of impact
factors and enables to calculate their interaction in the production of industrial enzymes. Eight
factors, viz. glucose, yeast extract, malt extract, inoculum, mineral solution, inducer (1 mM
CuSO4) and amino acid (L-asparagine) at three levels and pH at two levels, with an OA layout of
L18 (21 × 37) were selected for the proposed experimental design. The laccase yield obtained
from the 18 sets of fermentation experiments performed with the selected factors and levels
was further processed with Qualitek-4 software. The optimized conditions shared an enhanced
laccase expression of 86.8% (from 485.0 to 906.3 U). The combination of factors was further
validated for laccase production and reactive blue 221 decolorization. The results revealed an
enhanced laccase yield of 32.6% and dye decolorization up to 84.6%. This methodology allows
the complete evaluation of main and interaction factors.

Keywords: Laccase / Taguchi DOE methodology / Pleurotus ostreatus IMI 395545 / Optimization /
Dye decolorization

Received: March 14, 2010; accepted: April 29, 2010

DOI 10.1002/jobm.201000095

Introduction* applications like wastewater detoxification [5, 6], deter-

Laccase (benzenediol:oxygen oxidoreductase [EC.1.10.3.2])
belongs to a group of enzymes called blue copper oxi-
dases capable of oxidizing phenols and aromatic amines
by reducing molecular oxygen to water. Laccase is
widespread in nature and has been found in plants,
fungi, bacteria and insects [1]. Laccase plays an impor-
tant role in the global carbon cycle and could help in
degrading a wide range of xenoaromatics such as tex-
tile dyes [2], polychlorinated biphenyls, polycyclic aro-
matic hydrocarbons, pesticides and synthetic polymers
[3, 4]. Extensive studies made on fungal laccase have
proved its potential in the various fields of biotechnol-
ogy and created a great market demand for commercial
Correspondence: Dr. T. Palvannan, Department of Biochemistry, Peri-
yar University, Salem – 636 011, Tamil Nadu, India
E-mail: pal2912@yahoo.com
Phone: +91-427-2345766, -2345520
Fax: +91-427-2345124
gent manufacturing and transformation of antibiotics
and steroids [7]. The wide range of applications of lac-
case in the biotechnological and textile industries cre-
ates the need for large amounts of enzymes at low cost
to meet the market demand.
The main limitation for the extensive industrial ap-
plication of laccase is its high cost. To attain the pro-
duction of a large amount of enzyme at low cost, media
optimization plays a crucial role. The optimization of
fermentation media to generate a balanced proportion
of various nutrients is very important to get optimum
microbial growth and enzyme yield [8]. Laccase expres-
sion by fungi is found to be influenced by the applied
culture conditions, such as the nature of the carbon
source, the concentration of a carbon source, the pH of
the fermentation broth, the presence of inducers, the
presence of lignocellulosic materials and a nitrogen
source [5, 9 – 11]. It is essential to optimize all the cul-
ture conditions and the composition of the production

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Journal of Basic Microbiology 2010, 50, 548 – 556
medium to enhance the yield of enzyme. Considering
the potential applications of laccase in various biopro-
cesses and the market demand, the present study was
undertaken to explore a novel economic design of the
full-scale submerged fermentation process. Enzyme
overproduction can be achieved by optimizing all me-
dia parameters. To establish best possible conditions
numerous experiments have to be carried out with all
possible parameter combinations. This mode of media
engineering is uneconomical and practically not feasi-
ble.
Traditional methods of optimization have involved
changing one independent variable while fixing the
others at a certain level. This single-dimensional search
is laborious, time consuming, and incapable of reaching
a true optimum due to interaction among variables
[12]. A number of statistical experimental designs have
been studied for bioprocess optimization. The Taguchi
method of orthogonal array (OA) design of experiments
(DOE) involves the study of any given system by a set of
independent variables (factors) over a specific region of
interest (levels) [13, 14]. This methodology simplifies
the complicated optimization bioprocess to the sim-
plest one, which can easily identify the impact of indi-
vidual factors, establishing the relationship between
variables and operational conditions. Statistical signifi-
cance of the experimental results was validated with
ANOVA (analysis of variance) and makes the analysis
very precise. Hence, this methodology was greatly ap-
preciated for less production cost, time savings, high
standard and its systematic process for the optimiza-
tion of the near-optimum design parameters with lim-
ited experimental sets [15 – 17]. This methodology has
been used for various bioprocess applications [18 – 21]
and gives excellent results for the optimization of a few
biochemical techniques [22, 23].
The present work describes the optimization of sub-
merged-culture conditions for laccase production by the
newly identified species Pleurotus ostreatus IMI 395545,
using a methodological application of the Taguchi ex-
perimental design. The experimental setup was based
on an OA layout of L18 (21 × 37), selecting one factor
(pH) with two levels and the remaining seven factors
(glucose, yeast extract, malt extract, mineral solution,
inoculum, inducer and amino acid) with three levels.
Materials and methods
White-rot fungi
Pleurotus ostreatus IMI 395545(CABI UK Centre, Egham,
UK) was isolated from the deep forest of the Kolli hills
© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Optimization of laccase production by Pleurotus ostreatus 549
located in the Namakkal District, Tamil Nadu, India.
The isolated organism was systematically identified and
authorized by the Commonwealth Agricultural Bu-
reaux International (CABI), London, UK. The fungi were
maintained on malt extract agar plates, stored at 4 °C
and subcultured periodically [24].
Taguchi DOE methodology
DOE using the Dr. Genechi Taguchi approach attempts
to improve the quality defined as the consistency of
performance, to optimize the process designs and fin-
ished products, to study the effects of multiple factors
(i.e. variables, parameters, ingredients, etc.) on the per-
formance, and to solve production problems by objec-
tively laying out the investigative experiments [25]. It
was introduced in the USA in the early 1980s and can
economically satisfy the needs of problem solving and
product/process design optimization projects. The Ta-
guchi method of DOE analysis involves a large number
of experimental situations described as OA to reduce
experimental errors and to enhance their efficiency in
concordance with reproducibility of the experimental
results. The Taguchi method of DOE analysis helps us
to determine the relationship between variables of
medium components and to optimize their concentra-
tions, in four different phases [26, 27].
Experimental design
The first phase focused on the composition of the fac-
tors to be optimized in the culture medium that have a
critical effect on the laccase yield. Fungal laccase pro-
duction is influenced by many typical culturing pa-
rameters, such as medium composition, carbon and
nitrogen ratio, temperature, pH and aeration ratio [28].
The nature and type of the carbon and nitrogen sources
are among the most important factors that contribute
to any fermentation process [29]. Based on the obtained
experimental data from our initial studies, eight factors
were selected for the production of laccase by Pleurotus
ostreatus IMI 395545, which are shown in Table 1.
The second step was to design the matrix experiment
and to define the data analysis procedure. Taguchi pro-
vides many standard OA and corresponding linear
graphs for this purpose [26]. Three levels of factor va-
riation were considered and the size of experimenta-
tion was represented by the symbolic array L18. All the
factors except for pH (21) were assigned with three lev-
els, with a layout of L18 (21 × 37) as shown in Table 1.
The total degree of freedom is equal to the number of
trails minus 1, i.e. 17. In this study, the experiments
were carried out in cotton-plugged 250 ml Erlenmeyer
flasks containing 100 ml production medium (in g/100 ml
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550 R. Periasamy and T. Palvannan Journal of Basic Microbiology 2010, 50, 548 – 556

Table 1. Selected culture condition factors and assigned levels.

Serial no. Factors Level 1 Level 2 Level 3

1 pH 5.5 6.0
2 glucose [g/100 ml] 1.0 1.5 2.0
3 yeast extract [g] 0.250 0.375 0.500
4 malt extract [g] 0.175 0.262 0.350
5 mineral solution [ml/100 ml] 10 20 30
6 inoculum [ml] 0.1 0.2 0.5
7 inducer (CuSO4) [mM] 0.5 1.0 1.5
8 amino acid (L-asparagine) [mg/100 ml] 1.0 2.0 3.0

Table 2. L18 (2 × 3 ) orthogonal array of designed experi-

1 7
distilled water): glucose (1.0, 1.5, and 2.0); yeast extract
ments.
(0.250, 0.375, and 0.500); malt extract (0.175, 0.262, and
0.350); mineral solution (10, 20, and 30 ml/100 ml); Experiment 1 2 3 4 5 6 7 8 Laccase
amino acid L-asparagine (0.1, 0.2, and 0.3); and inducer no. activity [U]
Column (factors)
CuSO4 (0.5, 1.0, and 1.5 mM). The pH of the production
medium was adjusted to 5.5 or 6.0 with 2 N HCl prior 1 1 1 1 1 1 1 1 1 141.0 ± 0.4
2 1 1 2 2 2 2 2 2 263.6 ± 0.6
to sterilization. The composition of the mineral solu- 3 1 1 3 3 3 3 3 3 314.3 ± 0.5
tion was as follows (in g/L): K2HPO4 5; NaH2PO4 0.1; 4 1 2 1 1 2 2 3 3 355.0 ± 0.4
MgSO4 0.5; CaCl2 0.02; FeSO4 0.01; MnSO4 0.02; MnSO4 5 1 2 2 2 3 3 1 1 428.6 ± 0.4
0.02; ZnSO4 0.02; dissolved in 1 l distilled water. Pro- 6 1 2 3 3 1 1 2 2 569.6 ± 0.6
7 1 3 1 2 1 3 2 3 517.1 ± 0.7
duction medium and inducer were sterilized by auto- 8 1 3 2 3 2 1 3 1 687.3 ± 0.7
claving for 15 min at 121 °C with 15 lbs pressure. The 9 1 3 3 1 3 2 1 2 482.6 ± 0.5
inducer CuSO4 was added to the production medium 10 2 1 1 3 3 2 2 1 347.2 ± 0.3
11 2 1 2 1 1 3 3 2 388.0 ± 0.4
after 240 h of cultivation to reduce its effect during the 12 2 1 3 2 2 1 1 3 424.3 ± 0.4
initial phase of fungal growth during fermentation. 13 2 2 1 2 3 1 3 2 520.1 ± 0.6
14 2 2 2 3 1 2 1 3 589.0 ± 0.6
Inoculum preparation 15 2 2 3 1 2 3 2 1 722.6 ± 0.8
16 2 3 1 3 2 3 2 2 786.3 ± 1.2
The inoculum was prepared by fungal cultivation on a 17 2 3 2 1 3 1 2 3 624.0 ± 0.5
rotary shaker at 150 rpm in 250 ml flasks containing 18 2 3 3 2 1 2 3 1 570.5 ± 0.4
100 ml basal medium (in g/l): glucose 10; KH2PO4
0.8; NH4NO3 2; Na2HPO4 0.4; MgSO4 ⋅ 7 H2O 0.5; yeast
extract 2. The following microelements were added to which is used to determine the optimum culture condi-
the basal medium (in g/l): ZnSO4 ⋅ 7 H2O 0.001; tion for maximum enzyme production. The final step
FeSO4 ⋅ 7 H2O 0.005; CaCl2 ⋅ 2 H2O 0.06; CuSO4 ⋅ 7 H2O of the Taguchi method of DOE analysis is validation by
0.005; MnSO4 ⋅ 7 H2O 0.005. After 7 d of fungal cultiva- the potential of decolorizing the reactive dye.
tion, mycelial pellets were harvested and homogenized
with a Waring laboratory blender, three times for 20 s Dye decolorization experiment
with 1 min intervals [30]. The optimized culture medium was finally evaluated by
The laccase activity was determined by using the the dye decolorization potential of enzyme from the
guaiacol oxidation assay [31]. Enzyme activity was ex- culture. Crude enzyme was obtained from the opti-
pressed in units (U = μM/min/l), defined in terms of the mized culture filtrate on the 12th day when the maxi-
number of μM of guaiacol converted per minute per mum enzyme production was observed. Dye disappear-
liter (ε = 21,600 μM/min/l). ance was determined spectrophotometrically by moni-
toring the absorbance at the wavelength of maximum
Submerged-fermentation experiments absorbance for the dye. The reaction mixture contained
For this optimization methodology, the selected eight 100 mg/l dye in deionized water, 100 mM phosphate
factors and their levels are shown in Table 1. The de- buffer (pH 6.0) and fungal culture filtrate (2 U/ml)
tails of the individual combinations of the 18 experi- added in a total volume of 5 ml. A control in which the
mental trials and their obtained results for laccase en- enzyme was heat denatured was conducted in parallel.
zyme activity (U/l) are shown in Table 2. The obtained The assay was done in duplicate, with the experimental
results were analyzed by using “bigger is better” quality, error being below 3%.

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Journal of Basic Microbiology 2010, 50, 548 – 556
The percent decolorization of the dye was calculated
as follows:
Percent decolorization = (A – B)/A × 100
where A is the initial absorbance at 0 h and B is the
final absorbance after reaction with the enzyme.
Qualitek-4 software
The Qualitek-4 software (Nutek Inc., MI, USA) allows
designing experiments using any of the L-4 to L-81, L-16
and L-18 (modified) arrays. The experiments can be
designed to include as few as 2 two-level factor (L-4) or
as many as 63 two-level factors (L-64). The factors may
have two, three or four levels. Qualitek-4 offers two
options for experimental design. The present study
selects the automatic design option, which instructs
which array is to be used and when. Once the factors
and levels are described, the Qualitek-4 software auto-
matically selects the array-appropriate design and
places the factors in the correct column. In the manual-
design option, it is possible to control the design in
every step.
Results
Selection of a suitable substrate at an appropriate level
is a key factor in submerged fermentation. The compo-
sition of the culture medium and the quantities of the
components determine the production of laccase. Sub-
merged-fermentation experiments studied with the
designed experimental condition showed a significant
variation in laccase activity (Table 2). The average ef-
fects of the factors, along with the interaction at the
assigned levels, on the laccase production by Pleurotus
ostreatus IMI 395545 are shown in Table 3, in which the
inoculum shows the highest effect at level 1, whereas
pH, mineral solution, and glucose have a higher effect
at level 2. At level 3, the major effect was given by glu-
cose, followed by malt extract. The larger the difference
(L2-L1), the stronger is the influence. Among the factors
Table 3. Main effects of selected factors.
Serial no. Factor
1 pH
2 glucose [g]
3 yeast extract [g]
4 malt extract [g]
5 mineral solution [ml]
6 inoculum [ml]
7 inducer (CuSO4)
8 amino acid (L-asparagine) [mg]
© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Optimization of laccase production by Pleurotus ostreatus 551
and their levels studied on the laccase activity, glucose
showed a stronger influence (L2-L1) when compared
with other factors, viz. pH, mineral solution, yeast ex-
tract, inducer, L-asparagine, and malt extract.
The influence of each individual factor on the laccase
yield is shown in Table 3. Increases in the concentra-
tions of factors such as glucose, yeast extract, malt
extract, and inoculum resulted in increased enzyme
production. In the case of the mineral solution, the
inducer (CuSO4), and the amino acid (L-asparagine), the
laccase yield was higher up to level 2, but a subsequent
increase in the concentration (level 3) decreased the
laccase yield.
The severity indexes (SI) of the factors interacting at
various levels are shown in Table 4. The interaction
between two factors gives a better view for overall
process analysis. In culture, any individual factor may
interact with any or all of the other factors, creating
the possibility of a large number of interactions. The
results of the estimated interactions of the severity
indexes of two individual factors at various levels are as
follows: Inoculum and inducer CuSO4 (at levels 3 and 2;
column 1) interaction showed the highest interaction SI
(80.74%). CuSO4, which has the least impact factor,
when combined with inoculum showed a higher sever-
ity index. In the case of the amino acid L-asparagine
(lower impact factor), the combination with yeast ex-
tract resulted in a higher interaction SI (78.38%). It was
interesting to see that the two lowest impact factors,
i.e. the inducer CuSO4 and the amino acid L-asparagine,
in combination gave the third highest interaction SI
(66.92%). An SI of 14.41% was obtained when glucose
(strong impact factor) was combined with the amino
acid L-asparagine (with the lowest impact factor). On
the contrary, the SI between pH (second highest impact
factor) and amino acid L-asparagine (lowest impact
factor) showed a significantly lower interaction SI
(0.45%).
The influence of selected factors on the laccase pro-
duction at optimum performance is shown in Fig. 1.
Analysis of variance (ANOVA) was used to analyze the
Level 1 Level 2 Level 3 L2–L1
417.703 552.407 – –134.704
313.111 530.833 611.222 –217.721
444.444 496.777 513.944 – 52.332
452.222 453.944 548.999 – 1.722
462.444 539.888 452.833 – 77.444
494.388 434.611 526.166 – 59.777
475.333 507.388 472.444 – 32.054
482.833 501.722 470.611 – 18.888
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552 R. Periasamy and T. Palvannan Journal of Basic Microbiology 2010, 50, 548 – 556

Table 4. Estimated interaction of the severity indexes for different factors.

Serial no. Factors Columns SI [%) Reversed column Levels

1 inoculum × inducer 6×7 80.74 1 [3,2]
2 yeast extract × amino acid 3×8 78.38 11 [1,2]
3 inducer × amino acid 7×8 66.92 15 [1,2]
4 mineral solution × inducer 5×7 60.43 2 [2,1]
5 yeast extract × malt extract 3×4 55.14 7 [2,3]
6 malt extract × inducer 4×7 52.9 3 [3,1]
7 mineral solution × inoculum 5×6 52.86 3 [2,3]
8 yeast extract × mineral solution 3×5 52.07 6 [3,2]
9 inoculum × amino acid 6×8 50.5 14 [3,2]
10 malt extract × mineral solution 4×5 50.15 1 [3,2]
11 mineral solution × amino acid 5×8 43.39 13 [2,1]
12 yeast extract × inoculum 3×6 38.44 5 [2,1]
13 pH × malt extract 1×4 29.82 5 [2,1]
14 pH × yeast extract 1×3 29.78 2 [2,3]
15 pH × inducer 1×7 27 6 [2,1]
16 pH × mineral solution 1×5 21.76 4 [2,2]
17 malt extract × amino acid 4×8 19.39 12 [3,2]
18 malt extract × inoculum 4×6 18.63 2 [3,1]
19 glucose × inducer 2×7 15.74 5 [2,2]
20 glucose × malt extract 2×4 15.24 6 [3,3]
21 pH × inoculum 1×6 14.74 7 [2,3]
22 glucose × amino acid 2×8 14.41 10 [3,2]
23 glucose × inoculum 2×6 12.82 4 [3,1]
24 glucose × mineral solution 2×5 12.7 7 [3,2]
25 yeast extract × inducer 3×7 7.82 4 [3,2]
26 pH × glucose 1×2 1.49 3 [2,3]
27 glucose × yeast extract 2×3 1.25 1 [3,2]
28 pH × amino acid 1×8 0.45 9 [2,2]

results of the OA experiment and to determine how
much variation was contributed by each factor. From
the calculated ratios (F), it can be seen that all factors
and interactions considered in the experimental design
are statistically significant at the 90% confidence limit.
ANOVA with the percentage of contribution of each
factor to interaction is shown in Table 5. The optimum
conditions and their performance in terms of contribu-
tion for achieving higher laccase yield are shown in
Table 6. The contribution of selected factors to the lac-
case production at optimum performance is shown in
Fig. 2. The maximum contribution was given by glu-
cose, followed by pH, malt extract, mineral solution,
inoculum, and yeast extract. The optimization of lac-
case production by the above-mentioned process was
validated by dye decolorizing experiments. To validate
the potential of the Taguchi DOE methodology in me-
dium optimization, dye decolorization experiments
were conducted. Before the production medium opti-
mization, the dye decolorization experiment was car-
ried out with reactive blue 221, using the crude laccase
from unoptimized medium. The results revealed that
1 ml of the crude laccase at the peak production stage
decolorized the dye at up to 45% (Fig. 3). A similar ex-
periment was conducted by using the optimized en-
zyme, but without changing the unit volume. The dye

Table 5. ANOVA.

Serial no. Factors DOF Sums of squares Variance F ratio Pure sum Percentage [%]
1 pH 1 244960.598 244960.598 927.911 244696.607 16.861
2 glucose [g] 2 856413.243 428206.621 1622.048 855885.26 58.977
3 yeast extract [g] 2 47182.134 23591.067 89.363 46654.151 3.214
4 malt extract [g] 2 110424.907 55212.453 209.415 109896.925 7.572
5 mineral solution [ml] 2 82011.843 41005.921 155.33 81483.861 5.614
6 inoculum [ml] 2 77794.221 38897.11 147.342 77266.239 5.324
7 inducer (CuSO4) 2 13541.966 67770.983 25.648 13013.984 0.896
8 amino acid (L-asparagine) [mg] 2 8844.249 4422.124 16.751 8316.267 0.573
9 other/error 38 10031.666 263.991 – – 0.969
10 total 53 1451204.833 – – – 100.00

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Journal of Basic Microbiology 2010, 50, 548 – 556 Optimization of laccase production by Pleurotus ostreatus 553

Table 6. Optimum of culture conditions and their contribution.

Serial no. Factors Values Level Contribution

1 pH 6.0 2 67.351
2 glucose [g] 2.0 3 126.166
3 yeast extract [g] 0.500 3 28.888
4 malt extract [g] 0.350 3 63.944
5 mineral solution [ml] 20 2 54.833
6 inoculum [ml] 0.5 3 41.111
7 inducer (CuSO4) [mM] 1.0 2 22.333
8 amino acid (L-asparagine) [mg] 2 2 16.666

Total contribution from all factors = 421.292.

Current grand average performance = 485.055.
Expected result under optimum conditions = 906.347.

Discussion

Fungal laccase production is influenced by many typi-

Figure 1. Relative influence of factors and interactions.
was decolorized at up to 84.6% (Fig. 3). This shows that
the optimized medium produced more enzyme per unit
volume than the unoptimized medium.
cal culturing parameters, such as medium composition,
carbon and nitrogen ratio, temperature, pH, and aera-
tion ratio [28]. Optimum amounts of carbon and nitro-
gen in the medium enabled to reach high activities of
extracellular laccase [32, 33].
The variables (Table 1) selected for the present study
were identified based on a previous report and our
laboratory experiments. Table 2 reveals the variation in
laccase activity according to the experiments conducted
based on the Taguchi DOE method. The production
levels were found to be very much dependent on the
culture conditions. The average effects of the factors,
along with interactions at the assigned levels, on the
laccase production by Pleurotus ostreatus IMI 395545 are

Figure 2. Optimum performance with the contributions of the major factors.

© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

554 R. Periasamy and T. Palvannan
Figure 3. Comparison of the decolorization percentage of reactive
blue 221 by laccase produced before and after the optimization
process by the Taguchi DOE methodology.
shown in Table 3. It can be seen from Table 3 that,
among the factors studied, glucose (levels 2 and 3)
showed a stronger influence when compared to the
other factors, whereas the inoculum does not seem to
have any significant effect on the enzyme production.
However, as per the data in Table 4, the SI for inoculum
interaction with the inducer CuSO4 is highest (80.7%),
which was then followed by the interaction of the in-
oculum with mineral solution, amino acid, yeast ex-
tract, malt extract, and finally with glucose (12.82%).
The lowest SI interaction is shown between pH and
amino acid (0.45%). This reveals that the inoculum
cannot be omitted, based on the main effect of the
selected variables represented in Table 3, where the
inoculum shows negative values in the laccase enzyme
production. The percentage of contribution of each
factor with the respective variance is shown in Table 5.
From the ANOVA table, we statistically confirmed that
glucose is the main factor for the production of laccase.
In the present study, glucose contributed the highest
impact factor in the production of laccase, as shown in
Table 6. Increasing the glucose concentration from 5 to
20 g/l resulted in a more than fivefold increase of the
laccase activity. A further increase up to 40 g/l did not
enhance the laccase activity, but lower activities were
obtained [34]. This glucose repression is well known in
fungi, and is thought to be an energy-saving response
[35]. Among the carbon sources, glucose is a readily
utilizable substrate which would promote biomass
production. It has already been demonstrated that sub-
strates that are efficiently and rapidly utilized by the
organism result in high levels of laccase activity [36].
The culture pH condition is one of the important
parameters in fungal cultivation [26]. The obtained
result shows that the laccase yield was higher at pH 6.0
than at pH 5.5 (Table 3). It was proved that many Pleuro-
© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Journal of Basic Microbiology 2010, 50, 548 – 556
tus ostreatus species produced the maximum amount of
laccase enzyme when the initial pH of the medium was
adjusted to pH 6.0 in submerged culture [30]. The pH is
one of the operational parameters that influence the
metabolic activity of the organism, playing an impor-
tant role in the protocol optimization for any fermenta-
tion process [37].
The medium was supplemented with two types of
nitrogen sources, yeast extract and malt extract. Inor-
ganic nitrogen sources supported low levels of laccase
with sufficient biomass production, while the organic
nitrogen source gave high laccase yields with good fun-
gal growth. Yeast extract is one of the best nitrogen
sources that increase the yield of enzyme [38]. Moreover,
malt extract is rich in the aromatic amino acids trypto-
phan and tyrosine. Tryptophan is also produced de novo
by basidiomycetes and it functions as a precursor in the
synthesis of N-substituted aromatic secondary metabo-
lites of fungi [39]. The yield of laccase was increased by
supplementation of the medium with an additional
nitrogen source like the amino acid L-asparagine [37].
Nitrogen plays a key role in laccase production; the
nature and the concentration of nitrogen in the culture
media for growing white-rot fungi are essential for lac-
case production [40]. Usually, a high nitrogen concentra-
tion is required for optimal laccase production [41]. It
was evident that sufficient amounts of carbon and ni-
trogen in the medium increase the productivity of lac-
case two times higher than that obtained on the origi-
nal Lindbergh-Holm medium used as a control [37].
Besides the composition of the culture media includ-
ing the carbon substrates, all micronutrients are im-
portant for cell growth and specific enzyme production
[42]. The time point of cupric ion supplementation and
the cupric ion concentration were important for obtain-
ing increased laccase activity [37]. Another important
role for copper is regulating the laccase gene transcrip-
tion [43, 36]; at the same time, the copper concentra-
tion in the culture media had a clear effect during cul-
ture at low nitrogen concentrations, rather than during
culture with high nitrogen content [44]. This argument
well agreed with the results drawn by Schlosser et al.
[45].
The relative influence of the factors and their inter-
action at a chosen level are shown in Fig. 1. It can be
seen that glucose and pH play an important role in
influencing the laccase enzyme production. Moreover,
the studied strain produced increased laccase titers
without the addition to the culture medium of phenolic
and aromatic inducers related to lignin or lignin deriva-
tives, which are often used to stimulate enzyme forma-
tion in most other fungal species [46].
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Journal of Basic Microbiology 2010, 50, 548 – 556
It is evident from Table 6 that, upon considering the
optimum culture conditions from the designed experi-
ments, the laccase yield can be increased from 485.0 to
906.3 U/l; i.e., overall a 86.8% increase in enzyme pro-
duction can be achieved. To validate the proposed ex-
perimental methodology, production experiments were
conducted by applying the obtained optimized culture
condition as per Table 6. The obtained results con-
firmed an enhanced laccase yield of 820.5 U/l from
485.0 U/l (69.1% increase in laccase yield) with the Ta-
guchi DOE-optimized culture conditions. The increased
production of laccase was also confirmed by the dye
decolorization experiment, which showed an increased
decolorization of reactive blue 221 from 45% to 84.6%
in the same unit volume. The above experiment proved
that there is a linear correlation between the dye decol-
orization and the concentration of laccase. Many earlier
studies confirmed that, among the lignolytic enzymes,
laccase has a more active role in the decolorization of
synthetic dyes [47].
Conclusion
It can be concluded that optimization of the production
medium is one of the key factors to maximize the yield
of laccase. Traditional methods of optimization in-
volved changing one independent variable while fixing
the others at a certain level. This single-dimensional
search is laborious, time consuming, and incapable of
reaching a true optimum due to interactions among
variables. The Taguchi approach of OA DOE constitutes
a simple methodology that selects the best conditions
producing consistent performance. Hence, the produc-
tion medium for laccase was first optimized by the
Taguchi DOE methodology. The results obtained from
the dye decolorization experiment proved that the en-
zyme activity was increased after optimization, when
compared to the unoptimized conditions in the same
unit volume. In the case of inducers and enhancers,
further detailed studies have to be conducted to im-
prove the yield of laccase by identifying the right con-
centration and the time point for giving the dose for
the subjected strain. This step-by-step approach may be
very helpful to increase the yield of laccase in the case
of new strains.
Acknowledgement
The authors greatfully acknowledge R. K. Roy, Nutek
Inc., USA, for providing the Qualitek-4 software.
© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Optimization of laccase production by Pleurotus ostreatus 555
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