Sakthi et al.
EVALUATION OF AMYLASE ACTIVITY OF THE
AMYLOLYTIC FUNGI ASPERGILLUS NIGER USING
CASSAVA AS SUBSTRATE
S Siva Sakthi*, D Kanchana, P Saranraj, G Usharani
Department of Microbiology, Annamalai University, Annamalai Nagar,
Chidambaram – 608 002. E.mail: jpssivasakthi@gmail.com
Science Instinct Publications
Abstract
Fungal amylases are used for hydrolyzing carbohydrate, protein and other constitutes of
soybeans, wheat into peptides, amino acid, sugars and other low molecular weight compounds.
The present study was carried out to evaluate the amylase activity of the amylolytic fungi
Aspergillus niger using cassava waste as a feed substrate. The fungi were isolated from the soil
samples and the fungi isolated were identified as Aspergillus niger based on Lactophenol cotton
blue staining and plating on appropriate fungal growth medium. Amylase production by
Aspergillus niger was detected by the disappearance of blue colour in the Starch agar medium
around the microbial colonies after incubation. Cassava was used as the substrates for the
amylase production. Solid state fermentation was carried out for the production of amylase
using Aspergillus niger. The effect of different carbon sources, nitrogen source, Temperature
and pH was determined on enzyme production by Aspergillus niger. Amylase activity was
determined by four methods such as DNSA method, Dextrinizing activity method, decrease in
Starch-iodine color intensity and Plate assay.
Key words: Amylase, Aspergillus niger, Cassava and Solid state fermentation.
*
S. Siva Sakthi
Department of Microbiology,
Annamalai University, Annamalai Nagar,
Chidambaram – 608 002.
E.mail: jpssivasakthi@gmail.com
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Sakthi et al.
Introduction
M
icroorganisms in particular have been regarded as treasure of useful enzymes. There is
a great variation between various genera as to their ability to produce a specific
enzyme the production of particular enzyme varies with the particular medium and pH [1]. In
recent years the potential of using microorganisms as biotechnological sources of industrially
relevant enzymes has stimulated interest in the exploration of extracellular enzymatic activity
in several microorganisms [2, 3].
The first enzyme produced industrially was an amylase from a fungal source in 1894, which
was used for the treatment of digestive disorder [4]. At present Aspergillus and Rhizopus
species are considered to be the most important sources of industrial amylases. Amylases are
among the most important enzymes and are of great significance in present – day
biotechnology, having approximately 25% of the enzyme market. Agricultural substrate for
the production of amylase from amylolytic Aspergillus spp. Amylases represent a group of
enzyme of great importance to the food industry and other needs of life. Although amylases
can be obtained from several sources, such as plant and animals, the enzyme from microbial
sources generally meet industrial demand.
Agricultural and industrial wastes are among the causes of environmental pollution. Their
conversion into useful product may ameliorate the problems they cause. These wastes which
include cereals, straw, leaves, bran etc are highly underutilized in particularly microbes. In
most parts of the country, these materials are mainly used as animal feeds. A large quantity is
left on farmlands to be decomposed by microorganisms such as bacteria and fungi. The local
production of such enzyme using locally available agricultural wastes which can serve as
substrates may therefore reduce the cost of importation and encourage self reliance.
Cost of substrate on which amylases producing microbes can be cultivated has always been
an important factor in production cost [5]. They have abundant supply of agricultural wastes
that are generated from their rice mills, wheat and black gram bran processing, cassava
processing plants and other cottage industries annually. Exploitation of such wastes as
substrates for amylases production can be a viable option. Hundreds of different species of
fungi inhabit the soil, especially near the soil surface where aerobic conditions prevail. Such
fungi are active in degrading a wide variety of biological materials present in the soil. They
thrive on such material by secreting extra-cellular enzymes able to degrading large polymeric
plant molecules such as cellulose, starch and pectin, with subsequent assimilation of the
liberated nutrients.
Considering that a strain of Aspergillus niger isolated by soil during a screening program for
amylase producing microorganisms. Amylases are a group of enzymes that have been found
in several microorganisms like bacteria and fungi. Fungal source are confined to terrestrial
isolates, mostly to Aspergillus species. Among the microorganisms, many fungi had been
found to be good sources of amylolytic (amylase) enzymes. Studies on fungal amylase
especially in the developing countries have concerted mainly on Aspergillus niger probably
because of the ubiquitous nature and non – fastidious nutritional requirements of the
organisms [6]. The Aspergillus niger group is wide spread with many strains capable of
producing amylases.
Bacteria, yeasts and fungi can grow on solid states and find applications in solid state
fermentation process. Filamentous fungi are the best adopted for solid state fermentation.
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Sakthi et al.
The hyphal mode of fungal growth and their good tolerance to low water activity and high
osmotic pressure conditions make fungi efficient and competitive in natural microflora for
bioconversion of solid substrates. Amylases have been produced by submerged fermentation.
In recent years, however, the solid state fermentation (SSF) processes have been increasingly
applied for the production of this enzyme. Solid state fermentation compared to submerged
fermentation is more simple, require lower capital, has superior productivity, reduce energy
requirement, simple fermentation media and absence of rigorous control of fermentation
parameters, uses less water and produces lower wastewater, has easier control of bacterial
contamination and require low cost for downstream processing.
The amylase activity of Aspergillus niger decreased when the substrate concentration was
increased up to 2%. Besides the carbon source, it has been suggested that the nitrogen source
can also control amylase activity [7]. Agricultural waste (rice bran, wheat bran, Black gram
bran) have been used as substrates in solid state fermentation for the production of amylase
by Aspergillus niger. The present study was carried out to evaluate the amylase activity of
the amylolytic fungi Aspergillus niger using cassava waste as a feed substrate. Amylase
production by different organisms in submerged state fermentation has received more
attention and is found to be cost-prohibitive because of high cost of process engineering.
Materials and Methods
Substrates
Cassava was obtained from Salem, Tamil Nadu. The substrate was ground into coarse
powder with a blender.
Isolation and identification of Aspergillus niger strain
The Aspergillus niger were isolated from soil by serial dilution method. 1 g soil sample was
dissolve in 10 ml sterilized distilled water. The soil suspension was dilute up to 103 to 105.
The sample was inoculated on potato dextrose agar (PDA) for fungi. The inoculated media
were incubated at 30C for 3-5 days. Colony developments were observed after incubation
period. The young colonies of Aspergillus niger were aseptically picked up and transferred to
PDA slants. The slants were grown 30C for 7 days and stored at 4C. The isolate was subcultured for further studies.
Screening of microbial isolates for amylase
Primary screening was done by starch agar plate method. The isolate Aspergillus niger
inoculated on the starch agar medium. The plate incubated at 3 days. The iodine solution
flooded on the plate and then clear zone around the colonies. The plate showed a maximum
hydrolysis halo on this medium and was selected for further investigation.
Viable spore count
The total viable spore number on a Potato Dextrose Agar (PDA) slant was determined by
colony count technique. The spores were suspended in 10ml of distilled water with 0.1%,
using a sterile transfer needle and diluted serially. One ml of spore suspension was poured
onto sterile Petri-plates, containing sterile PDA medium and spread uniformly. The
inoculated Petri-plates were incubated at 30C for 48 hrs. A plate that developed between 7 to
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Sakthi et al.
200 colonies was selected for counting. The spore density was calculated as the count
multiplied by the dilution factor.
Solid state fermentation
Five gram of substrate (Cassava) was taken into a 250 ml Erlenmeyer flask and to this a
fungal amylase production containing (g/l) KH2PO4 – 1.4; NH4NO3 – 10; KCl – 0.5;
MgSO4.7H2O – 0.1; FeSO4.7H2O – 0.01; starch – 20 gm; and distilled water was added to
adjust the require moisture level. The contents of the flaks were mixed thoroughly and
autoclaved at 121C for 20 min. Solid state fermentation was carried at 30C with substrate
initial moisture content of 64% for 72 hours using 2 ml Aspergillus niger suspension as
inoculum. Studies were also performed to evaluate the influence of supplementation of
substrate with different carbon sources such as glucose, maltose, sucrose, lactose (3% w/v)
and nitrogen source such as peptone, casein, urea, yeast extract (3% w/v).
Optimization of culture condition
The effect of culture conditions the present study was carried out at different temperature (30,
40, 50 and 60°C), different pH (4, 5, 6 and 7), different carbon sources such as (glucose,
maltose, lactose and sucrose) and nitrogen sources such as (peptone, casein, urea, yeast
extract) were used to determine their effect on amylase production.
Enzyme extraction
22 ml of 0.1 M phosphate buffer (pH-6.5) was added to cultures, the mixture were shacked
for 30 min at 19C and 140 rpm on a rotary shaker. The mixture was filtered through cheese
cloth and centrifuged at 8000 rpm at 4C for 15 min. The supernatant was filtered through
Whattman Number-1 filter paper and the filtrate was used as the crude enzyme preparation.
Assay of enzyme activity
Dinitrosalicylic acid (DNSA) method
Assay system for amylase activity was carried out by measuring the amount of reducing
sugar according to the DNSA method [8]. Amylase activity was determined by incubating a
mixture of 1 ml of aliquot of each enzyme source and 1% soluble starch dissolved in 0.1 M
phosphate buffer, at pH 7, at 55C for 15 min. The reaction was stopped by adding 1 ml of 3,
5 Dinitrosalicylic acid, and then followed by boiling for 10 min. The final volume was made
up to 12 ml with distilled water and the reducing sugar released was measured at 540 nm [9].
One unit of amylase activity was defined as the amount of enzyme that releasing 1mol
glucose equivalent per minute under the assay conditions. Reducing sugar (Glucose or
maltose) concentration was determined from a standard curve under same condition using
glucose.
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Sakthi et al.
Calculation:
Microgram of glucose produced
Amylase activity (U/ml) =
Volume of enzyme solution x Incubation time
Dextrinizing activity method
Amylase was assayed by the iodine method described by Jones and Varner (1967)10, with
slight modifications. Activity was estimated in a reaction mixture containing 1.0 ml of 0.15%
soluble starch in 0.1M acetate buffer, pH 5.5, 200mM CaCl2 and 1.0 ml enzyme and
incubated at 65C for 10 min. After incubation, 1.0 ml of 1 M acetic acid, 1.0 ml of 0.2%
iodine, 2.0% KI solution and 15 ml of distilled water were added. The absorbance of the
diluted solution was measured at 620 nm. A blank was prepared under the same condition by
adding enzyme solution after the reaction had been stopped by the addition of 1.0M acetic
acid. One unit of dextrinizing activity was defined as the amount of enzyme which
hydrolyzes 1ml of starch/10minutes under the above conditions. Starch concentration was
determined from a standard curve under the same assay condition using soluble starch.
Calculation:
Microgram of glucose produced
Amylase activity (U/ml) =
Volume of enzyme solution x Incubation time
Decrease in starch-iodine color intensity
Starch forms a deep blue complex with iodine [11] and with progressive hydrolysis of the
starch, it changes to red dish brown. Several procedures have been described by various
groups for the quantitative determination of amylase based on this property. This method
determines the dextrinizing activity of amylase in terms of decrease in the iodine color
reaction.
Plate assay
The plate assay was performed using agar plates amended with starch. The agar plates were
prepared amended 2% of starch with 1.5% of agar. After agar solidification, around 10 mm
diameter of well was cut out aseptically with the help of cork borer. The well was filled with
the culture filtrate and incubated at 37C for overnight. 1% of iodine solution was over
layered on the agar and the observation was made to see the hydrolytic zone around the well.
The negative control was maintained by adding sterile water in the separate well.
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Estimation of protein content
The protein content was estimated by the method of Lowry et al.,. Bovine serum albumin
was used as standard solution to determine the protein in the crude enzyme [12].
Results and Discussion
In recent years the potential of using microorganisms as biotechnological sources of
industrially relevant enzymes has stimulated interest in the exploration of extracellular
enzymatic activity in several microorganisms. Starch degrading enzymes like amylase have
received great deal of attention because of their perceived technological significance and
economic benefits. This enzyme is also used for the commercial production of glucose.
Amylases are important enzymes employed in the starch processing industries for the
hydrolysis of polysaccharides such as starch into simple sugar constituent. Although
amylases can be obtained from several sources, such as plants and animals, the enzymes from
microbial sources generally meet industrial demand. Aspergillus niger was produced more
amylase yield on agricultural waste. Since agricultural waste is very cheap, easily available
source from mill factories and more suited for solid state cultivation of Aspergillus niger for
amylase production [13].
Many agro-industrial by products such as wheat bran, rice bran, molasses bran, black gram
bran and cassava wastes has been screened as low-cost solid substrates for microbial
production of amylase in solid state fermentation. In this study also agricultural waste cassava
was used for amylase production [14]. Agro-industrial reside which are generally used as
substrates for solid state fermentation. This agro-industrial residue in bioprocesses also solves
pollution problems, which their disposal may otherwise cause with the advent of
biotechnological innovations, mainly in the areas of enzyme and fermentation technology,
many new areas have opened for their utilization as raw materials for the production of value
added fine products. In addition, the microbial degradation of these residues by GRAs
(generally regarded as safe) strain may improve the substrate value as animal feed [15].
Growth conditions and nutrients promote high yields of microbial amylases. However,
carbon sources such as dextrin, fructose, glucose, lactose, maltose and starch are very
expensive for commercial production of these enzymes. These expensive products can be
replaced in the medium with economically available agricultural by-products or industrial
amylaceous substances as carbon substrates [16]. The bran, straws and flours of different
grains such as barley, corn, potato, rice and wheat have been used in the fermentation
medium to increase the productivity of amylases from bacteria and fungi. In the present study
cassava waste showed the highest enzyme production [17].
The fungi were isolated from the soil samples by serial dilution technique. The isolated fungi
were identified by macroscopic and microscopic observation. The fungi identified was
Aspergillus niger, it was used for further study. The fungi were identified different based on
their growth, pigmentation and colony morphology on potato dextrose agar at 300C for 72
hrs. Aspergillus niger were identified by the morphology (spore bearing heads which are
large) in Lactophenol cotton blue staining and the colony colour on potato dextrose agar
(Black or brownish black). The total viable spore count on a potato dextrose agar was
determined by Colony count technique. The spore well developed on the medium. A plate
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Sakthi et al.
that developed between 7 to 200 colonies was selected for counting. The spore density was
calculated as the count multiplied by the dilution factor.
Sumitra Ramachandran et al. (2004)18 carried out the Solid-state fermentation for the α–
amylase using Aspergillus niger. Different oil cakes such as coconut oil cake (coc), sesame
oil cake (soc), groundnut oil cake (Goc), Palm Kernal Cake (PKC) and olive oil cake (OOC)
were screened to be used as substrate for the enzyme production and also compared with
wheat bran (WB). Goc was found to be the best producer of the enzyme among these
combination of wheat bran and GOC resulted higher enzyme titres than the individual
substrates. The supplemented with lactose and ammonium nitrate (1% each) at 300C for 72hrs
using 2 ml spore suspension. Partial purification of the enzyme using ammonium sulphate
fractionation resulted in increase in the activity.
Amylase activity was preliminarily checked by plating the Aspergillus niger on the starch
agar medium, the medium was flooded using iodine indicator, clear zone around the colony
indicated the presence of amylase activity. Solid state fermentation was carried out with
substrate cassava compared with soluble starch for the production of amylase at room
temperature using Aspergillus niger for 72 hrs. Crude enzyme was separated from solid state
fermentation using enzyme extraction technique.
The effect of temperature on amylase production by Aspergillus niger using cassava waste as
substrate was carried out at various temperature viz., 30, 40, 50 and 60°C. The enzyme
production was maximum at 40°C (4.12 U/ml) and minimum at 30°C (3.12 U/ml) (Figure-1).
The effect of pH on amylase production by Aspergillus niger was carried out at various pH
viz.,4, 5, 6 and 7 . The enzyme production was maximum at pH 6 (4.11 U/ml) and minimum
at pH 4 (3.11 U/ml) (Figure -2).
The effect of various carbon sources like Glucose, Maltose, Sucrose and Lactose was tested
on amylase production by Aspergillus niger using cassava waste as substrate. The amylase
production was high in the medium containing Glucose (5.5 U/ml) (Figure-3). The effect of
nitrogen sources like Peptone, Yeast extract, Casein and Urea were tested for the amylase
production. Among the various nitrogen sources tested, peptone (4.8 U/ml) maximum
amylase production (Figure-4). Amylase activity was determined by Dinitrosalicylic acid
assay (5.9 U/ml), Dextrinizing activity (10.5 U/ml), Decrease in starch-iodine color intensity,
Plate assay (18 mm) (Table-1). The Protein concentration was determined by the Lowry’s
method. The crude extract contained protein, the protein levels were found to be 50µg/ml.
The increased production of glucoamylase by Aspergillus awamori through solid state
fermentation using wheat bran as the main carbon source and ammonium sulphate, Urea,
Pottassium Dihydrogen Phosphate (KH2PO4), glucose, maltose and starch as additional
nitrogen sources and carbon sources. The production of glucoamylase is strongly influenced
by N and C sources. A 100% increase was observed when the (NH4)2 SO4) ammonium
sulphate was replaced by Urea, with C/N=4.8, using maltose as the additional carbon sources
[19]. In our study, urea used as the nitrogen source and maltose used as the carbon source.
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Sakthi et al.
Fig. 1. Effect of temperature on amylase production by Aspergillus niger
Fig. 2. Effect of pH on amylase production by Aspergillus niger
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Fig. 3. Effect of carbon sources on amylase production by Aspergillus niger
Fig. 4. Effect of nitrogen sources on amylase production by Aspergillus niger.
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Table 1. Assay of amylase enzyme activity
S.No
Method
Enzyme activity (IU/ml)
1
Dinitro sacicyclic acid method
2
Dextrinizing activity
10.5
3
Plate assay (Diameter of zone in
mm)
18
5.9
Conclusion
Research on amylase has progressed very rapidly over the last five decades and potential
industrial applications of the enzyme especially in solid waste management have been
identified. Major impediments to exploit the commercial potential of amylase are the yield,
stability and cost of amylase production. Although amylase production by microbes have
been extensively studied by many researchers. The fungi Aspergillus niger was used for the
production of amylase and cassava wastes were used as a substrate.
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