Journal of Cell and Molecular Biology 9(2): 51-56, 2011
Haliç University, Printed in Turkey.
http://jcmb.halic.edu.tr
Research Article 51
Optimization of cellulase enzyme production from corn cobs using
Alternaria alternata by solid state fermentation
Amir IJAZ1*, Zahid ANWAR2, Yusuf ZAFAR3, Iqbal HUSSAIN1, Aish MUHAMMAD1,
Muhammad IRSHAD2 and Sajid MEHMOOD2
1
National Agriculture Research Center (NARC), Islamabad, Pakistan
Nawaz Sharif Medical College (NSMC), University of Gujrat, Pakistan
3
Biological Division PAEC Islamabad, Pakistan
(*
author for correspondence; [email protected])
2
Received: 3 August 2011; Accepted: 23 December 2011
Abstract
Cellulase is an important industrial enzyme which can be obtained from cheap agrowastes. Pakistan is an
agriculture country, producing tons of waste in the form of wheat straw, rice bran, sugarcane bagasee, corn
cobs, corn stover etc. The aim of the present study was to produce cellulase by using abundant agrowastes
like corn cobs. The conditions were optimized by using corn cobs and culturing Alternaria alternata with
solid state fermentation. Different incubation times (1-7days), temperatures (250C, 300C, 350C and 400C) and
pHs (3.0-9.0) were experimented for the production of cellulase. The optimum culture conditions were 96 hrs
of incubation at 350C and pH 6.0, giving enzyme activities of 15.06 µg/ml, 31.2406 µg/ml, 26.4106 µg/ml,
respectively.
Keywords: Cellulase, corn cobs, agrowaste, solid state fermentation, Alternaria alternata.
Katı hal fermentasyonu ile mısır koçanlarından Alternaria alternata kullanılarak selülaz enzimi
üretiminin optimizasyonu
Özet
Selülaz ucuz zirai atıktan elde edilen önemli bir endüstriyel enzimdir. Pakistan buğday samanı, pirinç kepeği,
şeker kamışı posası, mısır koçanı vb. şekillerde tonlarca atık üreten bir tarım ülkesidir. Bu çalışmanın amacı
mısır koçanı gibi bol tarım atıklarını kullanarak selülaz üretmektir. Bu nedenle koşullar mısır koçanı
kullanılarak ve Alternaria alternata katı hal fermentasyonu ile kültür edilerek optimize edilmiştir. Selülaz
üretimi çin farklı inkübasyon süreleri (1-7 gün), sıcaklıklar (250C, 300C, 350C ve 400C) ve pH’lar (3.0-9.0)
denenmiştir. Optimum kültür şartları 350C ve pH 6.0’da 96 saat inkübasyon olarak belirlenmiş ve bu
şartlarda enzim aktiviteleri sırasıyla 31.2406 µg/ml, 26.4106 µg/ml ve 15.06 µg/ml olarak tespit edilmiştir.
Anahtar Sözcükler: Selülaz, mısır koçanı, zirai atık, katı hal fermentasyonu, Alternaria alternata
Introduction
Agricultural waste is one of the major
environmental pollutants, their biotechnological
conversion is not only a remedy for environmental
problems but also the source of suitable microbial
byproducts like food, fuel and chemicals (Milala et
al., 2005). Agro-industrial wastes, e.g. wheat and
rice bran, sugar cane bagasse, corn cobs, citrus and
mango peel, are one of important wastes of food
industries
of
Pakistan.
Their
unchecked
accumulation on land serves as a source of
environmental pollution (Government of Pakistan,
2001). The most abundant renewable organic
compound in the biosphere is cellulose, which
accounts for 40-50% of plant composition and its
production is expected to be 1010 tones from cell
wall of plants per year (Thu et al., 2008). Pakistan
contributes about 50 to 60 agro-waste million tons
per year. An agricultural waste is a cheap source of
cellulose for the production of different useful
products all over the world (Ali and Saad, 2008).
Cellulase production from agrowastes is
52 Amir IJAZ et al.
economical as compared to production from pure
cellulose (Chahal, 1985). Three major structural
polymers combined to make up lignocellulose are
called cellulose (a homopolymer of ß-D-glucosyl
units), hemicellulose (a cluster of heteropolymers
which contain xylans, arabinans, mannans,
galactans), and lignin (an intricate polyphenolic
polymer) (Rajoka, 2005).
Cellulases are a group of enzymes that break
down cellulose into glucose monomers (Yi et al.,
1999). Bacterial and fungal cellulases are
traditionally separated into three classes:
Endoglucanases (EGs) (EC 3.2.1.4), exoglucanases
(EC 3.2.1.91), and ß-glucosidases (EC 3.2.1.21)
(Kim, 2008) based on the ability to degrade
carboxymethylated cellulose (CMC), whereas EGs
being the most efficient (Henriksson et al., 1999).
The endo-ß-glucanase is responsible for the scission
of the inner bonds in the cellulose chains yielding
glucose and cell-oligosaccharides. Exo-ß-glucanase
(cellobiohydrolases) cleaves non-reducing end of
cellulose with cellobiose as the main structure
(Be´guin, 1990; Tomme et al., 1995). The ßglucosidase (cellobiase) hydrolyses cellobiose to
glucose (Eveleigh, 1987).
Cellulase enzyme, having its importance due to
major role in industrial applications (Bhat, 2000). It
is used for bioremediation, waste water treatment
and also for single cell protein (Alam, 2005). It has
also importance in food sciences like food
processing in coffee, drying of beans by for
efficient purification of juices when used mixed
with pectinases, paper and pulp industry and as a
supplement in animal feed industry. This enzyme
helpful for plant protoplast isolation, plant viruses
investigations, metabolic and genetic modification
studies (Bhat, 2000; Chandara et al., 2005; Shah,
2007). This enzyme have also pharmaceutical
importance, treatment of phytobezons (a type of
bezoar cellulose existing in humans stomach) and a
key role in textile industry especially as its
detergent applications to recover properties of
cellulose related textiles and biofuels production
from cellulosic biomass(Ali and Saad, 2008).
Cellulases producing fungi include genra Aspergilli
(Ali and Saad, 2008) Aspergillus niger and
Aspergillus terreus, Rhizopus stolonifer (Pothiraj,
2006)
Trichoderma,
Penicillium,
Botrytis
Neurospora etc. (Pandey et al., 1999). Fungi are
capable of decomposing cellulose, hemicellulose
and lignin in plants by secreting multifarious set of
hydrolytic and oxidative enzymes (Abd Elzaher and
Fadel, 2010).
Solid State Fermentation (SSF) is a way of
fermenting substrate in the presence of excessive
moisture in growth medium in spite of large
amount of water being provided. SSF is an
environmental friendly (less waste water
production), low energy required and economical
technology in synthesizing cellulase enzyme in
response to submerged fermentation (Pandey,
2003). SSF from last decade has made its
importance in the production of value added
products i.e., secondary metabolites, alkaloids,
enzymes,
organic
acids,
bio-pesticides
(mycopesticides
and
bio-herbicides),
biosurfactants, biofuels, aroma compounds, biopulping, degradation of toxic compounds,
biotransformation, nutritional improvement of
crops, biopharmaceuticals and bioconversion of
agricultural waste (Pandey et al., 2000).
Pakistan has to spend about 106, 986.45 million
rupees per month to import organic chemicals
(Monthly Review of Foreign Trade, 2010). A huge
quantity agricultural waste is produced from agroindustries of Pakistan can be advantageous in
making useful by-products. A large amount of
money of our country is consumed in importing
various types of enzymes including cellulases for
local industries and research activities. The aim of
this study was to obtain a high yield of cheap
cellulase by using a local novel strain Alternaria
alternata through solid state fermentation and also
exploiting local agro-waste like corn cobs. This
study will help in proper disposal of agro-waste
resulting in resolution of the environmental
problems.
Materials and methods
Substrate selection
Agricultural waste/samples of corn cobs were
collected from local industry of Gujranwala district,
Pakistan, the substrate was dried in oven at 700C
and grinded mechanically with electric grinder to
make it in powdered form and sieve to 40 meshes.
Microorganism selection
Fungal strain of Alternaria alternata was selected
for production of cellulase enzyme. The strain was
obtained from fungal bank’s stock cultures of
Institute of Plant Pathology and Mycology, Punjab
University, Lahore.
Production of cellulase from corn cobs 53
Maintenance of Alternaria alternata
Strains of Alternaria alternata maintained on PDA
medium slants under sterilized conditions of LFH
and incubated at 300C for 72 hrs (Asgher et al.,
1999). T he p H o f me d i u m wa s ad j us ted to
4 . 8 wi t h 1 M H Cl /1 M N a O H a nd wa s
st eri liz ed a t 121 o C fo r 1 5 mi n ute s i n
au to c la ve. The spores of cultured Alternaria
alternata on PDA medium were isolated aseptically
using sterilized water with 0.1% Tween 80
followed by inoculation in PDA broth. Then
inoculated flasks were placed in shaker incubator at
370C and 150 rpm for 72 hrs and p H wa s
ad j u sted a t 5 .6 a nd wa s a uto cl a ved f o r 1 5
mi n u te s a t 1 5 lb / i n 2 i n a u to c la ve . After
specific incubation period inoculum of Alternaria
alternata was prepared. (Smith et al., 1996).
Composition of culture medium
Solid state fermentation was carried out in
Erlenmeyer duplicate flasks containing 5g of corn
cobs, moistened with 10 ml distilled water,
autoclaved at 1210C followed by inoculation with 3
ml sporulation medium of Alternaria alternata.
Substrate (5g), moisture level (10 ml), and fungal
inoculum (3ml) were kept constant for all
optimizing steps.
Selection of optimum conditions for cellulase
production under SSF
The strategy was adopted for optimizing the
engaged parameters enhancing cellulase yield was
to optimize one specific parameter and process it at
the optimized level in the next experiment
(Sandhya and Lonsane, 1994).
Cellulose determination
Optimization of incubation period
Raw cellulose contents of corn cobs were
determined by using Weendize method as described
previously (Henneberg, 1975) and were shown as a
schematic diagram Figure 1.
Duplicate Erlenmeyer flasks using corncobs
cultured with A. alternata were incubated at 300C
temperature for a period of 1-7 days to select the
optimum incubation period of A. alternata for the
production of cellulases. The growth was assessed
every 24 hrs and the best incubation period at
which employed strain would give maximum
cellulase activity was selected.
1g of sample in 200mL flask
Add 1.25 of 200mL of sulphuric acid
(Remove all glucid)
Boil for 30 minutes
Filter and wash several time with hot water
Temperature optimization
Duplicate flasks inoculated with A. alternata were
kept at 250C, 300C, 350C and 400C, respectively to
determine the optimum temperature at which said
strain would express high cellulase activity was to
select.
pH optimization
Add 200 ml sodium hydroxide 1,25%
(Remove proteins by hydrolysis and fats by saponification)
Boil for 30 minutes
Filter and wash several time with hot water
the assay is treated with ethyl alcohol
(remove dyes, tannins, fats marks, the raw ash complex).
Residue is dried at 105°C, cooled and weighed residue
Figure 1. Cellulose determination procedure
pH was optimized from 3.0-9.0 (50 mM) to select
optimum pH at which A. alternata would exhibit
hyper cellulase activity was selected.
Culture harvesting/ Isolation of crude cellulase
enzyme
The product of fermented cultures (cellulases) was
collected by simple contact method (Krishna and
Chandrasekaran, 1996) followed by addition of 100
ml distilled water due to neutral pH (except in case
of pH optimization where used 100 ml pH solutions
ranging 3.0-9.0 for each duplicate flask) shaking at
180 rpm in orbital shaker incubator for 45 min.
The shaked flasks were filtered and centrifuged
at 4000 rpm for 10 minutes to eliminate impurities
and insoluble materials. The supernatants were
54 Amir IJAZ et al.
carefully collected with the help of auto-pipette and
filtered through Millipore filter to make it spore
free.
Bioassay of cellulase (FPase)
Bioassay of cellulase (FPase) was performed by
taking 1ml of crude enzyme and 1ml of sodium
citrate buffer (pH 4.8) which were added in each
test tube containing 50 mg filter paper No. 1,
incubated at 500C for 30 min. Then, 500 µl enzyme
sample was boiled with 2.5 ml DNS 3, 5Dinitrosalicylic acid for 15 minutes, following
cooling, absorbance of sample was taken at 540 nm
(Mandel et al., 1976). The absorbance was
translated by plotting against regression equation to
get µg/ml/min of glucose by inserting into the
following formula to calculate units of enzyme
activity.
Enzyme activity = Absorbance of enzyme solution x Regression equation
(µg/ml/min)
Time of incubation
One unit of enzyme activity was defined as the
amount of glucose (μg) released per ml of enzyme
solution per minute.
Results
A. alternata under SSF are described in Figure 3.
The A. alternata accounted maximum cellulase
activity 31.24 ± 0.16 µg/ml at 350C, so, its
optimum temperature was 350C.
Figure 3. Optimum temperature for cellulase
cellulase production by Alternaria alternata
pH is also one of the main factors having direct
impact on cellulase production. Different pH (3.09.0) for cellulase production using corn cobs by A.
alternata is represented in Figure 4, the cellulase
activity was highest at an acidic pH 6.0 (26.41 ±
0.08 ug/ml) & lowest at pH 9.0 (11.84 ±
0.07ug/ml), indicating its optimum pH 6.0.
The cellulose contents in corn cobs were
determined to be 24.54 %. The incubation period is
directly associated with the production of enzyme
and other physiological functions up to a certain
extent. Incubation period for cellulase production
by Alternaria alternata under SSF is represented in
Figure 2, corn cobs and sugarcane bagasse showed
optimum day 3rd (72 hrs) with maximum cellulase
activity 15.06 ± 0.17ug/ml.
Figure 4. Optimum pH for cellulase production on
corn cobs by Alternaria alternata
Discussion
Figure 2. Incubation period
production by Alternaria alternata
for
cellulose
Temperature is also an important factor to affect
cellulase yield. Different temperatures (25-400C)
for the production of cellulase using corn cobs by
The cellulase activity trend concerning corn cobs
was gradually ascended from 1st day to 3rd day and
descended from 4th day to 7th day. The falling of
cellulase activity might be due to loss of moisture
and inactivation of enzyme resulting from
fluctuation in pH during fermentation (Melo et al.,
2007). Using banana waste culturing Bacillus
subtilis gave maximum cellulase activity after
72hrs of incubation (Krishna, 1999). Our results
can be correlated with the said results. The
cellulase activity increased gradually from 25-350C
and then fell at 400C. The mentioned strain
Production of cellulase from corn cobs 55
exhibited minimum cellulase activity (21.34 ± 0.06
µg/ml) at 250C. Using Trichoderma harzianum
T2008 grown on empty fruit bunches under SSF
exhibited maximum FPase activity (8.2 IU/g) at
32°C after 4 days of incubation in Erlenmeyer flask
(Alam et al., 2009). Our findings are in agreement
with the mentioned results. The cellulase activity
trend was increased gradually from pH 3.0-5.0 and
then settled down from pH 6.0-9.0 (showing acidic
nature of enzyme). The highest cellulase activity of
48.70 U/ mL was obtained by using bacillus strain
of BOrMGS-3 at an acidic pH or pH 5.0 (Tabao
and Monsalud, 2010). Our highest activity attained
at pH of 6.0 by showing that results were in
accordance with the mentioned results. Thus, the
maximum cellulase activity could be achieved in a
range of pH 5-6 culturing Trichoderma viride
strains; as pH increased up to 5.5, the hyper
activities
of
exoglucanase
(2.16
U/ml),
endoglucanase (1.94 U/ml) and β-glucosidase (1.71
U/ml) were observed (Gautam et al., 2010).
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