Al-Anbar J. of Agr. Sci., Vol.: 9 No.

(3 ), 2011 ISSN: 1992-7479

1
Extraction and Purification of Cellulase fromRuminants
Rumen Liquor
Rasha Mohammed Shaker AL-Rawi
College of Dentistry/ Univ. of Baghdad
Abstract
Producing cellulase by rumen cellulytic microorganisms was extracted and
purified by dialysis and DEAE-Sephadex A50 Ion-exchange chromatography 8.53
fold with a 40.02 % yield and a specific activity of 718.4 U/mg. It was optimally
active at pH 6 and 40 C while stable from pH 5 to 7 and 20 to 40 C. A 3, 5 - Dinitro
Salicylic Acid (DNSA) solution was used for measuring cellulase activity at 575 nm.



/


DEAE-Sephadex A50 8.53 40.0 %
718.4 \ .

6 40 5.0 - 7.0
20 - 40 .
3 5 - 575 .
Introduction
Cellulose is the most abundant organic compound in our earth. Its a linear
glucopolymer of anhydrous glucose chain, hydrolysis by acid and enzyme cellulase
(1). Ruminants can degrade cellulose by the living microorganisms in the rumen
which produce cellulase. So, Ruminants can eat and digest agricultural byproducts
like and finally degrade the huge amount of celluloses biomass which is widely
available in nature (2). Cellulose is the major constituent of raw materials like cotton
(over 94%) and wood (over 50%), its the primary structural component of the plant
cell wall and its offers energy and carbon source for microorganisms. Cellulose itself
cant pass through the membrane of microorganisms and has to be hydrolyzed by
cellulase. Cellulase hydrolyses 1,4--D-glucosidic linkages in cellulose and it is able
to decompose natural cellulose (e.g. filter paper) as well as modified celluloses such
as carboxymethyl cellulose or hydroxyethyl cellulose. Nowadays, all cellulolytic
enzymes are extracted from microorganisms like Clostridium acetobutylicum (3),
Bacillus spp. (4), Aspergillus niger (5) and some cellulytic microorganisms which live
in the rumen of ruminants. So the aim of this study is to find another cheap source of
cellulase and produce it using indigenous waste/byproducts by purification and
characterization of the enzyme from sheeps rumen liquor byproduct of
slaughterhouse.
Al-Anbar J. of Agr. Sci., Vol.: 9 No. (3 ), 2011 ISSN: 1992-7479
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Materials and Methods
1- Enzyme Extraction
Cellulase was extracted from rumen liquor of three Awassi male lambs. Their
average weights were approximately 20kg (live weight) and were 5 months old at the
beginning of the experiment. They were fed high roughage diets for three weeks
adaptation period. All rumen digest was mixed well after collection from
slaughtered lambs and filtered through sterilized cotton, then centrifuged (6), upper
layer was taken for measurement of activity and protein concentration and considered
as crude enzyme.
Determination of Cellulase Activity
The cellulase activity was determined by measuring the concentration of
glucose formed by cellulase activity. The assay contained a mixture of 0.9 ml of
substrate (consisted of 1gm cellulose dissolved in 0.1 M-sodium acetate buffer,
pH5.0, to a final concentration 10mg/ml ) with 0.1 ml crude enzyme (7), the reaction
was performed at 37C for 1hr, then was stopped by the addition of 1 ml of 3, 5-
dinitrosalicylic acid reagent (DNS). The mixture was heated for 5 minutes in a boiling
water bath then 10 ml of distilled water was added to each tube. A control tube was
prepared by adding denatured enzyme which was boiling for 5 minute before the
addition of substrate. Absorbance of the solution was measured at 575 nm. Enzyme
activity was obtained from a standard curve for the D-glucose.
[One unit of enzyme activity was defined as the quantity of enzyme liberates 1mole
of reducing sugar in 1hr under the assay conditions.]
Determination of Protein Concentration
Protein concentration was determined colorimetrically due to Lowrys method
(8) using bovine serum albumin as a standard.
2- Purification of Cellulase from Crude Extract
Two steps were used for partial purification of the enzyme as follows:
A- Dialysis
The Crude enzyme was dialyzed against phosphate buffer 0.01M pH 6 for 24hr,
with changing the buffer three times. After that the volume, activity and protein
concentration of the enzyme solution was measured.
B Ion - Exchange Chromatography on DEAE - Sephadex A50
The dialyzed enzyme was applied to a DEAE-Sephadex A50 column (2.2x 8
cm, flow rate of 60 ml/hr.) previously equilibrated with 0.01 M phosphate buffer (pH
6.0) and eluted with increasing concentration (gradient) of NaCl (0.1- 1.0 M). The
active fractions were pooled and subjected to further processes.
3- Characterization of the Purified Enzyme
A- Optimum pH for Activity
1. Cellulase activity was measured by using substrate prepared at different values of pH
ranged from 3 10 as described by (6).
B- Optimum pH for Stability
2. The enzyme incubated with buffer solutions at different pH values ranged from 4 - 10
for 30 min at 37 C as described by (6). Tubes were cooled by ice water bath and the
residual activity of cellulase was measured.
Al-Anbar J. of Agr. Sci., Vol.: 9 No. (3 ), 2011 ISSN: 1992-7479
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C- Optimum Temperature for Activity
3. Optimum temperature for enzyme activity was measured by ran the reaction at
optimum pH for activity at deferent temperatures (20, 30, 40, 50, 60, 70 and 80) C as
described by (6).
D- Optimum Temperature for Stability
4. Cellulase was incubated at different temperatures (20, 30, 40, 50, 60, 70 and 80) C in
optimum pH of stability for 1hr., tubes cooled by ice water bath and the remaining
activity of cellulase as % was measured as described by (6).
Results and Discussion
1- Enzyme Extraction
The cellulase was extracted from slaughtered sheep byproducts rumen
digesta. Slaughter was performed according to local Muslim practice by severing the
jugular vessels, the oesophagus and the trachea without stunning. The activity of the
crude enzyme was 41.66 U/ml with specific activity of 84.161U/mg.
2- Partial Purification of the Enzyme
Table (1) gives the summary of results when crude extract was subjected to partial
purification process. The crude enzyme had total enzyme activity 2083 U, protein
concentration 0.495 mg/ml and specific activity 84.161U/mg protein. The crude
enzyme was subjected to dialysis. Later, the enzyme was loaded on column packed
with DEAE - Sephadex A50 and fractions of 5mL were collected and analyzed for
enzyme activity. Purification fold of cellulase for dialyzed enzyme was 1.51 times
with yield recovery of 129.25 unit, causing increase in enzyme yield more than 100%
because of removal some low molecular weight compounds such as minerals which
inhibit enzyme activity. In ion-exchange chromatography step, the dialyzed crude
enzyme extract was first eluted with the buffer and then with increasing concentration
of NaCl (0.1-1M). Fig. (1) shows one peak for proteins which did not bind with
column in wash step without any peak for enzyme activity, that refer binding of
cellulase to DEAE sephadex because of opposite charged between them. In elution
step, the proteins were eluted as two major peaks and four minor peaks with two
peaks for activity A and B. That means, there were two types of cellulase. Rumen
includes a large variety of microbes such as bacteria and fungi species making the
rumen habitat very complex. These microbes throw their enzymes in the rumen, so the
enzymes may vary with the organism. For this reason, we have two different
cellulases. The high activity peak (A), fractions (34-45) were choice and pooled for
study the characterization of enzyme. The specific activity for chosen fractions was
718.4 U/mg, purification 8.53 fold and a yield of 40.02% (Table1). Each of the
purification step resulted in enhanced specific activity; and there was a decrease in
protein contents from 0.495 mg /ml (crude extract) to 0.025 mg/ml (after ion -
exchange chromatography on DEAE - Sephadex A50). This decrease in protein
content indicates the separation of protein. The results explicated that every step in the
enzyme purification resulted in the removal of undesirable protein. The results of the
study are in line with the findings of Khyami-Horani (9) when he observed that
cellulase purification from alkaline Bacillus spp. strain had a 10.6% yield, while
cellulase from thermophilic Actinomycetes which purified by ammonium sulphate
precipitation and Ion exchange chromatography, was 25.18-fold with a 23% yield
(10). Purification fold for cellulases from the wild-type of Pseudomonas fluorescens
were purified by ammonium sulphate precipitation, ion exchange chromatography on
DEAE sephadex A-50 and gel filtration on sephadex G-100 was about 36.2 fold with
a 11% yield (11), cellulase enzyme extracted from Aspergillus oryzae ITCC-4857.01
Al-Anbar J. of Agr. Sci., Vol.: 9 No. (3 ), 2011 ISSN: 1992-7479
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was purified by ion-exchange chromatography using DEAE-cellulose followed by gel
filtration, achieved 53 fold from the crude extract with a yield of 49 % (12).
Table 1. Summary of partial purification steps of cellulase from crude extract
enzyme of rumen liquor by dialyzed against phosphate buffer and ion-exchange
chromatography
Fig 1. Purification of cellulase by Ion-exchange chromatography using column of
DEAE Sephadex A 50(2.28cm) equilibrated with 0.01M phosphate buffer pH
6 and eluted using the same buffer with a linear gradient of NaCl (0.1 to1) M in
a flow rate 60 ml /hr. as 5ml for each fraction.
3- Characterization of the Partial Purified Enzyme
A- Optimum pH for Activity
The purified cellulase from rumen liquor can be considered as unsusceptible to
acidic and alkaline conditions, because it showed different activity in a broad range of
pH between 3.5 and 9.5 Figure (2). Increase or decrease of activity values over or
below the optimal value (pH 6.0) due to the effect of pH on the active side of enzyme,
substrate and enzyme - substrate complex (6). That mean enzyme active at natural
rumen environment (almost pH 7.0).
Purification
step
Volu
me
(ml)
Activity
(U/ml)
Total
activity (U)
Protein
con.
(mg/ml)
Specific
activity
(U/mg)
Purifi
cation
fold
Yield
%
Crude extract 50 41.66 2083 0.495 84.161 1 100
Dialysis 54.5 49.40 2692.3 0.388 127.31 1.51 129.25
Ion-exchange
by DEAE
sephadex A-50
(peak A)
60 17.96 1077.6 0.025 718.4 8.53 40.02
Al-Anbar J. of Agr. Sci., Vol.: 9 No. (3 ), 2011 ISSN: 1992-7479
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Fig 2. Effect of pH on the activity of purified cellulase using substrate prepared
at different pH from 3 10
The results are in agreement with those obtained from Aspergillus niger and
Sinorhizobium fredii which showed an optimal activity at pH 6-7 (1, 5, 13). On the
other hand, (14) show that cellulase in rumen liquor of cattle were more active on the
acid side (pH 4).
B- Optimum pH for Stability
The effect of various pH values on purified cellulase stability is depicted in
Figure (3). Graphic elaborates the remaining activity at different pH values, the
highest stability was found at pH 5-7. Important of pH values makes enzyme
potentially effective for use in industry. Generally, in comparison with other
cellulases were previously studied, the purified cellulase from Bacillus sp. was fairly
stable at a range of pH 6 to 10.5, that mean it was highly stable in alkaline conditions
(15). On the other hand, the cellulase from Alcaligenes faecalis was stable at pH
ranged 6.5 to 7.8 when the enzyme was incubated in different pH values(16).
However, cellulases from Thermotoga neapolitan showed stability at pH 6 - 6.6 (17).
Al-Ani (6) found that optimum pH for cellulase stability isolated from Aspergillus
spp. was between 5.0 and 7.0. While, Bakare (11)found the optimum pH for stability
of purified cellulases from Pseudomonas fluorescens was 6.5 7.0. It could be
observed that enzyme stability range near the neutral condition is larger than that in
acidic and alkaline conditions.
Fig3. Effect of different pH values on the stability of purified cellulase
for 30 min at 37 C
Al-Anbar J. of Agr. Sci., Vol.: 9 No. (3 ), 2011 ISSN: 1992-7479
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C- Optimum Temperature for Activity
The effect of various temperatures on the activity of purified cellulase was
presented in Figure 4. It was found that enzyme exhibited maximum activity at 40C.
When the temperature increased above 40C, enzyme activity was affected negatively
and gradually reduced. In the same way, when the temperature was below 40C, a
gradual decline in the cellulase activity was observed. Enzymes are proteins in nature
and sensitive to variations in temperature and particularly elevated temperature cause
denaturation for protein and thus resulting in decreased enzyme activity. General,
temperature significantly affected at enzyme activity when increase above 50C and
rapidly decreased the enzyme activity. In nutshell, the enzyme was most efficient at
40C as compared with other tested temperatures. The results are in close agreement
with the findings of Chen et al. (13) whom reported 35C as an optimum temperate
during the characterization of cellulase produced from Sinorhizobium fredii. The
results are also supported by Budiansyah et al. (14), they found 39C as a best
temperature at which the enzyme was most active. So, our results would be important
for new source of cellulase using in industrial applications.
Fig 4. Effects of temperature on the activity of purified cellulase by measuring at
optimum pH of activity (pH6) at deferent temperatures (20-80) C.
D- Optimum Temperature for Stability
The stability of enzymes has considerable importance from the application
viewpoint; as best effectiveness can be attained only when enzyme finds its favourite
conditions. The relationship of cellulase activity with the change in temperature is
shown in Figure 5. It is obvious from the graphic there was an inverse correlation
between temperature increase and the enzyme stability. The results revealed that the
maximum enzyme stability was found at 20-40 C, then remaining activity decreased
to 70% at 50C. At higher temperatures, above 50C stability of enzyme decreased
rapidly as the denaturation of protein. Generally, temperature has significantly
affected on the enzyme stability. Previously, Mawadzaa et al. (18) and Li et al.(19)
also reported a significant loss in cellulase activity from 50 to 80C that confirms
present results.
Al-Anbar J. of Agr. Sci., Vol.: 9 No. (3 ), 2011 ISSN: 1992-7479
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Fig. 5. Effect of temperature on the stability of purified cellulase. The enzyme
was incubated at different temperatures (20 to 80) C in optimum pH of stability
(pH 6) for 1hr.
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