全 文 ： 生 物 工 程 学 报 Chin J Biotech 2010, March 25; 26(3): 341–349
journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061
cjb@im.ac.cn ©2010 CJB, All rights reserved.


Received: September 7, 2009; Accepted: November 16, 2009
Supported by: Natural Science Research Program of the Educational Office of Anhui Province (No. 2010).
Corresponding author: Rong Jia. E-mail: ahdxjiarong@yahoo.com.cn
安徽省自然科学基金项目 (No. 2010) 资助。

环境生物技术
裂褶菌 F17锰过氧化物酶酶活力影响因素的响应面优化
查诚，荚荣，陶香林，姚祖亮
安徽大学生命科学学院，合肥 230039
摘 要: 锰过氧化物酶是真菌分泌的一种糖基化的含有血红素辅基的胞外蛋白, 在染料降解和脱色过程中起着重要作
用。本实验利用本实验室保存的的白腐真菌裂褶菌 Schizophyllum sp. F17 产锰过氧化物酶（MnP），研究 MnP 的酶学性
质，并对酶活条件进行优化。实验通过超滤浓缩、DEAE-纤维素、DE52 离子交换层析和 Sephadex G-75 凝胶过滤等步
骤，分离纯化得到电泳纯的锰过氧化物酶。该酶蛋白含量为 23 µg/mL，分子量大小为 49.2 kDa，在 0.1 mmol/L H2O2
中半衰期为 5~6 min。Mn2+、H2O2 以及酶的用量可以影响 MnP 酶促反应的效率，在单因子分析法的基础上，通过全因
子中心组合设计响应面分析表明：H2O2 以及 H2O2 与酶用量之间的交互作用对酶促反应的作用是最显著的。在优化条件
下，酶对偶氮染料金橙 G、刚果红显示出较强的脱色能力。
关键词 : 锰过氧化物酶 , 纯化 , 优化 , 相对酶活 , 全因子试验 , 偶氮染料
Optimization of process variables for the manganese
peroxidase of the white-rot fungus Schizophyllum sp. F17 by
full factorial central composite design
Cheng Zha, Rong Jia, Xianglin Tao, and Zuliang Yao
School of Life Science, Anhui Key Laboratory of Eco-engineering and Bio-technique, Anhui University, Hefei 230039, China
Abstract: White-rot fungus manganese peroxidase (MnP) that has great potential in degrading azo dyes is one of the extracellular
glycolsylated heme proteins. MnP from Schizophyllum sp. F17 was isolated and purified by Sephadex G-75 gel filtration
chromatography followed by DEAE-cellulose anion exchange chromatography. The molecular weight of the puried enzyme was
49.2 kDa, while the half-life of the MnP in the presence of 0.1 mmol/L H2O2 was 5−6 min. The efficiency of MnP-catalyzed reactions
were determined by three key factors: the concentrations of Mn2+, H2O2, and the amount of MnP. Using single factor analysis, an
optimized concentration of Mn2+, H2O2 and enzyme were optimized to be 1.2 mmol/L, 0.1 mmol/L, and 0.4 mL, respectively. A response
surface methodology (RSM) employing two-level-three-factor full factorial central composite design was used to optimize the catalytic
conditions. The result showed that the concentration of H2O2 and the interaction between H2O2 and MnP mostly affect the MnP catalytic
efficiency. Finally, we show that the azo dyes could be efficiently decolorized by the purified MnP under optimized conditions.
Keywords: manganese peroxidase, purification, optimization, relative MnP activity, full factorial central composite design,
azo dyes
DOI:10.13345/j.cjb.2010.03.003
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Introduction

Azo dyes are extremely versatile colorants and are the
largest group of synthetic known [1]. As a consequence,
they are also the most common group of synthetic
colorants released into the environment [2]. It is
estimated that about 10% and 20% of 0.7 million tons
of dyestuff being manufactured each year and used in
dyeing processed may be found in wastewater [3]. The
treatment of dye wastewater from textile and dyestuff
industries is one of the most challenging among
industrial wastewater [4]. Compared with physical
and/or chemical methods, microbial decolorization has
been claimed to be less expensive and less
environmentally intrusive alternative [5].
White-rot fungi have been shown to possess a
remarkable potential for degrading azo dyes because
they produce oxidase and peroxidase, which are highly
oxidative and substrate-nonspecific[6]. One of the most
understood enzymes is manganese peroxidase (MnP,
EC 1.11.1.13), first described in Phanerochaete
chrysosporium[7]. MnP oxidizes a wide range of
substance, rendering it an interesting enzyme for
potential applications. MnP is an extracellular
glycolsylated heme protein. MnP can catalyze the
H2O2-dependent oxidation of Mn2+ to Mn3+, which is
stabilized by chelators such as organic acid. Chelated
Mn3+ acts as a highly reactive, low molecular weight,
diffusible redox-mediator[2]. Thus, MnP is able to
oxidize and depolymerize its natural substrate, i.e.,
ligin and a range of diverse environmental pollutants
such as nitroaromatic compounds and textile dyes [8].
Although they have enormous potential, their
industrial application is hampered by their high price
and low operational stability[9]. Heme peroxidases are
swiftly inactivated in the presence of catalytic amounts
of hydrogen peroxide, which acts as an electron
acceptor during the catalytic cycle. Although the
inactivation mechanism is not completely explained,
several events such as heme destruction,
intermolecular crosslinking and oxidation of low redox
potential amino acid residues are known to lead to
activity loss[10]. The inactivation is considered a
suicide process, as the main inactivating species are
the enzymatic intermediates involved in the catalytic
cycle[11].
The MnP catalytic cycle process is a complicated
system. The three main factors that decide the enzyme
activity of MnP are concentrations of H2O2, Mn2+ and
enzyme itself. It is important to analyze the factors as
a whole and understand the combination interactions
among these three factors. The classic method of
determining optimum conditions by varying one
parameter while keeping the other at specified constant
level is a single-dimensional, laborious and time-
consuming method, often dose not guarantee
determination of optimal conditions [12]. In order to
overcome these problems, optimization studies have
been done using response surface methodology
(RSM), a statistically designed experimental protocol
in which several factors were simultaneously varied.
This multivariate approach has its advantages in terms
of reductions in the number of experiments, improved
statistical interpretation possibilities and reduced time
requirements from overall analysis. RSM has been
found to be much successful and economical during
optimization of various industrial processes [13]. Easy
way to estimate response surface, factorial designs are
the most useful schemes for the optimization of
variables with a limited number of experiments. A
variety of factorial designs are available to accomplish
this task. The most successful and best is the central
composite design (CCD), which is accomplished by
adding two experimental points along each coordinate
axis at opposite sides of the origin and at a distance
equal to the semi-diagonal of the hyper cube of the
factorial design and new extreme values (low and
high) for each factor added in the design. The model is
also used to predict the result by iso-response contour
plot and three-dimensional surface plots [14]. Here
two-level-three-factor full factorial central composite
design model was used to acquire deeper
understanding of the catalytic mechanism of MnP.
However, MnPs from different white-rot fungi may
have very different properties. For our studies on the
properties of MnP, we chose the MnP from
Schizophyllum sp. F17 which has been previously
shown potential for dye decolorization [15].

1 Materials and methods

1.1 Microorganisms and culture conditions
Schizophyllum sp. F17 was isolated from a decayed
wood chip pile in the vicinity of Hefei, China. The
fungus was cultured on potato dextrose agar (PDA)
slants for one week at 28°C. The grown mycelium mat
was washed with sterile water. Mycelia obtained from
several slants were blended with 100 mL of sterile
water in three cycles of 15 s. Five hundred millilitre
flasks containing a 100 mL liquid medium were
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inoculated with a mycelial suspension and grown at
28°C on a rotary shaker at 130 r/min for 2 days. The
liquid medium contained 20 g potato extrace, 2 g
dextrose, 0.3 g KH2PO4, 0.15 g MgSO4, 1 mg
thiamine, and was autoclaved at 121°C for 20 min.
Homogenized mycelia, which were obtained after
crushing the mycelial pellets in a Waring blender, were
used as the inoculums for 250 mL flasks containing 5 g
SSF medium. SSF medium containing 90% rice hull
and 10% soybean cake meal was humidified with 7.5 g
water and autoclaved at 121°C for 20 min [16].
Incubation was carried out at 28°C for 4 days.
To the culture flasks, 100 mL of sodium acetate
buffer (10 mmol/L, pH 5.9) was added. Contents were
gently beaten and incubated on the rotary shaker at
130 r/min for 30 min. The crude enzyme obtained was
then filtered and spun (10 000 r/min, 20 min, 4°C).
1.2 Enzyme activity assay of MnP
The level of MnP activity was determined by
monitoring the formation of the Mn3+-lactate complex
(ε240= 6500 M-1cm-1) at 240 nm at 25°C, during
oxidation of 1 mmol/L MnSO4 in 0.1 mol/L sodium
lactate(pH 4.5) in the present of 0.1 mmol/L H2O2.
One unit of MnP was defined as the amount of enzyme
producing 1 µmol of the Mn3+-lactate complex per
minute[17]. The relative MnP activity was defined as
the ratio of the enzyme activity and the corresponding
maximum activity. The maximum MnP enzyme
activity was 100%.
1.3 Enzyme purification and examination of
protein concentration
The crude enzyme was concentrated 20-fold by
ultrafiltration (20 kDa-cut-off polyethersultone
membranes, Model 8400, Millipore Corporation,
USA), then applied to a DEAE-cellulose anion
exchange column (Whatman DE52, England) (2.6 cm×
30 cm) previously equilibrated with sodium acetate
buffer (10 mmol/L, pH 5.9). The MnP was eluted with
a liner gradient of 0–0.5 mol/L NaCl in the same
buffer at a low rate of 100 mL/h. The fractions
containing MnP activity were collected and
concentrated 100-fold using Stirred Ultrafiltration
Cell Model 8010 (20 kDa-cut-off polyethersultone
membranes, Millipore Corporation, USA). The
concentrated samples were loaded onto a Sephadex
G-75 gel filtration column (Fluka, USA) (1.7 cm×
100 cm) equilibrated with sodium acetate buffer
(10 mmol/L, pH 5.9). The column was run using
sodium acetate buffer (10 mmol/L, pH 5.9) at a low
rate of 6 mL/h. MnP fractions were pooled and
concentrated with centrifugal ultrafiltration units, then
analyzed by SDS-PAGE. The enzyme solution was
kept at 4°C to be used for characterization
experiments.
Protein concentration was estimated using Bradford
with crystalline bovine serum albumin (BSA) as
standard[18]. Protein concentrations in the fractions
from the chromatography were determined from
absorbance values at 280 nm.
1.4 H2O2 sensitivity and the kinetics of MnP with
H2O2 and Mn2+
The inactivation of the enzyme in the presence of
H2O2 was studied as follows: MnP was incubated
with 0.1 mmol/L or 1 mmol/L H2O2 respectively and
the enzyme activities were monitored from 0 to 10 min
every minute and afterward, every 5 minutes up to
25 min.
The kinetics of MnP with H2O2 and Mn2+ was
studied as follows: The oxidation of Mn2+ was
measured at wavelengths of 240 nm. Reaction with all
substrates was quantitated in 0.1 mol/L sodium lactate
buffer, pH 4.5. The Km of Mn2+ and H2O2 were done in
0.1 mol sodium lactate buffer and 0.1 mL MnP using
fixed concentrations of H2O2 (0.1 mmol/L) and
varying Mn2+ concentrations (0.001–0.01 mmol/L) or
fixed concentrations of Mn2+ (1 mmol/L) and varying
H2O2 concentrations (0.004–0.04 mmol/L). The kinetic
data were analyzed using double-reciprocal plots of
rates versus substrate concentration.
1.5 The one-factor-at-a-time analysis
1.5.1 Effect of concentrations of Mn2+ on the MnP
activity
MnP activity was measured in 4 mL 0.1 mol/L
sodium lactate (pH 4.5) containing 0.1 mL MnP,
0.1 mmol/L H2O2 and a predetermined concentration
of Mn2+ (0.2–2 mmol/L), The reaction was carried out
for 5 min at 25°C. All data were the values for at least
three samples.
1.5.2 Effect of concentrations of H2O2 on the MnP
activity
To study the H2O2 dependence for the enzyme, MnP
activity was measured in 4 mL 0.1 mol/L sodium
lactate(pH 4.5) containing 0.1mL MnP, 1 mmol/L
MnSO4 and a predetermined concentration of
hydrogen peroxide H2O2 (0.02–0.5 mmol/L), The
reaction was carried out for 5 min at 25°C. All data
were the values for at least three samples.
1.5.3 Effect of amounts of MnP on the MnP activity
MnP activity was measured in 4 mL 0.1 mol/L
sodium lactate (pH 4.5) containing 0.1 mmol/L H2O2,
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1 mmol/L Mn2+ and different amounts of MnP
(0.05–0.6 mL), The reaction was carried out for 5 min
at 25°C. All data were the values for at least three
samples.
1.6 Design of experiments
A two-level-three-full factorial Central Composite
Design (CCD) was designed to assess the influence
of the main factors on the MnP activity, as well as
their interactions between those factors. The three
factors considered are (ⅰ) concentration of Mn2+(low
0.8 mmol/L and high 1.6 mmol/L ) (ⅱ) concentration
of H2O2(low 0.05 mmol/L and high 0.15 mmol/L ) (ⅲ)
amounts of MnP(low 0.25 mL and high 0.55 mL).
1.7 Azo dye decolorization by manganese
peroxidase
Dye decolorization was measured spectrophotometrically
at the following wavelengths: Orange G, 474 nm;
Congo red, 506 nm. The decolorization was carried out
directly in the spectrophotometer cuvette. The reaction
mixture contained sodium lactate buffer (pH 4.5,
100 mmol/L), dye (1 mmol/L) and optimized dosage
of Mn2+, H2O2 and purified MnP in a total volume of
3 mL. Data were noted every 5 min during the 60 min
reaction. Control samples, without H2O2, were done in
parallel identical conditions.

2 Results

2.1 Physicochemical properties of purified MnP
MnP was the main oxidoreductase produced in solid
cultures of Schizophyllum sp. F17. Additionally, the
fungus produced lower level of LiP and no laccase.
The crude MnP was fractionated by anion-exchange
chromatography at pH 5.9, then further purified by gel
filtration chromatography (Sephadex G-75). The
purified protein appeared to be homogeneous when
analyzed by SDS-PAGE. The molecular mass of MnP
was calculated to be 49.2 kDa under denaturing
conditions (Fig.1). The protein concentration of this
purified enzyme was 23 µg/mL estimated by Bradford
method. The enzyme solution was kept at 4°C to be
used for the next experiments.
The susceptibility of MnP to some concentrations
of H2O2 was studied. The half-life of the MnP in the
presence of 0.1 mmol/L or 1 mmol/L H2O2 were
shown below (Fig.2). When the H2O2 concentration
was 0.1 mmol/L, the MnP activity decreased by 50% in
the first 5–6 min and continued to decrease but at a lower
rate. When the H2O2 concentration was 1 mmol/L, the
MnP activity decreased rapidly by 80% at the

Fig. 1 SDS-PAGE of samples containing MnP from various
purification steps, which were stained with Coomassie blue. M:
protein standards molecular size markers; 1: purified MnP after
gel filtration; 2: concentrated samples after DEAE-Cellulose
DE52 column chromatography; 3: crude extract.


Fig. 2 Stability of MnP at two different concentrations of
H2O2. (a) 0.1 mmol/L H2O2. (b) 1 mmol/L H2O2.

beginning of the catalytic process, and remained the
same thereafter.
The kinetics of MnP with H2O2 and Mn2+ were also
studied. The Km for H2O2 and Mn2+ were 5.2 µmol/L
and 13.1 µmmol/L respectively. The Km of H2O2 was a
little higher than that of the MnPs from most
Bjerkandera species when the Km of Mn2+ was much
lower than that of Bjerkandera Species [19-22].
2.2 The one-factor-at-a-time analysis
In this section, the single-factor effects of H2O2,
Mn2+ and MnP dosage on MnP enzyme activity were
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investigated. The effect of Mn2+ on the MnP activity is
shown in Fig.3a. As the Mn2+ concentration reached
1.2 mmol/L, the MnP had the maximum activity then
slowly decreased over the concentration of 1.2 mmol/L.
MnP showed its strong activity (more than 80% of the
maximum) at a board concentration of H2O2 ranging
from 0.1 to 0.2 mmol/L. The maximum was observed
when the H2O2 was 0.1 mmol/L, but decreased sharply
at the concentration over 0.25 mmol/L (Fig.3b). The
optimum MnP dosage for the enzyme activity was
0.4 mL. The activity increased with the increase in
amount of purified enzyme used up to 0.4 mL,
however amounts above 0.4 mL decreased the MnP
enzyme activity (Fig.3c).



Fig. 3 The effect of single factor on MnP activity. (a) Mn2+.
(b) H2O2. (c) MnP.
2.3 Response surface factorial design for the
optimization of the MnP enzyme activity
In this study, the two-level-three-full factorial
Central Composite Design (CCD) experiment was
chosen to investigate the factors predominantly
affecting the MnP enzyme activity.
Table 1 shows the experiments performed according
to the experimental plan and the response thus
obtained for each combination of the variables; also in
this table is the predicted value from the model.
Table 2 shows the response surface regression
results, which give the coefficients for all the terms in
the model. The R2 value was 96.4%, which means
96.4% results of the total variations could be explained
by this model. Except square terms, all the P values were
very small (＜0.05) suggesting that these three factors
and there interactions may be important in this model.
Analysis of variance (ANOVA) utilized for
statistical testing is shown in Table 3. The p-values
(0.000) for Liner and Interaction terms meant these
three factors and their interactions had serious effect on
the activity of the MnP, and the model was applicable.

Table 1 Two-level-three-full factorial Central Composite
Design matrix and experiments results of dependent
variables (data processed by MINITAB software)
Run Mn2+ H2O2 Enzyme
Relative MnP
activity (%)
Predicted
value (%)
1 0.8 0.05 0.25 38.8 41.4
2 1.2 0.1 0.15 39.4 41.499
3 1.2 0.1 0.4 54.7 51.93
4 1.6 0.05 0.25 41.1 40.089
5 0.8 0.15 0.25 50.2 44.341
6 0.53 0.1 0.4 36.9 40.839
7 1.6 0.05 0.55 35.6 40.494
8 1.2 0.02 0.4 23.9 27.173
9 1.87 0.1 0.4 65.3 63.021
10 0.8 0.05 0.55 30.8 26.855
11 1.2 0.1 0.4 52.8 51.93
12 1.2 0.1 0.65 59.5 62.361
13 1.2 0.1 0.4 53.6 51.93
14 1.2 0.1 0.4 54.8 51.93
15 1.2 0.02 0.4 68.1 76.687
16 1.6 0.15 0.25 54.1 57.08
17 1.6 0.15 0.55 100 96.435
18 1.2 0.1 0.4 54.1 51.93
19 1.2 0.1 0.4 56.2 51.93
20 0.8 0.15 0.55 68.7 68.746
Central composite design: factor, 3; base runs, 20; base blocks,1;
replications, 1; total runs, 20; total blocks, 1. Two-level factorial: full
factorial: cube points, 8; center points in cube, 6; axial points, 6; center
points in axial, 0. alpha: 1.68179. Response Surface Regression: relative
MnP activity 100% versus Mn2+(mmol/L), H2O2(mmol/L), enzyme(mL).
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Based on the statistical analysis above, the
mathematical expression of relationship to the MnP
activity with variables Mn2+, H2O2 and enzyme is as
follows:
Relative MnP activity (%)=73.3358−21.0042×Mn2+−
264.820×H2O2−130.669×enzyme−2.07844×(Mn2+)2−
854.269×(H2O2)2−40.7023×(enzyme)2+175.625×
Mn2+×H2O2+62.2917×Mn2+×enzyme+1298.33×H2O2×
enzyme (1)
As the square terms were insignificant (5%), they
were deleted from the equation, hence a new
regression model as follows:
Relative MnP activity (%)=89.0738−25.9924×Mn2+−
435.674×H2O2–163.235×enzyme+175.625×Mn2+×H2O2
+62.2917×Mn2+×enzyme+1298.33×H2O2×enzyme (2)
The predicted values of relative MnP activity
obtained using Eq.(2) were close to the experimental
values(see Table 1) proving that the model was
applicable.
2.3.1 Residual plots for the relative MnP activity
The normal probability plot (Fig.4) showed that the
distribution of the residual value, which was defined as
the difference between the observed and the predicted,
could form a straight line, and these residual value
were normaly distributed on both sides of the line. It
showed that experimental point was reasonably
aligned with the predicted value.

Table 2 Estimated regression coefficient for relative MnP
activity
Term Coef SE Coef T P
Constant 54.2408 1.780 30.480 0.000
Mn2+ 6.5947 1.181 5.586 0.000
H2O2 14.7205 1.181 12.468 0.000
Enzyme 6.2023 1.181 5.523 0.000
Mn2+×Mn2+ −0.3326 1.149 −0.289 0.778
H2O2×H2O2 −2.1357 1.149 −1.858 0.093
Enzyme×enzyme −0.9159 1.149 −0.797 0.444
Mn2+×H2O2 3.5125 1.543 2.277 0.046
Mn2+×enzyme 3.7375 1.543 2.423 0.036
H2O2×enzyme 9.7375 1.543 6.312 0.000
R-Sq = 96.4%; R-Sq(adj) = 93.2%

Table 3 Analysis of variance for relative MnP activity
Source DF Seq SS Adj SS Adj MS F P
Regression 9 5120.62 5120.62 568.96 29.89 0.000
Liner 3 4078.63 4078.63 1359.54 71.41 0.000
Square 3 72.99 72.99 24.33 1.28 0.334
Interaction 3 969.00 969.00 323.00 16.97 0.000
Residual error 10 190.38 190.38 19.04
Pure error 5 6.77 6.77 1.35
Total 19 5311.00 5311.00

Fig. 4 The normal probability plot.

Fig.5 shows that the residuals distribute randomly
about zero, indicating that the regression terms were
incorrelated with one another. This plot rules out the
impact of order which may influence the results.

Fig. 5 Residual versus the order of the data.

2.3.2 Pareto chart for the effect of different factors
on the MnP activity
Mn2+, H2O2 and enzyme itself, as well as there
interactions among them are main factors that decide
the enzyme activity of MnP, In this work, we tried to
find out the most important factor for the enzyme
activity by Pareto chart (Fig.6). The Pareto Chart is a
vertical bar chart, the graph shows the relative
frequency of numerical way from left to right in
descending order. Plato can be effectively applied to
analysis of the primary concerns. Thus we found that
the concentration of H2O2 was the most important

Fig. 6 Pareto chart of the standardized effects. A: concentration
of Mn2+(mmol/L); B: concentration of H2O2(mmol/L); C:
amounts of enzyme(mL).
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factor for the MnP, as well as the interaction between
H2O2 and enzyme. The liner terms owned the stronger
ability to influence the MnP activity then the
interaction terms. According to the Pareto chart, the
effect of different factors from serious to minimal was:
H2O2> (H2O2×enzyme) >Mn2+> enzyme > (Mn2+×
enzyme) > (Mn2+×H2O2)
2.3.3 The three-dimensional response surface plots
and Interaction Plot for the mutual effect between
two variable factors
The three-dimensional response surface plots are the
graphical representations of the regression equation.
The main goal of response surface is to track
efficiently for the optimum values of the variables
such that the response is maximized [23]. The surface
plots (Fig.7a-c) were in 3D graphs in which the
relative MnP activity was represented by varying two
of the three main factors which have effect on the
enzyme activity.
It was shown that in different concentrations of
H2O2, the MnP had larger activity when the
concentration of Mn2+ was higher (Fig.7a). According
to the sharply slope, H2O2 had more significant effect
on the MnP activity compared with Mn2+.
From Fig.7b it was shown that in different amounts
of MnP, the MnP activity became larger when the
model had more Mn2+. However, when the
concentration of Mn2+ was low, the change of enzyme
activity was significant if the amounts of enzyme
increased.
Fig.7c showed the combined effect of varying
concentrations of H2O2 and amounts of MnP on the
MnP activity. From this response surface plot it was
shown that relative MnP activity could reach its
maximum if the concentration of H2O2 should be near
0.15 mmol/L and amounts of enzyme should be 0.55 mL.
The interaction plot shows the change of the one
factor if the other one is changed between the two
relative variables. It is very important to study the plot
because it can enlarge or eliminate the effect of the
main effect. Two variables’ interactions are the lines of
different slope. (Fig.8) The graph shown in Fig.6
confirmed the model of three-dimensional response
surface plots which reflected the relationship between
the variables.
2.4 Studies on the decolorization of azo dyes by
MnP
Azo dyes such as Orange G, Congo red were treated
with optimized dosage of Mn2 +(1.6 mmol/L),
H2O2(0.15 mmol/L) and purified MnP(0.42 mL). The



Fig. 7 (a) 3D surface plot of the combined effect of
concentration of Mn2+ and concentration of H2O2 on relative
MnP activity. (b) 3D surface plot of the combined effect of
amounts of enzyme and concentration of Mn2+ on relative MnP
activity. (c) 3D surface plot of the combined effect of amounts
of enzyme and concentration of H2O2 on relative MnP activity.
R: relative MnP activity; A: concentration of Mn2+ (mmol/L); B:
concentration of H2O2 (mmol/L); C: amounts of enzyme (mL).

Fig. 8 Interaction Plot for MnP activity. A: Mn2+ (mmol/L); B:
H2O2 (mmol/L); C: MnP ( mL).
348 ISSN1000-3061 CN11-1998/Q Chin J Biotech March 25, 2010 Vol.26 No.3

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results of decolorization are shown in Fig.9. The
decolorization for Orange G reached nearly 35% after
60 min of treatment as well as for the Congo red. The
purified MnP was able to play a role in the
decolorization of the azo dyes.


Fig. 9 Decolorization of azo dyes by MnP.

3 Conclusions

Statistically designed experimentation has been
applied in optimization of medium for MnP production
as well as in the enzymatic decolorization with MnP.
But to our knowledge little attention has been paid to
the interactions among those factors which had serious
effect on the MnP activity.
In this study, the statistically process analyzed the
key factors and their interactions in the biochemical
reaction, which was useful to investigate the
enzymatic mechanism of MnP. H2O2 was proved to be
the most important factor in the action, so did its
interactions with the amounts of the enzyme. But the
MnP is easily inactivated by H2O2 present in the
action. Meanwhile the half-life time of MnP presented
in H2O2 is too short to effectively apply to the
manufacture. However, this powerful oxidant[24] is
potentially valuable for some applications, such as in
the pulp and paper industries and the degradation of
environmental pollutants[25-27]. Therefore, investigation
of the effect of H2O2 on the MnP is desirable for
practical use. Here a model was set up by central
composite design which can predict the value of MnP
activity and show the effect of Mn2+, H2O2, amounts of
enzyme on the reaction, so do their inactions.
According to the result of decolorization
experiment, the purified MnP has a good potential for
the application in the optimized system.
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