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Optimization of Xylanase Production from Penicillium citrinum in Solid-State Fermentation G. Ghoshal,a,* U. C. Banerjee,b Y. Chisti,c and U. S. Shivharea aUniversity Institute of Chemical Engineering and Technology, Panjab University, Chandigarh – 160014, India bDepartment of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research, SAS Nagar-160062, Punjab, India cSchool of Engineering, Massey University, Private Bag 11 222, Palmerston North, New Zealand Solid-state fermentation of sugarcane bagasse by Penicillium citrinum MTCC 2553 was optimized to maximize the yield of xylanase. Preliminary experiments carried out with various lignocellulosic materials revealed sugarcane bagasse to be the most suitable substrate for producing xylanase. Response surface methodology was used in the optimization. Xylanase activity was maximized in a 5-day batch fermentation carried out under the following conditions: a substrate-to-moisture ratio of 1:5 by mass, an initial pH of 7.0 and an incubation temperature of 30 °C. Under the optimal conditions, the final activity of xylanase was 1645 U g–1 of dry substrate. Xylanase was recovered from an extract of the fermented solids by ammonium sulfate precipitation. The crude enzyme was further purified by dialysis. The activity of the enzyme was enhanced in the presence of Na+, Mg2+, Mn2+, Fe3+, Zn2+, Cu2+, Co2+ and Tween 80. The enzyme was inhibited by Hg2+, Ca2+ and the chelating agent ethylene diamine tetra acetic acid (EDTA). Key words: Xylanase, hemicellulose, sugarcane bagasse, solid-state fermentation, Penicillium citrinum Introduction The enzyme xylanase (EC 3.2.1.8) catalyzes the hydrolysis of the linear polysaccharide -1,4-xylan to xylose. Xylan is the major constituent of hemicellulose and therefore xylanases together with other hydrolytic enzymes are used in breaking down hemicellulose. Xylanases are potentially useful in processing of food, feed, pulp and paper.1–3 Many microorganisms are known to produce extracellular xylanases. Certain filamentous fungi are particularly good producers.4,5 Fungal xylanases can be produced both by submerged fermentation and solid-state fermentation (SSF).6 Production of extracellular enzymes by solid-state fermentation is generally less expensive compared to production by submerged fermentation. The fermented solids can be extracted with buffers to provide a preparation of crude xylanases7 that can be used directly without further processing. Low-cost agro-industrial residues such as wheat bran, rice bran, wheat straw, rice husk, sugarcane bagasse and corncobs have been effectively used as substrates for producing enzyme by SSF.8 Fungal xylanases have been previously reviewed.3 Substantial information exists on molecular structures and mode of action of certain xylanases.4,9 This work discusses the SSF production of xylanase by Penicillium citrinum. The fermentation process is optimized by a response surface method10 to maximize the activity of the enzyme. Optimization of substrate type was done by one factor at a time approach. The other parameters of the fermentation process were optimized with respect to the following factors: the initial pH of the fermentation; the initial moisture content in the substrate; the duration of the batch fermentation; and the incubation temperature using response surface methodology. As these factors influence the final enzyme activity interactively, the conventional optimization method of measuring the response (i.e. enzyme activity) in experiments involving variation of a single factor at a time is not a satisfactory strategy for identifying the conditions that maximize enzyme production. Statistical design of experiments in combination with the response surface method (RSM) has been successfully used in optimizing many fermentation processes.11–21 Although RSM has been used to optimize production of microbial xylanases15,22,23 less work has been reported on xylanase production by SSF using Penicillium citrinum. The enzyme produced by SSF G. GHOSHAL et al., Optimization of Xylanase Production from Penicillium citrinum …, Chem. Biochem. Eng. Q. 26 (1) 61–69 (2012) 61 Preliminary communication Received: January 2, 2012 Accepted: March 16, 2012
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G. GHOSHAL et al., Optimization of Xylanase Production from Penicillium citrinum ..., Chem. Biochem. Eng. Q. 26 (1) 61-69 (2012)
61
Optimization of Xylanase Production from Penicillium citrinum
in Solid-State Fermentation

G. Ghoshal,a,* U. C. Banerjee,b Y. Chisti,c and U. S. Shivharea
aUniversity Institute of Chemical Engineering and Technology,
Panjab University, Chandigarh - 160014, India
bDepartment of Pharmaceutical Technology, National Institute of Pharmaceutical
Education and Research, SAS Nagar-160062, Punjab, India
Preliminary communication
cSchool of Engineering, Massey University,
Received: January 2, 2012
Private Bag 11 222, Palmerston North, New Zealand
Accepted: March 16, 2012
Solid-state fermentation of sugarcane bagasse by Penicillium citrinum MTCC 2553
was optimized to maximize the yield of xylanase. Preliminary experiments carried out
with various lignocellulosic materials revealed sugarcane bagasse to be the most suitable
substrate for producing xylanase. Response surface methodology was used in the optimi-
zation. Xylanase activity was maximized in a 5-day batch fermentation carried out under
the following conditions: a substrate-to-moisture ratio of 1:5 by mass, an initial pH of
7.0 and an incubation temperature of 30 C. Under the optimal conditions, the final
activity of xylanase was 1645 U g-1 of dry substrate. Xylanase was recovered from an
extract of the fermented solids by ammonium sulfate precipitation. The crude enzyme
was further purified by dialysis. The activity of the enzyme was enhanced in the
presence of Na+, Mg2+, Mn2+, Fe3+, Zn2+, Cu2+, Co2+ and Tween 80. The enzyme was in-
hibited by Hg2+, Ca2+ and the chelating agent ethylene diamine tetra acetic acid (EDTA).
Key words:
Xylanase, hemicellulose, sugarcane bagasse, solid-state fermentation, Penicillium citrinum
Introduction
stantial information exists on molecular structures
and mode of action of certain xylanases.4,9
The enzyme xylanase (EC 3.2.1.8) catalyzes the
hydrolysis of the linear polysaccharide b-1,4-xylan
This work discusses the SSF production of
to xylose. Xylan is the major constituent of hemi-
xylanase by Penicillium citrinum. The fermentation
cellulose and therefore xylanases together with
process is optimized by a response surface method10
other hydrolytic enzymes are used in breaking
to maximize the activity of the enzyme. Optimiza-
down hemicellulose. Xylanases are potentially use-
tion of substrate type was done by one factor at a
ful in processing of food, feed, pulp and paper.1-3
time approach. The other parameters of the fermen-
Many microorganisms are known to produce extra-
tation process were optimized with respect to the
cellular xylanases. Certain filamentous fungi are
following factors: the initial pH of the fermentation;
particularly good producers.4,5
the initial moisture content in the substrate; the du-
ration of the batch fermentation; and the incubation
Fungal xylanases can be produced both by sub-
temperature using response surface methodology.
merged fermentation and solid-state fermentation
As these factors influence the final enzyme activity
(SSF).6 Production of extracellular enzymes by
interactively, the conventional optimization method
solid-state fermentation is generally less expensive
of measuring the response (i.e. enzyme activity) in
compared to production by submerged fermenta-
experiments involving variation of a single factor at
tion. The fermented solids can be extracted with
a time is not a satisfactory strategy for identifying
buffers to provide a preparation of crude xylanases7
the conditions that maximize enzyme production.
that can be used directly without further processing.
Statistical design of experiments in combination
Low-cost agro-industrial residues such as wheat
with the response surface method (RSM) has been
bran, rice bran, wheat straw, rice husk, sugarcane
successfully used in optimizing many fermentation
bagasse and corncobs have been effectively used as
processes.11-21
substrates for producing enzyme by SSF.8 Fungal
xylanases have been previously reviewed.3 Sub-
Although RSM has been used to optimize pro-
duction of microbial xylanases15,22,23 less work has
*
been reported on xylanase production by SSF using
Corresponding author; Tel: +91 172 253 4908; Fax +91 172 277 9173;
E-mail: gargighoshal@yahoo.co.in
Penicillium citrinum. The enzyme produced by SSF

62
G. GHOSHAL et al., Optimization of Xylanase Production from Penicillium citrinum ..., Chem. Biochem. Eng. Q. 26 (1) 61-69 (2012)
was partially purified. The temperature and pH for
or alkali for overnight. The pretreated substrate was
optimal activity of this enzyme were determined.
dried at 50 C to constant mass, ground to a particle
The influence of the various metal ions on the en-
size of 0.5 mm, and stored in polyethylene bags
zyme activity was established.
until use. The untreated substrate was used as con-
trol. All fermentations used the aforementioned
standardized inoculum.
Materials and methods
Experimental design and data analysis
Materials
Optimum operating conditions were deter-
All chemicals used were of analytical grade.
mined using the response surface method (RSM)
Potato dextrose agar (PDA) was obtained from
with 24 factorial design (Table 1) for the process
Hi-media (India). Birchwood xylan was purchased
variables (pH, temperature, substrate-to-moisture
from Sigma-Aldrich (Germany). Wheat straw, wheat
mass ratio, incubation time) with three levels each.
bran, rice straw, rice bran, sawdust, corncobs, and
Enzyme activity (U g-1 of dry substrate) after
sugarcane bagasse were purchased from the local
desired period of incubation was measured as the
market.
response. The response was modeled using the fol-
lowing polynomial:
Microorganism and culture condition
P = b
2
(1)
0 + a b X + b X
+ a b X X
The filamentous fungus Penicillium citrinum
i
i
ii
i
ij
i
j
MTCC 2553 (procured from Institute of Microbial
where P is predicted response (xylanase activity);
Technology, Chandigarh, India) was used through-
b is regression coefficient at the center of the re-
out. The microorganism was maintained on PDA
0
gression model; and b , b , b , are coefficients esti-
slants and stored at 4 C.
i
ii
ij
mated by the model for the linear, quadratic, and in-
In preliminary work, the aforementioned solid
teractive effects of the coded variables (A, B, C, D).
substrates were individually evaluated for produc-
In eq. 1 X and X are the process variables.
tion of xylanase by SSF. Thus, each substrate was
i
j
ground to a particle size of 0.5 mm. The ground
substrate (5 g) was mixed with a moistening me-
T a b l e 1 - Factor levels used in the experimental design
dium in a 250 mL Erlenmeyer flask such that the
Actual levels of coded factors
mass ratio of the substrate-to-medium was 1:5. The
Factor
Symbol
aqueous moistening medium contained the follow-
-1
0
+1
ing (g L-1): sucrose 30, yeast extract 5, K HPO 1,
2
4
pH
A
6.5
7.0
7.5
NaNO 30, KCl 5, MgSO 5 and FeSO 0.1. The
3
4
4
pH of the moistening medium was adjusted to 7.
Temperature (C)
B
25
30
35
The flasks containing the moistened substrate
Substrate-to-moisture
C
1:4
1:5
1:6
were then sterilized at 121 C, 15 min. The flasks
mass ratio
were cooled to 30 C and inoculated with a 1 mL sus-
Time (d)
D
4
5
6
pension of fungal spores in sterile water. The spore
suspension had been standardized to provide 108
spores g-1 of dry substrate. The inoculated substrate
Design Expert ver. 6.0.9 statistical software
was incubated at 30 C, for 5 days, in flasks plugged
(Stat-Ease Inc, Minneapolis, MN, USA) was used
with sterile cotton wool. The initial pH was 7.0.
for the statistical analyses. Data was analyzed to es-
The substrate that gave the maximum final
timate whether a given term had a significant effect
xylanase activity in the above study, was used for
(p < 0.05) on enzyme activity using the analysis of
all future work. In some cases, the selected sub-
variance (ANOVA) in combination with the Fischer
strate was washed with distilled water, dried and
test. Graphical and mathematical analyses were per-
pretreated prior to use in fermentations. The follow-
formed using the Design Expert program to deter-
ing pretreatments were tested: (1) alkali treatment
mine the optimum intensity level of the variables.
using 1 mol L-1 NaOH followed by neutralization
The overall second order polynomial relationship
with 1 mol L-1 HCl and repeated washing with dis-
between P and the variables could be represented
tilled water; (2) acid treatment with 1 mol L-1 HCl
by the following quadratic equation:
followed by neutralization with 1 mol L-1 NaOH
P = b0 + b A
1
+ b B
2
+ b C
3
+ b D
4
+
and washing with distilled water; and (3) steam
treatment of dry substrate at 121 C for 2 h. In
+ b A2 + b B 2
2
2
(2)
22
+ b C
33
+ b D
11
44
+
pretreatments (1) and (2), 100 g of dry substrate
was completely immersed in about 500 mL of acid
+b AB+b AC+b AD+b BC+b BD+b CD
12
13
14
23
24
34

G. GHOSHAL et al., Optimization of Xylanase Production from Penicillium citrinum ..., Chem. Biochem. Eng. Q. 26 (1) 61-69 (2012)
63
where, A, B, C, D are the coded variables for pH,
Determination of pH optimum,
temperature (C), substrate-to-moisture mass ratio,
temperature optimum, pH stability
and incubation time (d). In eq. 2, b , b , b , b , b ,
and thermostability of xylanase
0
1
2
3
4
b , b , b , b , b , b , b , b , b , b
are the co-
11
22
33
44
12
13
14
23
24
34
For determining the optimum pH of xylanase,
efficients for the various effects.
the activity was measured at 50 C in buffers of pH
values ranging from 3 to 11. The following buffers
Enzyme extraction
were used: citrate buffer for pH 3 to 6; phosphate
buffer for pH 6.5 to 8; and glycine NaOH buffer for
The crude enzyme from each fermentation
pH 8.5 to 11. The pH stability of the enzyme was
flask was extracted with 50 mL of 0.05 mol L-1 ci-
determined by incubating the enzyme in the above
trate buffer at pH 6.0. The suspended solids were
specified buffers at 302 C for 24 h. The activity
removed by filtering the slurry through a sterile
was then measured and compared to the initial ac-
muslin cloth. The solids remaining in the filtrate
tivity.
were removed by centrifugation (4 C, 10,000 g,
The optimum temperature was determined by
20 min). The resulting cell free supernatant was
measuring the xylanase activity for 5 min at incuba-
used as the crude enzyme preparation in subsequent
tion temperatures ranging from 25 to 90 C at pH 6
measurements.
(citrate buffer). The temperature stability was deter-
mined at 50 C by measuring the remaining activity
Total protein and enzyme assays
after incubation of the enzyme at the specified tem-
perature for 24 h, pH 6 (citrate buffer), in the ab-
The total protein in the crude enzyme extract
sence of the substrate. All measurements were done
was measured using the Lowry method24 with bo-
in triplicate.
vine serum albumin as the standard protein solu-
tion.
Effect of additives on xylanase activity
The activity of xylanase was measured at 50 C
using a 10 g L-1 solution of Birchwood xylan as the
Effect of various additives on xylanase activity
substrate in 0.05 mol L-1 citrate buffer, pH 6.0. The
was determined by incubating the xylanase solution
enzyme solution (0.2 mL) was incubated with the
(pH 6.0) with 10 mmol L-1 of the specified additive
substrate (1.8 mL) for 5 min. The reaction was then
at 302 C. Samples were withdrawn at 15 min in-
terminated by adding 3 mL of dinitrosalicylic acid
tervals and the xylanase activity was measured. The
solution and boiling for 5 min.25 Absorbance of the
additives were: Na+, Mg2+, Ca2+, Mn2+, Fe3+, Zn2+,
resulting mixture was read at 540 nm. One unit of
Cu2+, Co2+, Hg2+, Tween 80 and EDTA. The mea-
enzyme activity was defined as the quantity of en-
sured activity was compared to the activity of the
zyme liberating 1 mmol of xylose per min.26 Xylanase
control preparation (xylanase solution incubated as
activity was expressed as U g-1 of dry substrate.
above but with no additive). All measurements
were done in triplicate.
As cellulase is commonly produced together
with xylanase, the activity of cellulase in the crude
enzyme extract was also measured. Thus 0.5 mL of
Results and discussion
the crude extract was incubated with the substrate
(50 mg of Whatman no.1 filter paper suspended in
Effect of substrates on xylanase yield
0.5 mL of 0.05 mol L-1 phosphate buffer at pH 6.0)
for 1 h at 50 C. The reaction was stopped by add-
In preliminary batch fermentations (5-days,
ing 2 mL of dinitrosalicylic acid solution followed
30 C, initial pH of 7), the highest yield of xylanase
by boiling for 5 min. Absorbance of the resulting
(151.7 U g-1 of dry substrate) was obtained on
solution was measured at 540 nm. One unit of
sugarcane bagasse and only a negligible amount
cellulase activity was defined as the quantity of
of cellulase (0.06 U g-1 of dry substrate) was pro-
enzyme that released 1 mmol of glucose min-1.27
duced. In comparison, the xylanase yields on
Cellulase activity was expressed in U g-1 of dry
sawdust, corncobs, wheat bran, xylan, rice bran,
substrate. All measurements were done in triplicate.
rice straw, and wheat straw were 45.5, 44.6, 141.5,
128.5, 76.5, 20.5 and 12.2 U g-1 of dry substrate,
respectively. In view of the high yield, sugarcane
Partial purification of xylanase
bagasse was used in all future fermentations. The
The extracted crude enzyme was partially puri-
high xylanase yield on bagasse was likely due to a
fied by ammonium sulfate precipitation (60 %) fol-
relatively high level of hemicellulose in this sub-
lowed by dialysis overnight in 0.05 mol L-1 citrate
strate.28
buffer for desalting and used for characterization
As shown in Table 2, the acid, alkali and steam
studies.
pretreatments of sugarcane bagasse actually re-

64
G. GHOSHAL et al., Optimization of Xylanase Production from Penicillium citrinum ..., Chem. Biochem. Eng. Q. 26 (1) 61-69 (2012)
T a b l e 2 - Effect of different pretreatments of sugarcane
for solid-state fermentation. In view of the results
bagasse on xylanase activity
(Table 2), all subsequent fermentations used un-
treated bagasse as the substrate.
Xylanase activity
Pretreatment of substrate
(U g-1 ds)
Effect of process variables on xylanase activity
Untreated
151.7
Data on the final activity of xylanase produced
Alkali treatment (1 mol L-1 NaOH)
115.8
under different combinations of the initial pH
Acid treatment (1 mol L-1 HCl)
96.1
(pH 6.5-7.5), temperature (25-35 C), substrate-to-
-moisture mass ratio (1:4 to 1:6), and incubation
Steam treatment (121 C, 2 h)
108.5
time (4-6 d), are reported in Table 3. The combined
effects of the process variables on the final xylanase
activity could be expressed in the form of eq. 2, us-
duced the enzyme yield relative to the untreated
ing the coded variables (A-D, Table 1); thus:
substrate. This may have resulted from inhibitory
compounds being produced in some of the treat-
P = 1309.01 - 130.81A - 98.11B + 82.64C -
ments and/or leaching of nutrients from the sub-
- 98.11D - 124.08A2 - 178.89B2 - 51.61C2 -
strate as a consequence of the treatment process.
Alternatively, pretreatments may have altered the
- 94.03D2 - 35.61AB + 25.0AC + 6.89AD - (3)
porosity of the substrate29 to make it less suitable
- 35.50BC - 143.31BD - 140.0CD
T a b l e 3 - Experimental design and results of the central composite design for xylanase activity
Factors
Xylanase activity (U g-1 ds)
Run number
Block
A
B
C
D
observed
predicted
1
Block 1
7.5
25.0
1:6
6
1120
1114.8
2
Block 1
6.5
35.0
1:6
6
720
684.5
3
Block 1
7.5
25.0
1:4
6
990
984.8
4
Block 1
7.0
30.0
1:5
5
900
864.5
5
Block 1
7.5
35.0
1:4
4
1020
984.5
6
Block 1
7.0
30.0
1:5
5
1320
1314.8
7
Block 1
6.5
25.0
1:4
4
640
634.8
8
Block 1
7.0
30.0
1:5
5
890
854.5
9
Block 1
6.5
35.0
1:4
6
1330
1378.2
10
Block 1
7.5
35.0
1:6
4
1340
1378.2
11
Block 1
6.5
25.0
1:6
4
1330
1378.2
12
Block 1
7.0
30.0
1:5
5
1350
1378.2
13
Block 2
6.16
30.0
1:5
5
1080
1108.8
14
Block 2
7.0
30.0
1:5
5
640
668.8
15
Block 2
7.0
30.0
1:3.32
5
870
898.8
16
Block 2
7.84
30.0
1:5
5
540
568.8
17
Block 2
7.0
21.59
1:5
5
890
954.8
18
Block 2
7.0
30.0
1:6.68
5
1240
1232.8
19
Block 2
7.0
30.0
1:5
6.68
1110
1138.8
20
Block 2
7.0
30.0
1:5
3.32
780
808.8
21
Block 2
7.0
30.0
1:5
5
1350
1239.8
22
Block 2
7.0
38.41
1:5
5
1360
1239.8

G. GHOSHAL et al., Optimization of Xylanase Production from Penicillium citrinum ..., Chem. Biochem. Eng. Q. 26 (1) 61-69 (2012)
65
Fisher's test for analysis of variance (ANOVA)
tivity with respect to the individual variables and an
was used for statistical testing of the model (eq. 3)
inhibitory effect of the variable at other than the
and the results are shown in Table 4. The ANOVA
peak point.
revealed that the response surface quadratic model
A positive value of the linear coefficient for C
(eq. 3) was significant because the p-value for the
(eq. 3) indicated that the xylanase activity increased
model was less than 0.05 while the corresponding
with substrate-to-moisture ratio, a factor that is
F-value was 12.75 (Table 4). A insignificant value
known to profoundly affect many SSF processes.29
for the lack of fit indicated that the quadratic model
Too much moisture leads to water logging of
was valid for the present study.13 The R2 value for
the substrate and poor diffusion of oxygen. Too
the model was 0.967, indicating general good
little water means a water activity that may be too
agreement of the model and the data. The small
value of the coefficient of variation of 8.62 sug-
low to solubilize the substrate and support fungal
gested a high level of precision in the data (Table
growth.29
4).
Response surface plots were generated to iden-
The various p-values in Table 4 indicate the
tify the optimal levels of the process variables for
significance of the coefficients of the corresponding
maximizing the activity of xylanase (Figs. 1-2).
terms in eq. 3. The p-value of less than 0.05 for A,
The response surfaces were generated for the varia-
B, C, D, A2, B2, D2, BD and CD revealed these
tion of two independent variables while keeping
model terms to be significant. The negative qua-
other variables at fixed B levels. The response plots
dratic coefficient values (eq. 3) for all the variables
in Figs. 1-2 take into account the possible combina-
implied the existence of a peak point xylanase ac-
tions of independent variables.
T a b l e 4 - Analysis of variance for the response surface model (eq. 3)
Source
Sum of squares
DF
Mean of squares
F-value
p-value
Block
47345.61
47345.61
Model
1.425 * 106
14
1.425 * 106
12.75
0.003a
Significant
A
96800.0
1
96800.0
12.12
0.013a
B
54450.00
1
54450.00
06.82
0.040a
C
93271.85
1
93271.85
11.68
0.014a
D
54450.0
1
54450.0
06.82
0.040a
A2
2.372 * 105
1
2.372 * 105
29.71
0.002a
B2
4.93 * 105
1
4.93 * 105
61.75
0.000a
C2
41033.09
1
41033.09
05.14
0.064
D2
1.362 * 105
1
1.362 * 105
17.06
0.006a
AB
4201.92
1
4201.92
0.53
0.496
AC
5000.00
1
5000.00
0.63
0.459
AD
157.33
1
157.33
0.02
0.893
BC
8450.00
1
8450.00
01.06
0.343
BD
68058.78
1
68058.78
08.52
0.027a
CD
1.568 * 105
1
1.568 * 105
19.64
0.004a
Residual
47905.29
6
7984.21
Lack of fit
47580.29
2
23790.14
292.80
<0.0001
Insignificant
Pure error
325.00
4
81.25
Cor. total
1.52 * 106
21
R2 = 0.967, adjusted R2 = 0.896, C.V. = 8.62, mean = 1036.82
aSignificant model terms, C.V.: coefficient of variance, DF degree of freedom

66
G. GHOSHAL et al., Optimization of Xylanase Production from Penicillium citrinum ..., Chem. Biochem. Eng. Q. 26 (1) 61-69 (2012)
F i g . 1 - Combined effect of (a) media pH and incubation tem-
F i g . 2 - Combined effect of (a) incubation time and pH (b)
perature (b) incubation time and substrate to moisture ratio (c)
incubation time and temperature (c) substrate to moisture ratio
substrate to moisture ratio and temperature on xylanase activity
and pH of the fermentation media on xylanase activity

G. GHOSHAL et al., Optimization of Xylanase Production from Penicillium citrinum ..., Chem. Biochem. Eng. Q. 26 (1) 61-69 (2012)
67
Interactive effects of variables
response surface model. The values of the variables
on the xylanase activity
A-D in this confirmation experiment were set to
levels suggested by the Design Expert statistical
The p-value of >0.05 for AB (Table 4) in-
software and shown in Table 5. The three replicate
dicated that temperature and pH had not signifi-
fermentations yielded an average enzyme produc-
cantly interacted in influencing the xylanase titer,
tion of 1645.3 U g-1 of dry substrate, a value within
but had individually affected the xylanase titer.
20 % of the predicted value of 1369.6 U g-1 of dry
The negative values of coefficients for A, B, A2,
substrate (Table 5). Therefore, the response surface
B2, AB (eq. 3) indicated that an increase or decrease
model and its optimum point were verified. Statisti-
in pH and temperature from the optimal point re-
cal optimization of this fermentation increased the
duced xylanase titer. Fig. 1a shows the response
final activity from 151.7 U g-1 of dry substrate for
surface for the effect of pH and temperature on
unoptimized conditions to 1645.3 U g-1 of dry sub-
xylanase activity at constant values of the variables
strate for the optimized case. The, optimized condi-
C (= 1:5) and D (= 5 d). From Fig. 1a, the maxi-
tions enhanced the final titer of xylanase by nearly
mum response occurs in the vicinity of 30 C and
11-fold.
pH 7.
In view of p-value of <0.05 for CD (Table 4),
the variables of substrate-to-moisture ratio (C) and
T a b l e 5 - Predicted and measured values of maximum
incubation time (D) had a significant interactive ef-
xylanase activity
fect on xylanase titer. In the range of interest, the
Xylanase activity
xylanase titer increased with an increased incuba-
(U g-1 ds)
tion time and an increased level of moisture in the
Variable
Value
substrate (Fig. 1b). This is generally consistent with
predicted
measured
other
similar
observations
for
production
of
pH
7.0
1369.6
1645.3
xylanases by fungi.22
Temperature (B) and the substrate-to-moisture
Temperature (C)
30
ratio (C) did not have a significant interactive influ-
Substrate-to-moisture
1:5
ence on the enzyme titer (p > 0.05, Table 4). Both
mass ratio
these factors individually affected the enzyme titer.
Incubation time (d)
5.0
As shown in Fig. 1c, the maximum response
occurred in the temperature range of 29-30 C and
at a substrate-to-moisture ratio of 1:6.
It has been reported in literature that xylanase
The interactive effect of pH (A) and incubation
yield was lesser (approximately 20-100 U g-1 solid
time (D) on the enzyme titer was insignificant as
substrate) in SSF with Penicillium citrinum as the
p-value for AD was above 0.05 (Table 4). A pH of
producing organism.30-31 While, with Aspergillus
around 7 in combination of an incubation time of
niger, the xylanase yield was comparable with our
around 5 days maximized the xylanase titer (Fig.
results.29
2a) at fixed values of B (30 C) and C (sub-
strate-to-moisture ratio of 1:5).
The interactive effect of temperature (B) and
Partial purification of xylanase
incubation time (D) on xylanase titer was signifi-
The enzyme could be easily concentrated by
cant (p < 0.05 for BD in Table 4). Fig. 2b indicates
ammonium sulfate precipitation from the clarified
that the region of interaction of temperature with
extract of the fermented solids. Nearly half (51.1 %)
incubation time was in the temperature range of
27.5 to 32.5 C and a substrate-to-moisture ratio
of the xylanase activity was recovered in the pre-
range of 1:4 to 1:6.
cipitate after the crude extract was supplemented
with ammonium sulfate to 40-60 % of the saturation
The pH (A) and the substrate-to-moisture ratio
level. The precipitate was redissolved in 0.05 mol L-1
(C) had no significant interactive effect (p > 0.05
citrate buffer and dialyzed overnight against the
for AC in Table 4), but both these variable individu-
same buffer for desalting prior to activity measure-
ally affected the xylanase titer (Fig. 2c). Fig. 2c is
for a constant incubation temperature of 30 C and
ments. Precipitation concentrated the enzyme activ-
an incubation time of 5 days.
ity by 3.5-fold (Table 6). Similar purification levels
and concentration factors have been previously re-
ported for recovery of xylanases by ammonium sul-
Validation of the experimental model
fate fractionation from crude extracts and clarified
An additional fermentation was carried out in
broths of Aspergillus ochraceus32 and Aspergillus
triplicate to validate the optimal predictions of the
foetidus.29

68
G. GHOSHAL et al., Optimization of Xylanase Production from Penicillium citrinum ..., Chem. Biochem. Eng. Q. 26 (1) 61-69 (2012)
T a b l e 6 - Xylanase activity and yield during various steps of crude extract purification
Volume
Total xylanase
Total protein
Specific activity
Yield*
Purification
Purification step
(mL)
(U)
(mg)
(U mg-1)
(%)
factor
Crude extract
50
4230.6
135
31.3
100
1.00
Ammonium sulfate fraction
5.0
2160.6
19.9
108.8
51.1
3.5
Dialysis
9.5
2136.5
18.9
113.0
50.5
3.6
*Based on the total activity in the crude extract.
pH and temperature optima, pH
had strong activity reducing effects (Table 7).
and temperature stability
EDTA is a strong chelator of metal ions. Reduced
Xylanase of P. citrinum was stable in the pH
activity in the presence of EDTA suggests a require-
range of 3 to 8. Above a pH value of 8, the activity
ment for a metal ion for the action of xylanase, al-
reduced to 10 % of the initial value after 24 h of in-
though xylanases are not metaloproteins.36 The re-
cubation at 30 C. The optimum pH for activity was
quired ion is obviously not Hg2+ or Ca2+ as these
found to be 6. The optimum temperature for activ-
ions reduced activity (Table 7). Several other multi-
ity was 50 C. At 70 C, the activity was reduced to
valent metal ions enhanced activity (Mg2+, Mn2+,
20 % of the initial value after 24 h. Thus, the en-
Fe3+, Zn2+, Cu2+, Co2+) as did also the monovalent
zyme could be viewed as relatively thermostable.
Na+ (Table 7). Many xylanases are known to be in-
Often, enzymes are not stable for long at tem-
hibited by Hg2+,37,38 possibly because the catalytic
peratures of >40 C.33 In terms of pH stability, opti-
site of the enzyme contains histidine36,39 which ap-
mal pH, optimal temperature and thermostability,
pears to complex with Hg(II).40 Compared to other
the enzyme was generally comparable to the
metal ions, Hg(II) has in general a greater binding
xylanases of Bacillus pumillus,7 Rhizopus oryzae34
power for nitrogen ligands40 such as the ones found
and Streptomyces sp.35
in histidine residues. The activity stimulating effect
of some of the other metal ions may be linked to
Effect of metal ions on xylanase activity
their weaker interactions with the active site lig-
ands, although no direct evidence for this appears to
Relative to control (no additive), the activity of
exist.
xylanase was enhanced or reduced depending on
the additive used (Table 7). EDTA, Hg2+ and Ca2+
Conclusions
T a b l e 7 - Effect of additives on xylanase activity relative
to control
Of the substrates tested, sugarcane bagasse
Additives
Activity (%)
proved to be the best for producing xylanase by
solid-state fermentation using the microfungus
None (control)
100
Penicillium citrinum MTCC 2553. The optimal
Co2+
189
conditions for producing the enzyme in 5-day batch
fermentation
were:
substrate-to-moisture
mass
Cu2+
165
ratio of 1:5, initial pH of 7 and incubation tem-
perature of 30 C. Under these conditions, the final
Zn2+
142
activity of xylanase was 1645 U g-1 of dry sub-
Fe3+
132
strate. During hydrolysis of Birchwood xylan, the
partly purified xylanase was shown to have tem-
Mn2+
124
perature and pH optima of 50 C and 6, respec-
Na+
123
tively. The enzyme was inhibited by 10 mmol L-1
Ca2+, Hg2+ and EDTA, but was activated by
Mg2+
113
10 mmol L-1 Co2+, Cu2+, Zn2+, Fe3+, Mn2+, Na+ and
Mg2+.
Tween 80
108
Ca2+
56
ACKNOWLEDGEMENTS
Hg2+
21
GG and USS are thankful to AICTE, New
EDTA
14
Delhi, for providing financial support for this work.

G. GHOSHAL et al., Optimization of Xylanase Production from Penicillium citrinum ..., Chem. Biochem. Eng. Q. 26 (1) 61-69 (2012)
69
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