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Biochemical Engineering Journal 34 (2007) 82–86
Short communication
Statistical optimization of xylanase production from new isolated
Penicillium oxalicum ZH-30 in submerged fermentation
Yin Li a,∗ , Zhiqiang Liu b , Hui Zhao a , Yingying Xu c , Fengjie Cui d
a
Department of Plant Science, North Dakota State University, Fargo, ND 58105, USA
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China
c Department of Cereal and Food Science, North Dakota State University, Fargo, ND 58105, USA
d School of Food Engineering and Biotechnology, Jiangsu University, Zhenjiang 2120013, PR China
b
Received 19 June 2006; received in revised form 25 September 2006; accepted 13 November 2006
Abstract
This research aimed at optimizing fermentation condition (initial pH and temperature) of xylanase production from Penicillium oxalicum ZH-30
by statistical analysis using response surface methodology. Statistical analysis of results showed that the linear, quadric terms and interaction of
these two variables had significant effects. The optimal conditions for higher production of xylanase were: initial pH 7.38 and temperature = 31.1 ◦ C.
The predicted and verified xylanase activities under optimal condition were 14.33 and 14.50 U/mL, respectively. The temperature range suitable
for the industrial application of xylanase from P. oxalicum ZH-30 was 50–60 ◦ C.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Penicillium oxalicum ZH-30; Optimization; Response surface methodology; Xylanase
1. Introduction
Xylanase (1,4-␤-d-xylan-xylanhydrolase, EC 3.2.1.8) catalyzes the hydrolysis of xylan, the major component of
hemicellulose in plant cell walls, to xylo-oligosaccharides and
xylose. A variety of microorganisms, including bacteria [1,2],
yeast [3] and filamentous fungi [4,5], have been reported to produce xylanases. The potential applications of xylanase, with
or without concomitant use of cellulase, include biocoversion
of ligocellulose to sugar, ethanol and other useful substances,
degradation of arabinoxylans in brewing [6], clarification of
microflitration membrane [7], and nutritional value improvement of silage and green feed [8].
The xylanase production by microorganisms is strongly influenced by many factors, such as nutritional sources [9–11] and
cultivation condition [12–14]. Classical experimental design
requires that only one variable be changed at a time to determine
its effect. Such work is extremely laborious and time-consuming
using the conventional techniques such as ‘one-factor-at-a-time’
method, and moreover, it does not guarantee the determina-
∗
Corresponding author. Tel.: +1 701 231 7737.
E-mail address: yin.li@ndsu.edu (Y. Li).
1369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2006.11.011
tion of optimal conditions, and often fails to consider the
combined effects of all involved factors [15]. Response surface methodology (RSM) is a collection of mathematical and
statistical technique useful for analyzing the effects of several independent variables. Usually, this process employs a
low-order polynomial equation in a pre-determined region of
independent variables, which is later analyzed to locate the
optimum values of the independent variables for the best
response. Recently, different statistical designs for fermentation condition optimization regarding xylanase production
have been reported, among which factorial experiments and
response surface methodology (RSM) are included [16–18].
These statistical methods have proved to be powerful and useful.
The objective of the present work was to apply statistical
methods to optimize fermentation parameters for enhancing
the xylanase production by Penicillium oxalicum ZH-30. Two
variables, initial pH and temperature, were selected as process (independent) variables while xylanase production was the
response (dependent variable). An empirical model including
the effects of independent variables has also been developed
through SAS software to represent the response surface. The
thermal stability of xylanase from P. oxalicum ZH-30 was
reported in this work as well.
Y. Li et al. / Biochemical Engineering Journal 34 (2007) 82–86
2. Materials and methods
2.1. Microorganism
The P. oxalicum ZH-30 strain was isolated from soil and identified according to the morphology and comparison of ITS rDNA
gene sequence (DQ473437). It was maintained at 4 ◦ C on potato
dextrose agar (PDA) in our laboratory. Spores suspensions were
prepared from 6-day-old cultures that had been grown on PDA
slopes at 30 ◦ C. Sterile distilled water was aseptically added to
each slope and a suspension of the spores was made by lightly
brushing the mycelium with a sterile wire loop. The suspension
was diluted with sterile distilled water to give a final spore count
of 1 × 107 spores/mL.
2.2. Media and cultivation
The medium used for xylanase production was composed
of (g/L): NH4 Cl 9; KH2 PO4 1; NaNO3 1; MgSO4 ·7H2 O 1;
CaCl2 ·2H2 O 0.3; yeast extract 1. The agricultural waste wheat
bran (10 g/L) was added to the medium. The pH was adjusted
according to the experimental design. The media was then
autoclaved for 20 min at 121 ◦ C. Erlenmeyer flasks (250 mL)
containing 75 mL of medium were inoculated with 1 mL of
diluted spore suspension and then were maintained at 30 ◦ C on
a rotary shaker under 150 rpm for 15.4, 48, 96, 144 or 176.6 h,
according to the experimental design. At the end of fermentation, the mycelium was separated from the enzyme-containing
broth by centrifugation at 10,000 × g for 15 min to obtain the
crude enzyme.
2.3. Experimental design
Response surface methodology (RSM) was used to optimize
the fermentation conditions for enhanced xylanase production.
Central composite design (CCD) with two factors and five levels,
including five replicates at the center point, was used for fitting
a second-order response surface. The CCD contained an imbedded factorial or fractional factorial matrix with center points and
“star points” around the center point that allow estimation of the
83
curvature [19]. One unit was designated to the distance from the
center of the design space to a factional point, while α unit was
designated to the distance from the center of the design space to
a star point. The star points represent new extreme values (low
and high) for each factor in this design. If the factorial is a full
factorial then
1/4
α = [2k ]
(1)
In this study k = 2 factors (pH and temperature), so α = 1.414.
Table 1 gives the factors and their values, and the experimental design, respectively. This methodology allows the modeling
of a second-order equation that describes the process. Xylanase
production was analyzed by multiple regressions through the
least squares method to fit the following equation:
β i xi +
βij xi xj +
βij xi2
(2)
Y = β0 +
where Y is the measured response variable; β0 , βi , βij , βii are constant and regression coefficients of the model, and xi , xj represent
the independent variables in coded values.
Data from the central composite design for the optimization of xylanase production was subjected to a second-order
multiple regression analysis using the least squares regression
methodology to obtain the parameter estimators of the mathematical model. The regression analysis and analysis of variance
(ANOVA) were carried out using the RSREG procedure of the
SAS statistical package (Version 8.1, SAS Institute, Cary, NC,
USA) to fit second-order polynomial equations for all response
variables. Canonical analysis, which was used to predict the
shape of the curve generated by the model, was carried out as
well. Response surface was made by the fitted quadratic polynomial equation obtained from RSREG analysis.
2.4. Analytical method
The xylanase activity was determined by measuring the
release of reducing sugars from oat spelt xylan (1%, w/v) using
the dinitrosalicylic acid method [20]. The reaction mixture containing 1 mL of a solution of 1% oat spelt xylan in a citrate buffer
50 mM, pH 5.0 plus 1 mL of the diluted crude enzyme, was incu-
Table 1
Box-Behnken experiments design matrix with experimental and predicted values of xylanase production by Penicillium oxalicum ZH-30
Runs
1
2
3
4
5
6
7
8
9
10
11
12
13
Coded setting levels
Actual levels
Xylanase production (U/mL)
X1 (pH)
X2 (temperature)
X1 (pH)
X2 (temperature)
Experimental
Predicted
−1
−1
1
1
−1.414
1.414
0
0
0
0
0
0
0
−1
1
−1
1
0
0
−1.414
1.414
0
0
0
0
0
7
7
9
9
6.6
9.4
8
8
8
8
8
8
8
25
35
25
35
30
30
23
37
30
30
30
30
30
7.76
11.82
1.94
1.25
13.11
1.68
1.72
7.20
11.64
13.10
12.48
11.58
12.86
7.30
12.46
1.54
1.94
13.03
1.51
2.38
6.32
12.33
12.33
12.33
12.33
12.33
84
Y. Li et al. / Biochemical Engineering Journal 34 (2007) 82–86
bate for 30 min at 50 ◦ C. One unit of xylanase was defined as the
amount of enzyme required to released 1 ␮mol of xylose from
xylan in 1 min under the assay condition.
2.5. Partial purification of xylanases by ammonium
sulphate fractionation
A calculated amount of solid ammonium sulphate was added
to the culture supernatant with constant stirring at 10 ◦ C to
achieve 40% saturation. After centrifugation at 8000 × g at 4 ◦ C
for 20 min the precipitate was discarded and the supernatant
was subsequently adjusted to 40–70% saturation by addition
of calculated amounts of ammonium sulphate. The precipitate
was dissolved in a small volume of citrate buffer (50 mM, pH
5.0). The enzyme solution was subjected to dialysis for about
18–24 h at 10 ◦ C against 50 mM citrate buffer (pH 5.0) fortified
with 100 ppm sodium azide, with three intermittent changes of
the buffer. Xylanase activity and protein estimation were carried
out as well.
2.6. Protein assay
Protein quantitative analysis was determined by the Bradford
method [21] with bovine serum albumin as a standard.
2.7. Determination of themostability of partially purified
xylanase
The thermal stability was determined at the temperatures 50,
55, 60, 65 and 70 ◦ C after incubation of suitably diluted enzyme
samples in absence of substrate for 0, 15, 30, 45 and 60 min.
3. Results and discussion
3.1. Xylanase activity optimization
For response surface methodology (RSM) based on the central composite design, which was used to optimize the cultivation
conditions for xylanase production, 13 experimental runs with
the combination of two factors were carried out (Table 1). The
variables used in the factorial analysis were initial pH and
temperature named X1 , X2 in this design, respectively. The maximum xylanase production was 13.11 U/mL in run 5, while the
minimum xylanase production was 1.25 U/mL in run 4. This
result suggested that the xylanase from P. oxalicum ZH-30 had
a higher enzyme activity at neutral pH.
Statistical testing of the model was done by the Fisher’s statistical test for analysis of variance (ANOVA) and the results are
shown in Table 2. The computed F-value (91.91) was much
higher than the F-value in statistic tables [19]. Usually, the
higher the value of CV, the lower the reliability of experiment
is. Here, a lower value of CV (9.60) indicated a greater reliability of the experiments performed. The goodness of a model can
be checked by the determination coefficient (R2 ) and correlation coefficient (R). The determination coefficient (R2 ) implies
that the sample variation of 98.5% for xylanase production is
attributed to the independent variables, and only about 1.5% of
the total variation cannot be explained by the model. The closer
the value of R (correlation coefficient) to 1, the better the correlation between the experimental and predicted values. Here the
value of R (0.992) for Eq. (3) being close to 1 indicated a close
agreement between the experimental results and the theoretical
values predicted by the model equation. Linear, crossproduct
and quadratic terms were significant at the 5% level. Therefore,
the quadratic model was selected in this optimization study.
The Student’s t-distribution and the corresponding values of
the variable estimation are also shown in Table 2. Significance
of coefficients has been reported to be directly proportional to
t-value and inversely to P-value [22]. The parameter estimates
and the corresponding P-values suggest that the independent
variables X1 (pH) and X2 (temperature) have a significant effect
on xylanase production. Positive coefficient for X2 indicated
a linear effect to increase xylanase production, while negative
coefficient for X1 was observed to decrease xylanase production
in a linear effect. The quadric term of these two variables also
had a significant effect. The model clearly revealed a significant
interaction between pH and temperature (P < 0.0207). In this
case, X1 , X2 , X12 X22 , X1 X2 are significant model terms.
By applying multiple regression analysis on the experimental
data, the following second-order polynomial equation was found
Table 2
Analysis of variance for the response of xylanase production
Model term
Degree of freedom
Estimate
Standard error
Sum of squares
F-value
P-value
X1
X2
X1 2
X1 X2
X2 2
Linear
Quadratic
Crossproduct
1
1
1
1
1
2
2
1
−4.07
1.39
−2.53
−1.19
−3.99
0.28
0.28
0.30
0.40
0.30
132.47
15.46
44.42
5.64
111.01
147.93
139.58
5.64
208.45
24.32
69.90
8.88
174.67
115.96
109.41
8.84
<0.0001*
0.0017*
<0.0001*
0.0207*
<0.0001*
<0.0001
<0.0001
0.0207
Total model
Total error
5
7
293.15
4.47
91.91
<0.0001
Coefficient of variation (CV) = 9.60; coefficient determination (R2 ) = 0.985; correlation coefficient (R) = 0.992.
* Significant at 5% level (P < 0.05).
Y. Li et al. / Biochemical Engineering Journal 34 (2007) 82–86
Fig. 1. Response surface plot of the combined effects of pH and temperature on
the xylanase activity by Penicillium oxalicum ZH-30.
to explain the xylanase production:
Y = 12.33 − 4.07pH + 1.39T − 2.53pH2 − 1.19pH × T
−3.99T 2
(3)
where Y is the predicted response (xylanase production).
The fitted response surface plot and their corresponding
counter plot for the xylanase production from P. oxalicum ZH30 by the above model were given in Figs. 1 and 2, respectively.
The contour plots affirmed that the objective function is unimodal in nature showing an optimum in the boundaries. The
Fig. 2. Contour plot of the combined effects of pH and temperature on the
xylanase activity by P. oxalicum ZH-30.
85
boundary optimum point was evaluated using gradient method
in the direction of steepest ascent. The contour plots clearly
revealed that there were no saddle points within the experimental region. Fig. 2 depicts the contour plot showing the effect of
pH and temperature on the xylanase production. As shown in
Fig. 2, decreasing the pH within the tested range was beneficial to improvement of xylanase production under submerged
fermentation. The study of Fig. 2 indicates that the maximum
xylanase production could be obtained in the range of pH 7–7.5
and temperature 30–32.5 ◦ C.
The statistical optimal values of variables are obtained when
moving along the major and minor axis of the contour and
the response at the center point yields maximum xylanase production. These observations were also verified from canonical
analysis of response surface. Canonical analysis revealed a minimum region for the model. The stationary point presenting a
maximum xylanase had the following critical values: initial pH
7.38, temperature = 31.1 ◦ C. The predicted xylanase activity for
these conditions was 14.33 U/mL.
A repeat fermentation of xylanase by P. oxalicum ZH-30
under optimal conditions was carried out for the verification
of optimization. The maximal xylanase level obtained was
14.50 U/mL. This value was found to be 2.7% less than the
predicted value. This discrepancy might be due to the slight
variation in experimental conditions. The optimization resulted
in 10.6-fold increase of xylanase production, compared with the
lowest xylanase production of 1.25 U/mL at run 4 in central
composite design.
3.2. Thermal stability of xylanase from P. oxalicum ZH-30
Ammonium sulphate fractionation (40–70% saturation) of
crude xylanase yielded 80.3% of the enzyme with 3.54-fold
purification. Thermal stability is a very important aspect of enzymatic bioreactors. Utilization of enzymes in industrial processes
often encounters the problem of thermal inactivation of the
enzyme. In fact, enzymes containing a number of extremophilic
characteristics may be of the most use in industry.
Thermal stability assessment of partially purified xylanase
from P. oxalicum ZH-30 were carried out by preincubating the
enzyme up to 60 min at 50, 55, 60, 65, 70 ◦ C, respectively
(Fig. 3). The xylanase retained 86.1 and 82.4% activity at 50
and 55 ◦ C, respectively, after 60 min preincubating. The enzyme
was sensitive at 65 ◦ C, retaining 45.1% activity after 15 min
and only 15.5% activity left after 60 min. At 70 ◦ C, the enzyme
activity was completely inactivated (0.9%) within 15 min. These
results clearly indicated that the suitable temperature range for
industrial application for xylanase from P. oxalicum ZH-30 was
50–60 ◦ C. Belancic et al. [23] reported that xylanases from Penicillium purpurogenum lost about 40% of their activities when
kept for 3 h at 60 ◦ C. Sinitsyna et al. [24] also reported the similar
properties of xylanase from Penicillium canescens. The properties of partially purified xylanase from P. oxalicum ZH-30
indicated its possible use in processed at moderate temperature
which may include preparation of baked cereal food products
and degradation of arabinoxylans in brewing. At the beginning of
mashing, the mashing-in temperature was around 50 ◦ C, which
86
Y. Li et al. / Biochemical Engineering Journal 34 (2007) 82–86
Fig. 3. Themostability of partially purified xylanase from P. oxalicum ZH-30.
was a favorable temperature for xylanase to degrade arabinoxylans polymers into xylo-oligosaccharides [6]. The enzymatic
degradation of these polymers would increase wort filterability and reduce haze in the final product. The applications of
xylanase from P. oxalicum ZH-30 in brewing science are now
in progressing.
4. Conclusion
To the best of our knowledge, there are no reports of optimization of xylanase production by P. oxalicum ZH-30 using
statistical experimental design. Statistical optimization of cultivation conditions using central composite design appears to be
a valuable tool for the production of xylanase by P. oxalicum
ZH-30. The predicted and verifiable xylanase activities under
optimal conditions were 14.33 and 14.50 U/mL, respectively.
The suitable temperature range for industrial application of
xylanase from P. oxalicum ZH-30 was 50–0 ◦ C.
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