Isolation, purification and characterization of carboxymethyl cellulase (CMCase) from endophytic Fus

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: refazahmad@gmail.com

2Department of Botany, University of Pune, Pune, Maharashtra, India

Received 28 June 2013; revised 15 August 2013; accepted 2 September 2013

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

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

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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