Journal of Scientific & Industrial Research

454
Vol. 69, June 2010, pp. 454-459 J SCI IND RES VOL 69 JUNE 2010

Production and optimization of cellulase from Fusarium oxysporum

by submerged fermentation
G Ramanathan*, S Banupriya and D Abirami
Department of Microbiology, V H N S N College, Virudhunagar 626 001, India

Received 27 July 2009; revised 25 February 2010; accepted 03 March 2010

Fusarium oxysporum, isolated from infected tomato plant parts, produced maximum cellulase at optimum parameters (pH,
6.0; temp., 50C; and incubation period, 12 d) in cellulase enzyme production broth having 1% CMC (carboxy methyl cellulose)
as a cellulose substrate. Activities of purified cellulases (mol wt, 24, 29 and 45 kDa) were stimulated by concentrations (0-70 mM)
of Na+ and Mg++, while EDTA inhibited enzyme activity at all concentrations.

Keywords: Cellulose, CMC, Fermentation, Fusarium oxysporum, Glucosidase

Introduction Broth cultures were incubated on a rotarty shaker

Cellulose is most common carbohydrate on earth1.
Utilization of cellulose as a nutrient source requires
enzymes that cleave - 1, 4- glycosidic bonds between
constituent sugars 2 . Cellulase is associated with
pathogenicity of a number of microorganisms3. Fusarium
oxysporum, a cosmopolitan soil borne filamentous
fungus, is an anamorphic species that includes numerous
filamentous plant pathogenic strains causing wilt disease
of a broad range of agricultural and ornamental host
plant species. It produces several enzymes that act upon
pectic and cellulose components of cell walls4. This study
presents production and optimization of cellulose enzyme
from F. oxysporum by submerged fermentation.
Experimental Section
Sample Collection and Assessment of Cellulase Activity
Infected parts (leaf, stem) of diseased tomato plant
were collected and aseptically transfered to laboratory
for further isolation of fungal pathogen. Fungal colonies
were isolated in potato dextrose agar (PDA) plates.
Cellulose degradation potential was assessed of selected
strains by growth and zone formation in carboxy methyl
cellulose (CMC) agar medium.
Liquid State Fermentation
Cellulase enzyme production medium and inoculated
spore suspensions of F. oxysporum were prepared.
*Author for correspondence
E-mail: san_than2002@yahoo.co.in
(150 rpm) at 30C. Fermented samples were withdrawn
at two days (2, 4, 6, 8, 10, 12 days) interval. Reducing
sugar was estimated by DNS (Dinitro Salicylic acid)
method5 and protein content by reported method6.
Assay of Cellulase Enzyme
Activity of cellulase was assayed by reported method7.
Fermented sample was centrifuged at 3000 rpm for
10 min. Supernatant was used as enzyme source. For
CMCase activity, suitably diluted enzyme solution (1 ml
diluted equally with 0.2 M acetate buffer, pH 4.8) was
incubated with 1 ml CMC (10 mg/ml) in 0.2 M acetate
buffer at 60C for 30 min. For FPase activity, suitably
diluted enzyme solution (1 ml diluted equally with 0.2 M
acetate buffer) was incubated with 50 mg filter paper
(What man No.1) in 1 ml of 0.2 M acetate buffer (pH 4.8)
for 1 h at 60C. For -glucosidase activity, suitably diluted
enzyme solution (1 ml diluted with 0.2 M acetate buffer)
was incubated with 1 ml of salicin (10 mg/ml) solution in
0.2 M acetate buffer (pH 4.8) at 60C for 30 min. Amount
of reducing sugars released in CMCase, FPase,
-glucosidase assay after incubation was estimated by
DNS method5. Cellulase was estimated8 and its activity
was expressed in International Unit (IU).
Effect of Cations and EDTA on Cellulase Activity
Effect of cations on cellulase activity was determined
by using two cations (Na+ and Mg++; conc., 0, 10, 20, 30,
40, 50, 60 and 70 mM). Substrate cation mixture was
incubated at room temperature (RT) for 1 h before it was
 RAMANATHAN et al: CELLULASE FROM FUSARIUM OXYSPORUM
used in enzyme assay. Effect of ethylene diamine tetra
acetic acid (EDTA) at various molar concentrations
(0, 10, 20, 30, 40, 50, 60 and 70 mM) on activity of cellulase
was determined. Substrate chemical compound mixture
was incubated at RT for 1 h before it was used in enzyme
assay.
Optimization of Culture Conditions
Optimum culture conditions [pH (4, 5, 6, 7, 8 and 9),
temperature (30, 40, 50, 60, 70 and 80C), incubation
period, carbon (glucose, lactose, sucrose and cellulose;
1%w/v) and nitrogen (peptone, yeast extract, urea,
diammonium sulphate; 1%w/v) source requirements]
were determined for maximum growth of F.oysporum
and cellulase activity were recorded.
Crude Protein Extraction by Ammoniumsulphate Precipitation
Ammonium sulphate (60%) was added to filtrate slowly
with continuous stirring at low temperature (in an ice
bath/beaker) for 5-10 min and left overnight in
refrigerator. Mixture (salt+filtrate) was centrifuged at
12000 rpm for 20 min at 4-6C, then pellet was collected
and dissolved in 0.2 M sodium phosphpate buffer
(pH 7.0) using two volumes of buffers. During collection
of enzymes, tubes and funnel were washed with same
buffer and collected; process was repeated thrice. Volume
should not be 20% of total volume of filtrate. Enzyme
salt solutions were dialysed with same buffer, which was
changed 3-5 times for removing ammonium salt9.
Double-Layer Plate Assay
To detect crude enzyme activity, a bottom layer
[15 ml of 0.7% (w/v) agarose and 50 mM potassium
phosphate-citric acid buffer (pH 5.2)] was overlaid with
5 ml of CMC (0.2%; w/v) and agarose (0.5%; w/v).
Plates were inoculated with protein extract (40 l) at
30C for 24 h. To detect CMCase activity, plates were
flooded with congored solution (1%; w/v) for 30 min and
then rinsed several times with 1 M NaCl. This procedure
revealed distinct hydrolysis regions10.
Purification by Ion- Exchange Chromatography on DEAE -
Sephadex A - 50
DEAE sephadex A 50 column was packed into a
vertically mounted column (1.5 cm x 40 cm) at a flow
rate of 30 ml/h. Column was equilibrated with 3 bed
volumes of 0.02 M sodium phosphate buffer, pH 7.0
containing 1 mM 2-mercaptoethanol. Enzyme concentrate
obtained from ammonium sulphate precipitation was
redissolved in minimal amount of buffer and dialysed for
455
24 h against 0.02 M sodium phosphate buffer, pH 7.0
containing 1 mM EDTA and 1 mM 2-mercaptoethanol
with two changes of buffer per hour. Undissolved
precipitate was removed by centrifugation and clear
supernatant was layered on prepared column. Column
was washed to remove all unbound proteins and a linear
gradient of 0-0.5 M NaCl in 0.02 M sodium phosphate
buffer (pH 7.0) was used to elute any bound proteins.
Fractions containing cellulase activities were pooled
together and precipitated with ammonium sulphate.
Precipitate was collected by centrifugation at 4000 g in a
cold centrifuge at 4C for 30 min, redissolved in 2.5 ml of
0.02 M sodium phosphate buffer (pH 7.0) and dialysed
against buffer (pH 7.0) for 6 h11.
Molecular Mass Determination
Molecular weight of cellulase was determined by SDS-
PAGE using selectable markers12.
Results and Discussion
Assessment of Cellulase Activity in CMC Agar Medium
Selection of over producing cellulase strains of
F. oxysporum were based on diameter of clearing zone
surrounding small well in plate screening medium. Size
of clearing zone diameter for selected strain was recorded
for different incubation period as follows: 2 days, 12 mm;
4 days, 24 mm; 6 days, 32 mm; and 8 days, 36 mm.
Enzyme activity of F. oxysporum was found as follows:
CMCase, 1.92 U/ml; FPase, 1.34 U/ml; and
-glucosidase, 1.78 U/ml.
Cellulose
Cellulose degradation was assessed on 8th day of
fermentation by selected fungal strains. F. oxysporum
utilized 32.68% of cellulose on 8th day of incubation (Fig.
1a). Similar results have been reported13 with studies on
cellulosic substrates.
Reducing Sugar
Initial content of free reducing sugar in cellulase
production broth was found to be 2.38 mg/ml. A gradual
and steady increase in reducing sugar content was
observed in cellulase production broth supplement with
CMC. A tremendous (1.28 fold) increase in content of
reducing sugar was observed during 8 th day of
fermentation period by F. oxysporum (Fig. 1b). Amount
of reducing sugar increased with time of incubation with
presence of enzyme when cellulose rich agro waste
supplemented as a substrate14.
456 J SCI IND RES VOL 69 JUNE 2010

50

45
a)
a)
40
Cellulose utilization, %

35

30

25

20

15

10

0
2 4 6 8 10 12

Fermentation period, days

1.4

b)
1.2 b)
Reducing sugar, mg/ml

0.8

0.6

0.4

0.2

0
2 4 6 8 10 12

Fermentation
Fermentationperiod,
period,days
days
0.8

c)
0.7 C)
0.6
Protein content, mg/ml

0.5

0.4

0.3

0.2

0.1

0
2 4 6 8 10 12

Fermentation period, days

Fig. 1Liquid state fermentation media inoculated with F.oxysporum and supplemented with CMC: a)
Cellulose utilization; b) Reducing sugar content; and c) Protein content
 RAMANATHAN et al: CELLULASE FROM FUSARIUM OXYSPORUM 457

Fig. 3Effect on cellulase activity of F.oxysporum during 8th day

of fermentation of: a) EDTA; b) Mg concentration; and
c) Na concentration
Protein
Initial protein content of cellulase enzyme production
broth was 0.35 mg/ml. Protein content gradually
increased till 8th day of fermentation. F. oxysporum could
produce 0.71 fold increase in protein content in same
period. After incubation, protein content declined
(Fig. 1c).
Enzyme Assay
Fermentation time (8 days) had optimum effect of
F. oxysporum on enzyme production as follows: CMCase
activity, 1.92 0.005; filter paper degradation activity,
1.34 0.003; and -glucosidase activity, 1.78 0.005
(Table 1).

Effect of pH, Temperature, C & N Sources on Cellulase Enzyme

Fig. 2Effect on cellulase activity of F.oxysporum during 8th Production
day of fermentation of: a) pH; b) Temperature; At pH 6 and 7, selected fungal strains of F. oxysporum
c) Carbon sources; and d) Nitrogen sources showed heavy growth and higher cellulase activity
458 J SCI IND RES VOL 69 JUNE 2010
Table 1Cellulase enzyme activity of Fusarium oxysporum in liquid state fermentation system supplement
with carboxy methyl cellulose
Enzymes activity, Duration of fermentation, days
U/ml
2 4
CMCase 0.63+0.007 0.98+0.001
FPase 0.51+0.008 0.73+0.005
-Glucosidase 0.45+0.001 0.61+0.006
(Fig. 2a). Maximum growth and enzyme production were
recorded at 50C liquid state fermentation (Fig. 2b). At
50C, confluent growth and better enzyme activity was
noticed15. -Glucosidase activity (0.769 U/ml), observed
in F. oxysporum when lactose supplemented as a sole
carbon source, was higher than FPase (0.654 U/ml) and
CMCase (0.542 U/ml) activity (Fig. 2c). Cellulase
biosynthesis required residual inducer in culture medium
for 2 days, when cellulose and lactose were used as growth
substrate 12. In F. oxysporum, enzyme activity got
influenced when urea supplemented as a nitrogen sole
source (Fig. 2d).
Effect of Cations and EDTA on Cellulase Enzyme Production
CMCase, FPase and -glucosidases activity were
inhibited with increasing concentration of EDTA (Fig.
3a). Cellulase activity was observed with decreasing
concentration of Mg++ ions by F. oxysporum (Fig. 3b).
Maximum cellulase activity was observed without
addition of sodium for selected fungal strains16. Drastic
reduction of enzyme activity was observed with
increasing concentration of sodium ions (Fig. 3c).
Extraction of Crude Cellulase Enzyme
Crude cellulase enzyme of F. oxysporum, recovered
following 60% saturation of culture supernatant with
ammonium sulphate, showed an increase of specific
activity.
Molecular Weight
Molecular weight of partially purified cellulase seemed
to be 24, 29 and 45 kDa in F. oxysporum. Purified
enzymes of F. oxysporum (three distinct bands) have
been observed with the molecular weight of 24, 29 and
45 kDa17.
6 8 10 12
1.62+0.003 1.92+0.005 1.16+0.006 0.98+0.004
0.26+0.007 1.34+0.003 0.87+0.003 0.54+0.007
1.04+0.001 1.78+0.005 0.59+0.004 0.32+0.005
Conclusions
Production and optimization of cellulase has been found
suitable for F. oxysporum and optimization of pH,
temperature, incubation time, carbon and nitrogen sources,
EDTA and cations are limiting factors for maximum
cellulase enzyme production as well as enzyme activity.
Therefore, F. oxysporum is a potential fungal strain for
production of cellulase enzyme.
Acknowledgement
Authors thank management and Department of
Microbiology, V H N S N College, Virudhunagar, to carry
out this study.
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