I.J.S.N., VOL.

6 (3) 2015: 474-479 ISSN 2229 – 6441

ISOLATION AND PURIFICATION OF CELLULASE

*Megha, S.V., Maragathavalli, S., Brindha, S., Karthikeyan, V., Annadurai, B. & #Gangwar, S.K.
Research and Development Centre, Bharathiyar University, Coimbatore-641 041, TN
#
College of Medical and Health Sciences, School of Veterinary Medicine, Wollega University, Nekemte, Ethiopia.
Corresponding author email: [email protected]

ABSTRACT
The production of cellulase enzymes on carboxymethyl cellulase (CMCase, endoglucanase activity) by an isolated fungus
Aspergillus terreus evaluated. The intracellular cellulase from A. terreus was isolated purified and some properties were
characterized. Employing a purification scheme consisting to the following steps isolated it: Crude extract after
centrifugation, Ammonium sulphate precipitation & DEAE – cellulase chromatography and ultra gel column
chromatography. By adopting these step a fold purification of 270 with 22.11 % overall yield of were obtained. PAGE,
SDS- PAGE, immunodiffusion and immunoelectrophoresis and isoelectric focusing confirmed the homogeneity of the
enzyme. The extracted fractions analyzed based on soluble protein amounts and enzymes activities. The cellulase enzymes
from isolated A. terreus showed 80% (according to CMCase). The best enzyme activities were in pH 4-7 and the
temperatures between 40°C to 50°C. By the purification process, 40% of protein, 77% of CMCase activities extracted from
concentrated enzyme solution from A. terreus.

KEY WORDS: Cellulase, Aspergillus terreus, DEAE cellulase, Ultrogel, Immunodiffussion.

INTRODUCTION possible purity is required and the strategy for enzyme

Cellulases (1,4)-(1,3:1,4)-/ J-D-glucan 4-glucanohydrolase purification good source for the enzyme production good
(EC 3.2.1.4) or the enzyme essential for the Carbon cycle method of homogenization and a perfect method of
in nature. These cellulases are commercially used in the separation is required. Hence in this chapter cellulases is
food industry for clarification of fruits and juices and purified with LKB column (Sweden) with Carboxy CM-
pharmaceuticals industry. It is almost necessary to purity trisacryl, ultrogel column with maximum yield and the
with 100% purity and yield. In the last decayed much chart is presented. The purified enzyme is tested for its
attention is given on cellulase degradation, because of its purity with the Ouchterloney immunodiffusion technique,
purpose as a renewable energy alternative for fossil fuel. It Rocket electrophoresis, Immunoelectrophoretic technique,
is the abundant biomass on earth. Cellulase is composed of PAGE, SDS-PAGE and isoelectric focusing.
Beta-1-4 linked D-glucose units which could be
hydrolysed by cellulases resulting in fermentable sugar for MATERIALS & METHODS
bioethanol bioconversion. The widely accepted Purification of Cellulase
mechanism of enzymatic cellulase hydrolysis involved the Crude enzyme preparation
synergistic action of 3 types of cellulases including endo- In the present study, the culture medium which was
beta 1,4-glucanase (EC 3.2.1.4), Exoglucanase (EC standardized earlier (chapter I) was used for culturing the
3.2.1.91) and beta Glucosidase (EC 3.2.1.21), Which step mold. To obtain maximum quantity of enzyme one
by step nicked the intermolecular beta 1,4 glycosidic 1000ml of medium in 5 litre Haffkin’s Flask. This was
bonds cleaved cellulase chain in to release of cellobiose autoclaved in bars lead (Boston) autoclave at 15 lb
units and cut cellobiose and oligosaccharides to produce pressure for 20 minutes. After sterilization the flasks were
glucose. Many fungal and microbe sources where known cooled to 32±1°C. Then a slant of A. terraeus was
for decomposing cellulase, the available application of inoculated into medium in a sterile chamber. After 16 days
them was far from satisfactory because the properties and of incubation at 32± 1°C the contents were taken out by
production of their known cellulases could not conform to adding 500 ml of 0.25M NaCl. This was blended in
the specified requirements such as high catalytic efficiency waring blender for 5 second at 4°C the blended juice was
of insoluble cellulosic substrate, superior stability at filtered through 2 layers of cheese cloth. The filtrate was
elementary temperature and at a certain pH, and strong centrifuged at 20,000 rpm for minutes at 4°C. The clear
tolerance to end product inhibition (Zhang et al., 2006). supernatant was dialyzed against 2 liter. 0.01 M phosphate
Since, Cellulases are commercially and medically used buffer at pH 7.0 for 48 hours with two changes at 4±1°C.
enzyme high purity with maximum yield is required. The dialysed crude enzyme extract was used for
Various laboratories attempted to purify cellulases. No ammonium sulphate precipitation.
single method was satisfactory for any purification Ammonium sulphate precipitaion
biochemist should concentrate and maximum possible To 1 liter of dialysed supernatant solution ammonium
yield, the maximum catalytic activity and the maximum sulphate (AR) was added slowly with a constant stirring in

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 Isolation and purification of cellulase

a magnetic stirrer for 4 to 6 hours at 4°C to give 0-40 %, was loaded on the ultrogel column. 3 ml fractions per
40-80%, 80-95% saturation. After the desired percentage tube at the flow rate of 9ml/ hour were collected. The
of saturation the contents of each step was centrifuged at individual fractions were analysed for Endoglucanase
20, 000 rpm for 20 minutes at 4°C. The supernatant activity. The Endoglucanase active fractions were pooled
solution was taken for the next (NH4)2SO4 precipitation. and lyophilized. The lyophilized and dialyzed
The precipitate obtained from each step after Endoglucanase from DEAE column was loaded on to the
centrifugation was dissolved in 0.01 M phosphate buffer at ultrogel column (1.6 X 88.0 cm). 3 ml fractions per tube
pH 7.0 containing 0.1 M NaCl. The dark brown color were collected at the flow rate of 9 ml per hour using the
solution thus obtained was dialysed at 4ºC for 24 hours same buffer. The individual fraction was analysed 8p8 for
against the same buffer with at least three changes of 3 cellulase activity. The Cellulase active fractions were
liter buffer. The activity of endoglucanase and protein pooled and lyophilized.
content were estimated. Estimation of protein
Chromatographic procedures Protein content was determined by adopting the procedure
The sample containing maximum cellulase activity was of Lowry et al. (1951) described under general Materials
selected for further purification by chromatography. The and methods with crystalline bovine serum albumin (BSA)
entire chromatography operations were carried out at 4°C. as standard. The fractions collected after chromatographic
Addition of sample to the column, elution of proteins and column were directly read at A80 in a shimadzu
maintenance of column flow rate were performed using spectrometer for monitoring the protein.
peristaltic pump (LKB model 2132 micro perpex Test for purity of the enzyme
pump).The column effluents were continuously monitored The homogeneity of the purified Cellulase was determined
at 280 nm using a uv monitor (LKB uv cord S II model by polyacrylamide gel electrophoresis, SDS-
2338). The recording of absorbance was simultaneously polyacrylamide gel electrophoresis, isoelectric focusing,
done by LKB model 2210 recorder. Constant volume immuno diffusion and immuno electrophoretic techniques.
fractions were collected by fraction collector (LKB Polyacrylamide gel electrophoresis (PAGE)
Redirac moderl 2112). Measurements of absorbance of Polyacrylamide gel electrophoresis was carried out
collected fractions (A280) were carried out in a shimadzu according to the method of Davis (1964). The detailed
spectrophometer uv-260 japan with 1 cm cuvette at 280 procedure has been given in general material and methods.
nm. SDS- Polyacrylamide gel electrophoresis (SDS- PAGE)
Cellulase activity was quantified by measuring the Sodium dodecyl sulphate polyacrylamide gel
microgram of reducing sugar released per ml of enzyme in electrphoresis was carried out to determine the
one hour from carboxy methyl cellulase using reducing homogeneity of purified cellulase according to procedure
sugar method of Miller by Di Nitro Salicylic acid. One of Laemmli (1970). The detailed procedure is explained
unit of Cellulase activity is defined as micro gram of in general materials and methods.
reducing sugar released from carboxy methyl cellulase per Isoelectric focusing
mg of enzyme in one hour at 32±1°C under assay The isoelectric focusing was carried out in a semi
conditions. preparative manner using polyacrylmide gel rods (11
DEAE column chromatography X165 mm) by adopting the method of Wrigley (1971) at
The DEAE–cellulase column chromatography was carried 4°C. The procedure is explained under general materials
out according to the following procedure. A DEAE and methods.
cellulase (Sigma chemicals fine grade) column of 3 X 25 Immunological studies
cm was prepared after pretreatment with 0.5 N NaOH and To study the homogeneity of the enzyme, double immuno
0.5 N HCl. The column was equilibrated with 0.01M diffusion technique was carried out according to the
phosphate buffer pH 7.0 50 ml of the dialysed 80- 95% method of ouchterlony and Nilsson (1973) and immuno
(NH4)2 SO4 precipitation solution which shows high electrophoresis was performed according to the technique
cellulase activity was loaded in the column and washed of Graber and Burtin (1964).
with the same buffer till the washing showed the Determination of Molecular weight of cellulase
absorbance almost zero at 280nm. The elution of the The molecular weight of the purified cellulase I and
enzyme was carried with 0.01 M phosphate buffer cellulase II was determined by gel filtration on sephadex
containing stepwise gradient of 0.05 M, 0.1 M and 0.2 M G-100 by the procedure which was adopted by Andrews
NaCl. The flow rate was 30 ml per hour. 6 ml of fractions (1964). The column was standardized by using low
were collected at the interval of 12 minutes. molecular weight kit containing ribonuclease (MW 43,
All the individual fractions obtained from DEAE column 000), Albumin (BSA) (Mw 67,000) and Blue dextran
were analysed for Cellulase activity. Both fractions of 2000. The Kav values of standard protein were plotted
peak I and peak II were found of possess the cellulase against corresponding molecular weight in semi
activity. Fractions of Endoglucanase and Endoglucanase II logarithmic graph. The straight line obtained was used for
were individually pooled lyophilized dissolved in buffer the determination of molecular weight of purified cellulase
pH 7.0 containing 0.05M NaCl. I and cellulase II.
Ultrogel Column Ultraviolet absorption spectrum of cellulase
Further purification of Cellulase I fractions was carried out The purified lyophilized sample of cellulase I and
by gel filtration on ultrogel ACA 44 (LKB) column. The cellulase II was dissolved in distilled water (2 mg/ 1 ml) in
column (1.6 X 88cm) was equilibrated with 0.01M the quartz cell of Shimadzu spectrophotometer model UV
phosphate buffer pH 7.0 containing 0.05 M NaCl.5 ml of 260 and the absorbance was scanned from 190 nm to 300
the lyophilized and dialysed fraction of Endoglucanase I

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nm. A print out of the scanned diagram on the screen of RESULTS & DISCUSSION
shimadzu spectrophometer was obtained. Table 1, Shows the precipitation of cellulase activity at
different percentages of ammonium sulphate
concentration. The pellet obtained shows maximum
cellulase activity.

TABLE 1. Ammonium sulphate precipitation of crude enzyme preparation of Cellulase from natural onion medium
Sl No Percentage of Volume cellulase activity Protein Specific activity
(NH4)2 SO4 (%) (ml) units up of reducing content in units/µg
sugar rel/ml/1 hr g/ml
1 Crude enzyme 900 56.4 71.2 0.79
2 0-20 130 1.32 26.4 0.05
3 20-40 100 1.85 25.8 0.07
4 40-60 80 2.04 29.1 0.07
5 60-80 60 976.4 76.9 12.69
6 80-100 50 345.6 73.5 4.7
Values given are the mean value (X ) of 4 datas, d.f. = degrees of freedom = n-1, Significance ++ = p < 0.001
+ = p < 0.05, NS = Not significant

Fig 2 shows the elution profile of DEAE Cellulase column Elution profile of the purification of Endoglucanase from
chromatography of cellulase. The precipitate obtained crude enzyme fraction of 80 to 95% ammonium sulphate
between 80- 95% (NH4)2SO4 fraction was dialyzed and fraction by ion exchange chromatography on DEAE
applied on DEAE cellulase column. The stepwise gradient cellulase column. Fractions 6 ml per tube were collected at
elution shows that first fraction of EG activity (designated the flow rate of 30 ml per hour in DEAE cellulase column
as cellulase eluted at the concentration of 0.1M NaCl (3 X 25 cm). Eluted with 0.2 M sodium acetate buffer at
eluted at the concentration of 0.2 M NaCl. pH 7.0 containing 0.05 M to 0.2 Na Cl gradient.

------ = absorbance at 280 nm, ------ = cellulase activity

FIGURE 2: Elution profile of DEAE-cellulase column chromatography for the purification of Cellulase

Elution profile of EG I from DEAE cellulase column after same buffer and loaded on ultrogel column (Fig 3) shows
pooling and dialysis against 0.01 M phosphate buffer (pH two protein fraction but cellulase activity is seen only in
7.0) was lyophilized. A small amount was dissolved in the 1st fraction, ie from 36th to 52nd fraction.

FIGURE 3: Elution profile of ultrogel column for cellulase purification

Elution profile of the purification of cellulase from DEAE concentrated and rechromatographed on ultrogel column
cellulase on ultrogel column. Fractions corresponding to (1.6 X 88 cm). Fractions of 3 ml / tube were collected at
peak I of DEAE cellulase column were pooled together,

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 Isolation and purification of cellulase

the flow rate of 9 ml / hour eluted with sodium acetate ulrogel column. The eleution profile (Fig. 4) shows only
buffer. the one peak, which contains both protein and cellulase
-------- = protein absorbance at 280 nm activity. The cellulase activity is seen. A summary of
-------- = cellulase activity purification presented in Table 5 reveals that cellulase is
The second fraction of DEAE column after conducting the purified to 245 fold with 26% yield. cellulase is purified
same procedure as described above was loaded to the to 270 fold and with 22% yield.

TABLE 5. Purification of Cellulase

Steps Volume Total Total cellulase Specific activity Purification Yield
(ml) Protein Activity units Units/g protein fold (%)
(g) (g of gal. acid rel)
Culture filtrate
900 64080 50760 0.79 1 100
(Supernatant)
80-95% (NH4)2
50 3845 48820 12.7 16.0 96.18
SO4 Precipitation
DEAE-column
138 201 21618 107.55 135.5 42.58
CXI-fraction
CX II - fraction 40 162 2085 128.54 161.34 41.01
Ultrogel column
34 68 13285 195.36 245.18 26.16
CX 1 –fraction
CX II – fraction 16 52 11226 215.88 270.93 22.11
From each step of purification the enzyme samples were tested for homogeneity using PAGE and SDS-PAGE. After final
purification CX I show only one band in PAGE (Fig 6) and SDS – PAGE (Fig 7). Similarly CX II also shows single band
in PAGE (Fig 8) and SDS – PAGE (Fig 9).

Determination of isoelectric point in a semi preparative cellulase I and cellulase II (Fig 12 and 14).The highly
manner of CX I shown in Fig 10. It indicates a single purified enzyme gave a single immunoprecipitin line in
peak of cellulase activity between 11th and 12th fractions at reflected light when reacted with its rabbit antiserum
isoelectric pH 06.7. The semi preparative isoelectric showing the presence of only one component. Graber and
focusing of cellulase II is shown in Fig 11. It shows a Burtin qualitative analysis by immuno electrophoresis also
single of CX activity between fractions 16 and 17 and its confirmed the homogeneity of the enzyme as depicted in
isoelectric pH is 7.1.The outchterlony’s double Fig 13 and 15.
immunodiffusion pattern indicates the homogeneity of the Determination of isoelectric point of cellulase

Cellulase activity is expressed in units as µg of polyacrylamidegel rods (11 X 165 mm) based on the
galacturonic acid released /ml/30 minutes. Iso electric method of Wrigley (1971) at 4C.
focusing was carried out in semipreparative manner using

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Determination of isoelectric point of cellulase Ouchterony’s double diffusion patterns of purified

Isoelectric focussing was carried out in a semi preparative cellulase
manner using polyacrylamide gel rods (11 X 165 mm)
based on the method of Wrigley (1971)

The central wall contained antiserum of highly purified enzymes. The peripheral walls 1,2,3,4 and 5 contained 6 micro liter
antigens.
Graber and burtin immunoelectophoresis pattern of purified cellulase

The through contained antiserum of highly purified homogeneous. Isoelectric focussing pattern (10 and 11)
cellulase I and the wells contained 3 micro liter of the also confirmed the previous findings of the homogeneity
antigen. The central wall contained antiserum of highly of the enzyme. The presence of single protein band
purified enzymes. The peripheral walls 1, 2, 3,4 and 5 suggests that cellulase I and cellulase II consists of a
contained 6 micro liter of antigen. single polypeptide chain (Cervone et al., 1977). The
immuno diffusion and immno electrophoresis results
DISCUSSION showing single precipitation line confirm the homogeneity
In the previous study, we reported the isolation of A. of the purified endoglucanase.
terreus and the effects of the cellulase enzymes of this
fungus on the biodegradation of lignocellulosic wastes REFERENCES
(Emitiazi, G. 2001). In the present study an optimization Cervone, F. Scala, A. Foresti, M. Cacacea, M.G. &
of cellulase activity of this fungus due to the best pH and Noviello, C. (1977) Endopolygalacturonase from
temperature, showed an elevated cellulase production Rhizoctonia frgariae. Purification and characterization of
(80% for CMCase) by A. terreus. The fungi showed the two isoenzymes. Biochim Biphys acta 482, 379-385.
best enzyme production in pH 4-7 and temperatures 40-
50°C. Coral et al., 2002 extracted the carboxy methyl Chinedu, N.S., Nwinyi, O.C. & Okochi, V.I. (2008)
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specific activity. Similar affinity column was used for the terreus,” Biodegradation, vol 12, pp. 259-263, 2001.
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 Isolation and purification of cellulase

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