Vol.1, No.4, 91-96 (2013) Advances in Enzyme Research
http://dx.doi.org/10.4236/aer.2013.14010
Isolation, purification and characterization of
carboxymethyl cellulase (CMCase) from endophytic
Fusarium oxysporum producing podophy llotoxin
Refaz Ahmad Dar1*, Iram Saba1, Mohd. Shahnawaz2, Manisha Kashinath Sangale2,
Avinash Ba purao Ade2, Shabir Ahmad Rather1, Parvaiz Hassan Qazi1
1Microbial Biotechnology Division, Indian Institute of Integrative Medicine (CSIR), Sanatnagar, Srinagar, Jammu and Kashmir,
India; *Corresponding Author:
2Department of Botany, University of Pune, Pune, Maharashtra, India
Received 28 June 2013; revised 15 August 2013; accepted 2 September 2013
Copyright © 2013 Refaz Ahmad Dar et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Endophytic fungus Fusarium oxysporum is a
rich source of cellulases. In the present study,
the highest activity was reported at 28˚C, pH 5.6
with 2% Carboxymethyl cellulose (CMC) as car-
bon source. CMC was purified using Sephadex G
and DEAE cellulose chromatography to 15.9
folds and the molecular weight was determined
to be 84 kDa by SDS-PAGE analysis and was
subsequently characterized. The purified en-
zyme was stable over the pH range from 4.0 to
8.0 and at temperatures belo w 50˚C. The enzyme
was highly active on CMC and reduced or no
activity on Avicel, cellobiose and it was sug-
gested to be CMCase/endoglucanase. The activ-
ity of endoglucanase was enhanced in the
presence of MgCl2, CoCl2, FeCl3, CaCl2, FeCl2
and intensive to HgCl2. The purified enzyme
showed its optimum activity at pH 5.0 - 6.0 and
was quite stable at 50˚C for 30 min and retained
45% of original activity.
Keywords: Endophyte; Fusarium oxysporum;
Juniperus recurva; Podop hyllotoxin; CMCase
1. INTRODUCTION
The most important source of carbon on this planet is
cellulose, and is being synthesized by both land plants
and marine algae at the rate of 0.85 × 1011 tonnes per
annum [1,2]. The combined and co-operative action of
endocellulases, exocellulases (cellobiohydrolases and
glucanohydrolases) and beta-glucosidases (β-D-gluco-
side glucohydrolase), leads to the degradation of cellu-
lose into glucose [3,4]. The random hydrolysis of inter-
nal glycosidic linkages due to endocellulases leads to
diminishing the length of the polymer followed of gradual
increase of reducing sugar concentration. Whereas the
removal of cellobiose from reducing or the non-reducing
ends due to hydrolysis of cellulose by exocellulase re-
sults in rapid release of reducing sugars, the polymer
length experiences a little change. The synergetic action
of endocellolose and exocellulose on cellulose leads to
the production of cellooligosaccharides and cellobiose.
The beta-glucosidase cleaved both the cellooligosaccha-
rides and cellobiose and results in the production of glu-
cose [5,6]. Cellulases have been widely used in various
industries such as agriculture, bioconversion, detergents,
fermentation, food, pulp and paper and textile for bio-
mass and genetic engineering [7-9]. Throughout the
biospshere these cellulases are distributed and are being
manifested in microbes such as bacteria, fungi and ac-
tinomycetes [10-12]. In literature there are various re-
ports on degradation of cellulose by fungi [13,14]. The
cellulolytic microbes utilize cellulosic substrates during
the growth phase and produce a complex array of glycol-
sacyl hydrolases and all the components of this multien-
zyme system including its specification and mode of
action are reported in Trichoderma sp. (filamentous
fungi) [15]. At present, many research groups around the
globe are screening new cellulases to identify the en-
zymes with high specific activity and stability which
have significant biotechnological applications [16,17].
Various workers reported that the production costs of
cellulases are highly associated with the microbial strains
capable of its production [18,19]. Various factors such as
pH, temperature, incubation period, cations, carbon, and
nitrogen sources are responsible for the yield of enzyme
production [20,21].

R. A. Dar et al. / Advances in Enzyme Research 1 (2013) 91-96
92
Podophyllotoxin, a well-known naturally occurring
aryltetralin lignin occurs in few plant species and is used
as a precursor for the chemical synthesis of various anti-
cancer drugs like etoposide, teniposide and etopophose
phosphate [22]. In the present study known, endophytic
fungal strain producing podophylotoxin was used for
production of cellulase. Since it was identified to be the
Fusarium oxysporum producing podophyllotoxin and it
was shown to possess cellulase enzyme complexes as
well. In the present study an attempt has been made to
determine some other factors that would be responsible
for the optimal production of cellulase by the fungus.
2. MATERIALS AND METHODS
2.1. Organism and Culture Conditions
The strain of the endophytic fungus Fusarium ox-
ysporum producing podophyllotoxin was procured from
the fungal repository of Indian Institute of Integrative
Medicine (CSIR), Srinagar, Jammu and Kashmir State,
India, which had earlier been isolated from Juniperus
recurvaa (a medicinal plant). The strain was cultured at
28˚C and was routinely maintained on potato dextrose
agar (PDA) medium by periodic transfers. The spore
suspension of Fusarium oxysporum was transferred to
500ml Erlenmeyer flasks containing 100 ml of sterilized
medium composed of following composition: glucose
1.0%; yeast extract 1.0%; KH2PO4 0.6%; K2HPO4 0.04%;
MgSO4·7H2O, 0.05%; urea 0.05% and the pH was ad-
justed to 5.6. The flasks were incubated in a rotator
shaker (180 rpm) at 28˚C [23].
2.2. Isolation of Enzymes
Extra cellular enzymes were isolated by filtering the
culture through Whatman No. 1 filter paper. The Car-
boxymethyl cellulase (CMCase), cellobiase (1,4-β-glu-
cosidase) and filter paper activity (Fpase) were measured
using the methods described by Ghose (1987). One unit
of enzyme activity (IU) was defined as the amount of
enzyme that released 1 µmol of glucose per ml per min-
ute. The protein content was determined by Bradford
method [24].
2.3. Optimization of Growth Conditions
To determine the optimum temperature and pH of en-
zymes, the crude CMCase activity was measured under
standard assay conditions, the temperature was varied
from 25˚C to 50˚C and the pH was adjusted within the
range of 4.0 to 8.0. All the experiments were performed
in triplicates.
2.4. Purification of the Enzyme
All extraction steps were performed at 4˚C. Cellulase
was purified from the fungal filtrate after concentration
by freeze drying and storing at 4˚C. The enzyme protein
was bulk precipitated by (NH4)2SO4 (70%) and dissolved
in a minimum volume (100 ml) of 0.1 M citrate phos-
phate buffer (pH 5.0). The enzyme was dialyzed against
the same buffer for 24 h at 4˚C. 50 ml of enzyme protein
was loaded onto a column chromatogram of 100
Sephadex G (18 × 2 cm) pre-equilibrated with 50 ml
buffer. The column was eluted with the same buffer at 20
ml·h−1 and 5 ml fraction was collected. Fraction was ana-
lyzed for protein and activity of CMCase. The most ac-
tive fractions were pooled, concentrated by freeze-drying
and dialyzed as before. The pooled fraction was loaded
(25 ml) onto column chromatograms of DEAE-cellulose
(Diethyl-amino-ethyl-cellulose). The column was eluted
with gradient of 0 - 0.8 M NaCl at a flow rate of 10
ml/h−1 and 5 ml fraction was collected and dialyzed once
again to remove Na+ and Cl−.
2.5. Determination of Molecular Weight
The apparent molecular weight of the purified cellu-
lase from fugal isolate was determined by sodium dode-
cyl sulfate-polyacryamide gel electrophoresis (SDS-
PAGE) according to the method of Weber and Osborn
[25] with protein molecular weight ladder. The gels were
stained with coomassie brilliant Blue R-250 and de-
stained in acetic acid: methanol: water (1:4:5 v/v).
2.6. Effect of Cations on Endoglucanase
Activity
The effect of different metal ions on production of the
enzyme was determined by the addition of the corre-
sponding ions at a concentration of 10 mM to the me-
dium. The enzyme production was studied in the pres-
ence of (MgCl2, CoCl2, FeCl3, CaCl2, FeCl2 and HgCl2).
The activity is expressed as a percentage of the activity
in the absence of metal ion.
2.7. Stability of the Purified Enzyme
The thermal and pH stability of the purified enzyme
was monitored by incubating the enzyme at various
temperatures ranging from 25˚C - 50˚C for 30 min and
pH ranging from 4 - 8. Then the treated enzyme was as-
sayed for activity.
3. RESULTS AND DISCUSSION
3.1. Selection of Cellulase Producing Fungal
Strain
The cellulase degrading activity of the endophytic
fungal strain was assessed through agar diffusion assay
Figure 1. The strain Fusarium oxysporum JRE1 pro-

R. A. Dar et al. / Advances in Enzyme Research 1 (2013) 91-96 93
duced the clear hydrolytic zone and therefore was main-
tained on PDA slants for further use.
3.2. Effect of Different Carbon Sources for
Enzyme Production
Different carbon sources used for the cellulase produc-
tion were wheat straw, wheat bran, crystalline cellulose
and carboxymethyl cellulose (CMC). The best cellulase
activity was found in the CMC, but extracellular en-
zymes were higher in shaking cultures than in static cul-
tures. Different concentrations of the carbon sources
were used. Among them the best CMCase activity was
recorded with 2% CMC (16.8 U/ml) followed by 2%
wheat bran (11.5 U·mg−1) where as wheat straw and
crystalline cellulose does not showed any marked activ-
ity (Figure 2).
3.3. Purification and Electrophoresis of
Enzyme
The Supernatant from 7-day-old submerged culture of
the Fusarium oxysporum grown on 2% CMC, 1% solu-
ble starch, and 0.4% yeast extract was used for endo-
cellulase purification. Fractionation of concentrated, di-
Figure 1. Agar plate showing
clear zones of hydrolysis formed
by F. oxysporum.
Figure 2. Production of cellulase production by F. oxysporum
on different carbon source.
alysed culture filtrate using ion-exchange chromatogram-
phy on DEAE-Sepharose separated three peaks of endo-
cellulase activity. Fractions of the first and second peak
had major amounts of the enzyme activity (over 75%).
The major endocellulase component was further purified
on a Phenyl-Sepharose column (Table 1). The major
endocellulase component was purified 15.9 fold with a
yield of 27.7% to a specific activity of 2.9 U·mg−1 of
protein. A single protein band was observed by SDS-
PAGE (Figure 3), indicating that the major endocellulase
had been purified to homogeneity.
3.4. Effect of Metal Ions
Among different metals examined the endoglucanase
production was enhanced in the presence of metal
cations like MgCl2, CoCl2, MnCl2 and CaCl2 and to some
extent by FeCl2. The result also shows that endogluca-
nase was insensitive to HgCl2 (Table 2).
3.5. Effect of pH
The enzyme was active over a broad range of pH (4.0
- 8.0), showing maximum activity at pH 5.6. The effect
Table 1. Details of the purification of the enzyme CMCase.
Step
Total
protein
(mg)
Total
activity
(U)
Specific
activity
(U·mg−1)
Recovery
(%)
Purification
(fold)
Crude extract 188.7203.4 0.9 100 1
70% (NH4)2SO441.6290.8 8.3 90.6 4.9
DEAE-Sepharose14.2170.6 16.8 34.1 10.5
Phenyl-Sepharose2.9 98.2 27.7 16.3 15.9
Figure 3. SDS-PAGE (8%) analysis of
cellulase b purified from F. oxysporum.
M: Protein ladder; L: Purified bands of
CMCase.

R. A. Dar et al. / Advances in Enzyme Research 1 (2013) 91-96
94
of pH on stability of the enzyme was studied by using
CMC as a substrate under the standard assay condition.
The pH-stability profile of the CMCase was determined
by the residual activity measurement showed 75% of its
original activity was retained between pH 4.0 - 7.0 (Fig-
ure 4).
3.6. Effect of Temperature
The enzyme displayed significant activity within a
temperature range of 25˚C - 37˚C with maximum activity
at 28˚C and at pH 5.6. In order to examine the
temperature stability of the CMCase, the purified
cellulase was maintained after 30 min incubation at
temperatures ranging from 25˚C to 50˚C and pH 5.6. The
enzyme retained more than 70% of its maximum activity
after 30 min exposure to temperatures of 25˚C - 37˚C
and 45% after 30 min exposure at a temperature of 50˚C
(Figure 5).
A CMCase enzyme was purified from this strain and
biochemically characterized. The podophylotoxin
Table 2. Effect of different metal ions on CMCase production
by F. oxysporum.
Metal ion Final conc. (mM) Relative activity (%)
None 0 100
CaCl2 10 100
CoCl2 10 109
HgCl2 10 43
MgCl2 10 116
MnCl2 10 158
FeCl2 10 95
Figure 4. Cellulase activity at different pH (4 - 8).
Figure 5. Cellulase activity at different temperature (25˚C -
50˚C).
producing endophytic fungus already isolated and
identified as Fusarium oxysporum was subjected to
submerged fermentation using different residues as raw
material. The enzyme was highly active on carboxy-
methyl cellulose (CMC) but much reduced or no activity
was observed on Avicel, cellobiose and it was suggested
to be a CMCase/endoglucanase. Independent researchers
have also reported the CMCase from the same strain of
the fungus, but here we are reporting for the first time the
endophytic fungal strain producing podophylotoxin that
is a well known anticancer drug. Kumar et al. [26] also
reported highest CMCase activity with CMC as a carbon
source in Paenibacillus polymyxa. A similar trend has
also been reported in Bacillus sp. [27]. The enzyme was
stable over a broad pH range and was in agreement with
the previous reports [27-30]. The temperature also plays
an important role in activity and the stability of the en-
zymes. Over a broad range of temperature, the enzyme
was also found stable and was also supported by many
workers around the globe [27-30]. Finally, the micro-
organism is promising for industrial application since it
grows quickly in submerged condition in simple and of
low cost substrates and secretes the enzymes extra-
cellularly and shares features which are frequently re-
quired for industrial application.
To the best of our knowledge no one has yet reported
CMCase from the endophytic fungi (F. oxysporum) from
the Sonamarg Area of Kashmir valley, India. This is the
first report of CMCase production from the F. oxysporum
from this region.
4. CONCLUSION
Based on the study carried out, it can be concluded that
the enzyme was highly active on carboxymethyl cellu-
lose (CMC) but much reduced or no activity was ob-
served on Avicel, cellobiose and it was suggested to be a
CMCase/endoglucanase. The enzyme was stable over a
broad pH range and temperature and can be produced
commercially at the cheapest cost. Thus we conclude that

R. A. Dar et al. / Advances in Enzyme Research 1 (2013) 91-96 95
a single endophytic fungal strain which is a source of
anticancer molecule (podophyllotoxin) will be a very
good source for bioethanol production from low cost
substrates.
5. ACKNOWLEDGEMENTS
The authors are thankful to the Council of Scientific and Industrial
Research (CSIR), Government of India for financial support for this
work. The authors would also like to pay thanks to Dr. Ram A. Vish-
wakarma, Director, Indian Institute of Integrative Medicine (CSIR), for
providing the laboratory facilities. MS and MKS also thankful to
UGC-BSR for providing fellowship.
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