Continental J.
Biological Sciences 5 (1): 37 - 41, 2012 ISSN: 2141 - 4122
© Wilolud Journals, 2012
201 http://www.wiloludjournal.com
Printed in Nigeria doi:10.5707/cjbiolsci.2012.
sci.2012.5.1.37.41
ISOLATION, SCREENING AND MEASUREMENT OF AMYLASE AND CELLULASE ACTIVITIES OF
SOME MICROORGANISMS
Nwagu K.E., Ominyi M. C., Nwoba G.E.,
Department of Biotechnology, Ebonyi State University. P.M.B. 053, Abakaliki.
ABSTRACT
The amylase and cellulase production abilities of some microorganisms isolated from cassava
processing site, refuse dumping ground, rice straw and saw dust dumps were assessed. Bacterial
Bact and
fungal strains were isolated using Potato Dextrose Agar (PDA)/Sabouraud Dextrose Agar (SDA) and
Nutrient Agar (NA)/Deoxycholate Citrate Agar (DCA) media respectively. The screening of the
isolates for enzyme production and measurement of enzyme activity activity were carried out using
carbohydrate degradation test and spectrophotometric method respectively and the isolates were
identified using microbiological and biochemical standard methods. There was significant (P<0.05)
variation in enzyme activity between
between the bacterial and fungal isolates. The fungal isolate, Aspergillus
specie (AS-100)
100) exhibited the highest glucoamylase activity (0.083±0.003)
(0.083 0.003) after 96 hours of incubation
while the peak cellulase activity (0.079±0.002)
(0.079 was seen in Rhizopus specie (RS-200).
(RS Among the
bacterial isolates, Bacillus specie (BS-88)
(BS had the highest glycoamylase (0.025±0.002)
0.002) and cellulase
(0.061±0.001)
0.001) activities. The result showed that microorganisms especially fungi (RS-200
(RS and AN-100)
are host of polysaccharide degrading enzymes which are highly invaluable in bio-refining
bio refining industries.
KEYWORDS: Amylase, Cellulase, Bacteria, Fungi, Activities.
INTRODUCTION
Biomass resources such as starch and lignocellulose materials are the most abundant renewable resources and
good raw materials on earth for the microbial production of food, fuel and chemicals (Coughlan, 1983). They
need to be hydrolyzed to simpler products in order to yield valuable resources such as ethanol and food.The
hydrolysis step is often the limiting step;
step; thus, there is need for good sources of these enzymes. These enzymes
are relatively expensive especially in Nigeria and other developing countries because no company is producing
them. However, tropical climate as in Nigeria, harbours varieties of microorganisms
microorganisms that produce these
enzymes.
Amylase are starch (a heterogenous polysaccharide composed of a polymer of glucose residues) degrading
enzymes while cellulases are cellulose (a homogenous polysaccharide composed of amylose and amylopectin)
ing enzyme. These enzymes act by hydrolyzing the α–1,4 and β–1,4
degrading 1,4 glycosidic bonds present in starch
and cellulose respectively to yield a lower molecular weight sugar (Prasanna, 2005). In the early 1970’s, it was
considered that plant and animal materials were the best sources of enzymes, but nowadays, microbial enzymes
en
are becoming increasingly important owing to their technical and economical advantages (Cherry et al., 2004).
In the present day biotechnology, the potential of using microorganisms as biotechnological sources of
industrially important enzyme has triggered
triggered renewed cynosure in the exploitation of extracellular enzymatic
activity in several microorganisms (Akpan et al., ., 1999; Buzzini and Martin, 2002). A number of
microorganisms including fungal and bacterial species are associated with the production of o amylase and
cellulase. Extracellular amylase (α,, β and glucoamylase) and cellulase (exoglucanase, endoglucanase and β–
glucosidase) have been reported to be produced by various species of bacteria such as Bacillus subtilis, B.
licheniformis, Escherichia species,
pecies, Clostridium species and fungi such as Trichoderma, Aspergillus,
Aspergillus Rhizopus,
Penicillium and Candida species (Pothiraj et al., 2006; Syu and Chen, 1997).
The isolation, screening and measurement of the activities of these microorganisms require a suitable
culture/fermentation media or condition that will favour their growth and consequently high yield of enzymes.
They require an optimal temperature of about 25oC – 31oC for fungi and 35oC and above for bacteria since most
are thermostable. Enzymess of fungal origin can utilize different kinds of bacteria and fungal agar media but
preferably, a medium containing a substrate (starch and cellulose) in its composition.
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Nwagu K.E et al.,: Continental J. Biological Sciences 5 (1): 37 - 41, 2012
AIMS OF THE STUDY
1. To isolate microorganisms capable of hydrolyzing starch and cellulose
2. To compare the amylase and cellulase activities of some microorganisms
MATERIALS AND METHODS
Sample Collection
Samples were aseptically collected from cassava processing site, refuse dumping ground, rice straw, and saw
dust dump in Abakaliki metropolis of Ebonyi State, south-east Nigeria on 8th September, 2010. The upper
layers of the dumps were removed with a sterilized spatula and the samples beneath were collected using sterile
universal container.
Isolation of Microorganisms
Each of the samples from different sources was prepared by serial dilution and pour plate technique. Potato
Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA) media was used for the culturing of fungi while
Nutrient Agar (NA) and Deoxycholate Citrate Agar (DCA) media was used for the culturing of bacteria, all
prepared according to the manufacturers specifications. The pure cultures were identified by their morphology
and colony characteristics. The organisms were maintained on PDA and NA slant for fungi and bacteria
respectively and stored at 4oC
Screening of Isolates for Amylase Production
This was carried out using microbial carbohydrate degradation test. The isolates were inoculated in a media
containing 3ml of soluble starch broth prepared by a mixture of 1% tris buffer, pH 6.5, and 1.2% sterilized
soluble starch. This was followed by an addition of 2-3 drops of phenol red indicator, and incubation at 30oC for
3-4 days with daily monitoring. Observation of colour change from red to yellow depicts that the isolate was
metabolizing the starch and thus there was amylase production/activity.
Screening of Isolates for Cellulase Production
The isolates were grown in Carboxymethylcellulose (CMC) agar flooded with Congo red at 37oC for 2-3 days.
A clear zones around the colonies of the isolate are an indication that the isolate is metabolizing the substrate
and thus, there is enzyme production/activity.
Cultivation of the Isolates for Amylase and Cellulase Production
The isolates were dispersed on a medium (100ml) containing 0.05% peptone, 0.5% commercial soluble starch
and cellulose for starch and cellulose broth respectively and 0.lml liquid fertilizer in Erlenmeyer flasks of 250ml
capacity. The cultivation was carried out for four (4) days but the enzyme activity of each isolate was assayed
daily. The cultures were left at room temperature with manual shaking at intervals to enhance mass-transfer and
avoid cell sedimentation. The enzyme activities of the isolates were determined by measuring the activities of
crude enzyme in the culture broth.
Measurement of Glucoamylase Activity
The glucoamylase activity of each of the isolate was determined by the method of Trinder (1959) called the
glucose oxidase/ peroxidase (GOP/POD) method.
Measurement of Cellulase Activity
Cellulase activity of the isolate was assayed using filter paper assay method of Stephen et al. (2003). This is a
combined assay for endo and exo β-1,4 glucanases.
Identification of Bacterial Isolates
The bacterial isolates were presumptively identified by means of macroscopic, microscopic and some
biochemical characterization and then compared using “Bergey’s manual of determinative bacteria” (Buchanan
and Gibbons, 1974)
Identification of Fungal Isolates
The fungi isolates were identified by morphological characteristics according to the taxonomic key of
Alexopoulos et al. (1990).
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Nwagu K.E et al.,: Continental J. Biological Sciences 5 (1): 37 - 41, 2012
RESULTS
The result showed that ten (10) isolates with carbohydrate degrading activities were isolated. Among these
isolates, five (5) isolates were fungi and the other 5, bacteria. All the fungal and bacterial isolates were able to
produce amylase and cellulase except the bacterial isolate, EC-30 that produced only amylase as shown in figs
1-4.
The order of glucoamylase production by the fungal and bacterial isolates was AN-100>MR-55>AV-72>RS-
200>PM-22 and BS-88>EA-45>EC-30>PP-92 respectively.
The order of cellulase production by the fungal and bacterial isolates was RS-200>AN-100>MR-55>AV-
72>PM-22 and BS-88>EA-45>BM-199>PP-92.
Among the fungal isolate in figure 1, AN-100 had the highest glucoamylase activity (0.083 + 0.003) followed
by MR-55 (0.051 + 0.004) after 96 hours of incubation while the lowest glucoamylase activity was seen in PM-
22 (0.022 + 0.001). The isolate, BS-88 had the highest glucoamylase activity (0.025 + 0.002) among the
bacterial isolates followed by BM-199 (0.021 + 0.002) as shown in figure 3. The glucoamylase activity of all
the fungal isolates were higher than that of the bacterial isolates except the fungal isolate PM-22 which had a
lower activity than the bacterial isolate, BS-88. In the measurement of cellulase activity, RS-200 had the highest
cellulase activity (0.091 + 0.002), followed by AN- 100 (0.071 + 0.002) after 72hours of incubation as shown in
figure 2. In the case of the bacterial isolates, BS-88 produced the highest cellulase (0.061 + 0.001) as depicted in
figure 4 but the activity was lower than those produced by RS-200 and AN- 100 fungal isolates.
Figs 1-4 show the result of the identified bacterial and fungal isolates, the enzymes produced, and their
activities.
Aspergillus Specie
(AN – 100)
Aspergillus Specie
(AV – 72)
Mucor Specie
(MR – 55)
Penicillium Specie
(PM - 22)
Rhizopus Specie
(RS - 200)
FIG 1.: Glucoamylase activities of the fungal isolates
Aspergillus Specie
(AN – 100)
Aspergillus Specie
(AV – 72)
Mucor Specie
(MR – 55)
Penicillium Specie
(PM - 22)
Rhizopus Specie
(RS - 200)
FIG 2: Cellulase activities of the fungal isolates
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Nwagu K.E et al.,: Continental J. Biological Sciences 5 (1): 37 - 41, 2012
Enterobacter specie
(EA – 45)
Escherichia specie
(EC – 30)
Bacillus specie
(BM - 199)
Bacillus specie
(BS – 88)
Pseudomonas specie
(PP - 92)
FIG 3: Glucoamylase activities of the bacterial isolates
Enterobacter specie
(EA – 45)
Bacillus specie
(BM - 199)
Bacillus specie
(BS – 88)
Pseudomonas specie
(PP - 92)
FIG. 4: Cellulase activities of the bacterial isolates
DISCUSSION
Both fungi and bacteria have been heavily exploited for their abilities to produce a wide variety of cellulase and
amylase. Most emphasis have been placed on the use of fungi because of their capability to produce copious
amount of cellulases and amylases which are secreted to the medium for easy extraction and purification. This
study investigated the isolation of microorganisms, screening for the production of amylase/ cellulase and the
measurement of their enzyme activity.
According to the study, the fungal isolate, Rhizopus specie (RS-200) had a higher cellulase activity than
Aspergillus specie (AN-100). This result is in conformity with the findings of Pothiraj et al (2006) who reported
that Rhizopus stolonifer cultivated on cassava waste gave a higher cellulase activity than Aspergillus niger
cultivated in the same cassava waste. Similar observation has been expressed by Vipan et al (1994). Aspergillus
niger has been used mostly for the production of cellulase and amylase (Ray et al., 1993). However, in the
present investigation, the cellulase activity of Aspergillus specie (AN- 100)was surpassed by the activity of
Rhizopus specie (RS-200). AN- 100 produced more glucoamylase than RS- 200 which is also in conformity
with other findings.
The higher cellulase activity of RS-200 might be due to the good growth of its mycelial biomass in its source,
that is, rice straw, saw dust, refuse dump and cassava processing site which led to higher enzyme activities. It
could also be as a result of their pH, aeration and temperature tolerance.
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Nwagu K.E et al.,: Continental J. Biological Sciences 5 (1): 37 - 41, 2012
CONCLUSION
Finally, this study clearly demonstrated that Rhizopus specie has a great potential for use in cellulase and
amylase production order than Aspergillus specie. The factors such as incubation time, pH, aeration, agitation,
temperature and energy source need to be considered in the cultivation of these microorganisms since it affects
their rate of growth and enzyme production. These enzyme producing microorganisms are better sourced from
sites where there is huge deposit of cellulosic or starchy materials because their adaptation in such site is an
evidence that it can metabolize such material and thus, produce these enzymes
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Received for Publication: 12/11/2011
Accepted for Publication: 08/03/2012
Corresponding author
Nwagu K.E.,
Department of Biotechnology, Ebonyi State University. P.M.B. 053, Abakaliki.
Correspondence: [email protected]
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