Advances in Bioscience and Bioengineering
ISSN 2201-8336
Volume 3, Number 1, 2015, 1-11
Effect of Carbon Source of Growth on α-Amylase Production by a Strain of
Penicillium funiculosum Thom. Isolated from Irish Potato (Solanum tuberosum Linn):
Comparative Studies
Adekunle Odunayo Adejuwon1 and Olusola Abiola Ladokun2
Department of Microbiology, Faculty of Information Technology and Applied Sciences, Lead City
1
University, Ibadan, Nigeria
Department of Biochemistry, Faculty of Information Technology and Applied Sciences, Lead City
2
University, Ibadan, Nigeria
Corresponding author: Adekunle Odunayo Adejuwon, Department of Microbiology, Faculty of
Information Technology and Applied Sciences, Lead City University, Ibadan, Nigeria
Abstract. Irish potato (Solanum tuberosum Linn.) is cultivated in the tropical middle belt of Nigeria. It is
infested by a range of phytopathogens including Penicillium funiculosum Thom. In the present
investigation, irish potato tubers and a defined growth medium were inoculated with pure culture of
Penicillium funiculosum Thom. The composition of the defined medium was potassium dihydrogen
sulphate, magnesium sulphate, calcium sulphate, hydrated iron sulphate, manganese sulphate, copper
sulphate, zinc sulphate, thiamine, biotin, sodium nitrate as nitrogen source and varied carbon sources.
Proteins which exhibited α-amylase activity were expressed and partially purified daily by ammonium
sulphate fractionation followed by dialysis to remove small molecular weight impurities. The results
showed that, optimum α-amylase activity was induced by the ninth day of inoculation of the potato
tubers. Only traces of the α-amylase were observed in uninfected irish potato tubers. Similarly, starch,
glucose, lactose, galactose, glucose and maltose as carbon source in the defined medium induced αamylase activity varyingly. Highest value of induction was expressed when starch was carbon source
followed by irish potato, then maltose glucose, sucrose, lactose, galactose consecutively. Earliest
induction was with glucose with expression starting on the second day of inoculation of the defined
growth medium. Inspite of being harmful to cultivation and storage of irish potatoes in Nigeria, West
Africa, Penicillium funiculosum Thom. is a potential source of industrial production of α-amylase.
Key words: α-Amylase; Penicillium funiculosum Thom.; irish potato (Solanum tuberosum Linn.)
© Copyright 2015 the authors.
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1. Introduction
α-Amylases are starch degrading enzymes that catalyze the hydrolysis of α-1, 4O-glycosidic bonds in polysaccharides with the retention of α-anomeric configuration
in the products. Most of the α-amylases are metalloenzymes which require calcium ions
(Ca2+) for their activity, structural integrity and stability (Bordbar et al., 2005). Amylases
are one of the most important industrial enzymes that have a wide variety of
applications ranging from conversion of starch to sugar syrups, to the production of
cyclodextrins for the pharmaceutical industry. These enzymes account for about 30% of
the world’s enzyme production (van der Maarel et al., 2002). The α-amylase family can
roughly be divided into two groups: the starch hydrolyzing enzymes and the starch
modifying or transglycosylating enzymes (Satyanarayana et al., 2005).
α-Amylases are produced by bacteria and fungi (Oh et al., 2005; Hashim et al.,
2005). In earlier studies, we have observed the induction of polygalacturonase and
cellulase in Penicillium funiculosum Thom. by tomato fruits during infection (Adejuwon
et al., 2006; Adejuwon et al., 2009).
Irish potato is a starchy staple food of the indigenes of the middle belt in Nigeria.
In this current investigation, we envisaged the possibility of the production of
hydrolytic enzymes such as α-amylases in irish potatoes infected with Penicillium
funiculosum Thom. We engaged in this study since the pythopathogen is able to infest
irish potatoes during storage, causing substantial loss to producers as observed in Jos,
Nigeria and the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria.
2. Materials and Methods
2.1 Materials
All the chemicals used in this research were of analytical grade and were
purchased from Sigma Chemical Company St. Louis, MO, USA or BDH Poole, England.
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3. Methods
3.1 Source and Identification of Fungi
The isolate of Penicillium funiculosum Thom. used in this research was from
deteriorated irish potato (Solanum tuberosum Linn.) and identified at the Seed Health
Unit of the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria, with
the aid of the illustrated Handbook of Fungi (Hanlin, 1990).
3.2 Culture Conditions and Inocula
The isolate was cultured and maintained on Potato Dextrose agar plates. The
fungus was further subcultured into test tubes of the same medium and incubated at
25oC. Sixty-hr-old culture of Penicillium funiculosum was used in this investigation.
Based on the method of Olutiola and Ayres (1973), culture was grown in a defined
medium of the following composition: Na2NO3 (9.9g), MgSO4.7H20 (0.1g), KH2PO4 (0.5g),
CaSO4 (0.1g), MnSO4 (1mg), CuSO4 (1mg), ZnSO4 (1mg), biotin (0.005mg), thiamine
(0.005mg) and FeSO4.7H20 (1mg) with added carbon (10g) sources (Sigma) in 1 litre of
distilled water. The carbon source was varied as: starch, glucose, lactose, galactose,
glucose and maltose. The nitrogen source was sodium nitrate. One hundred millilitre of
growth medium in conical flasks (250 ml) was inoculated with 1 ml of an aqueous spore
suspension containing approximately 6x105 spores per ml of isolate. Spores were
counted using the Neubauer counting chamber (Olutiola et al., 1991). Controls were
uninoculated growth medium. Experimental and control flasks were incubated without
shaking at 250C (Olutiola and Nwaogwugwu, 1982).
On a daily basis, the contents of each flask were filtered using glass fibre filter
paper (Whatman GF/A). The protein content of filtrate was determined using the Lowry
et al. (1951) method. Filtrate was assayed for α-amylase activity using the method of
Pfueller and Elliott (1969).
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3.3 Inoculation of Irish Potato (Solanum tuberosum Linn.) Tubers
Irish potato (Solanum tuberosum Linn.) tubers were bought at the Bodija Market,
Ibadan, Nigeria and identified at the Herbarium of the Department of Botany,
University, Ibadan, Ibadan, Nigeria. The potatoes were surface sterilized in 3% mercuric
chloride for 15 minutes. Using red heat sterilized cork borer, 3mm tissue disc was
removed from each potato tuber and inoculated with tissues disc of sixty-hr-old culture
of isolate grown on potato dextrose agar plates. They were kept in sterile petri dishes on
sterile glass plates under bell jars with the rims of the jars sealed with vaseline to ensure
air tight contact with the glass plates. This was done to avoid contamination. Controls
were potato tubers inoculated with discs of sterile potato dextrose agar in plates.
Incubation of both experimentals and controls was at 25oC. The potato tubers were
observed daily for fungal growth and deterioration.
3.3.1 Enzyme extraction
On a daily basis, infected irish potato tubers were carefully weighed, mercerated
and homogenised with cold (4oC) 0.5M NaCl in 0.02 M citrate phosphate buffer pH 6.0
to extract enzyme. The homogenate was filtered using four layers of muslin cloth and
clarified using glass fibre filter paper. The protein content of the crude filtrate was
determined using the method of Lowry et al. (1951). α-Amylase activity was
determined using the method of Pfueller and Elliott (1969).
3.4 Enzyme and Protein Assays
3.4.1 α-Amylase
α-Amylase activity was determined using the method of Pfueller and Elliott
(1969). The reaction mixtures consisted of 2 ml of 0.2% (w/v) starch in 0.02 M citrate
phosphate buffer, pH 6.0 as substrate and 0.5 ml of enzyme. Controls consisted of only 2
ml of the prepared substrate. The contents of both experimental and control tubes were
incubated at 35oC for 20 min. The reaction in each tube was terminated with 3 ml of 1 N
HCl. Enzyme (0.5 ml) was then added to contents of each control tube. Two millilitre of
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the mixture from each of the sets of experimentals and controls was transferred into
new sets of clean test tubes. Three millilitre of 0.1 N HCl was added into the contents of
each test tube after which 0.1 ml of iodine solution was added. Optical density readings
were taken at 670nm. One unit of enzyme activity was defined as the amount of enzyme
which produced 0.1 percent reduction in the intensity of the blue colour of starch-iodine
complex under conditions of the assay. Specific activity was expressed in enzyme units
per mg protein.
3.4.2 Protein concentration determination
Protein concentration was determined by the method of Lowry et al. (1951).
Serial dilutions of egg albumin were used to plot a standard graph. The unknown
protein value in each test sample was intrapolated from the standard calibration graph.
3.5 Ammonium Sulphate Fractionation
On the days of optimum α-amylase activity of inoculation medium, the crude
enzymes were treated with ammonium sulphate (analytical grade) at 90% saturation.
Precipitation was allowed at 4oC for 24 h. Mixtures were centrifuged at 6,000 g for 30
minutes at 4oC using a cold centrifuge (Optima LE-80K Ultracentrifuge, Beckman, USA).
The supernatant were discarded. The precipitate was reconstituted in 0.02M citrate
phosphate buffer, pH 6.0. α-Amylase activity was determined using the method of
Pfueller and Elliott (1969). Protein content was determined using the Lowry et al. (1951)
method.
3.6 Dialysis
Using acetylated dialysis tubings (Visking dialysis tubings, Sigma) (Whitaker et
al., 1963) and a multiple dialyser, the enzyme preparations were dialysed under several
changes of 0.02M citrate phosphate buffer, pH 6.0 at 4oC for 24 h. α-Amylase activity
was determined using the method of Pfueller and Elliott (1969) as described earlier.
Protein content was determined using the method of Lowry et al. (1951).
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4. Results and Discussion
Within ten days, the infected irish potato tubers were completely covered with
greenish spores from mycelia of the isolate. Substantial α-amylase was produced during
the infection process. Only traces of the enzyme were observed in uninfected irish
potato tubers. This is evidenced with traces of activity at days 1 and 2 in infected potato
tubers and absence of activity at days 1 of the carbon sources of inoculated growth
medium (Table 1). From data, highest protein value was with sucrose as carbon source
as observed on the fifith day of inoculation of growth medium. The least protein value
was with galactose as observed on day 1 of inoculation of medium (Table 1). Optimum
α-amylase activity was observed with: with irish potato as 279 units/mg protein on the
ninth day; starch as 432 units/mg protein on the tenth day; maltose as 251 unit/mg
protein on the tenth day; sucrose as 142 unit/mg protein on the ninth day; lactose as 87
unit/mg protein on the tenth day; glucose as 208 unit/mg protein on the ninth day; and
galactose as 39 unit/mg protein on the tenth day (Table 2). Starch as carbon source of the
growth medium induced highest α-amylase activity. The value is expressed as 432
units/mg protein and is on the tenth day of inoculation of medium (Table 2). α-Amylase
induction was earliest with glucose as carbon source and expressed on day 2 of
inoculation of medium as 12 units/mg protein. In Figure 1, we have the protein
concentration standard curve with egg albumin as standard protein.
When rish potato tubers were infected with Penicillium funiculosum Thom.,
proteins which exhibited α-amylase activity was expressed. By the ninth day, optimum
activity had been induced. Similarly, starch; glucose; lactose; galactose; glucose; and
maltose in the composition of a defined growth medium induced α-amylase (Adejuwon,
2013). Starch is expected to induce α-amylase if the gene for such expression is found on
the genome of such fungus. Induction by other carbon compounds used in this study
apart from starch clearly suggests constitutive enzyme production since α-amylase
catalyses degradation of starch to maltose and smaller dextrins. Poor induction was
observed when galactose was carbon source. Inspite of Penicillium funiculosum Thom.
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being harmful to cultivation of irish potatoes in tropical Nigeria, the data in this study
clearly suggests that α-amylase could be produced industrially using this isolate with
conditions stated for production.
Table 1:
Effect of carbon source on α-amylase activity produced by Penicillium
funiculosum Thom. showing unit activity and total protein
Days
Carbon
source
Irish potato
Starch
Maltose
Sucrose
Lactose
Glucose
Galactose
1
2
3
4
5
6
7
8
9
10
Activity
(Units)
0.001
0.001
0.025
0.065
0.10
0.325
0.32
0.58
0.535
0.54
Protein
(OD 600nm)
0.27
0.20
0.25
0.275
0.295
0.30
0.345
0.32
0.35
0.455
Activity
(Units)
0.00
0.00
0.00
0.00
0.00
0.01
0.47
0.395
0.34
0.45
Protein
(OD 600nm)
0.14
0.265
0.165
0.165
0.185
0.155
0.15
0.235
0.21
0.25
Activity
(Units)
0.00
0.00
0.035
0.01
0.03
0.55
0.675
0.58
0.58
0.525
Protein
(OD 600nm)
0.35
0.385
0.37
0.33
0.32
1.13
0.925
0.51
0.62
0.65
Activity
(Units)
0.00
0.02
0.005
0.02
0.01
0.26
0.405
0.565
0.53
0.46
Protein
(OD 600nm)
0.84
0.875
0.87
0.88
1.45
0.815
0.925
0.68
0.725
0.85
Activity
(Units)
0.00
0.015
0.025
0.01
0.02
0.03
0.45
0.025
0.025
0.10
Protein
(OD 600nm)
0.255
0.21
0.195
0.195
0.18
0.205
0.32
0.315
0.285
0.235
Activity
(Units)
0.00
0.03
0.045
0.05
0.05
0.115
0.69
0.565
0.56
0.495
Protein
(OD 600nm)
0.33
0.405
0.46
0.43
0.425
0.475
0.385
0.26
0.47
0.58
Activity
(Units)
0.00
0.01
0.06
0.03
0.04
0.015
0.05
0.01
0.02
0.055
Protein
(OD 600nm)
0.13
0.225
0.23
0.25
0.26
0.385
0.445
0.355
0.24
0.25
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Table 2: Effect of carbon source on α-amylase activity produced by Penicillium
funiculosum Thom. showing specific activity in units/mg protein, derived from
Table 1
Days
Carbon
Source
1
2
3
4
5
6
7
8
9
10
Irish
potato
0.05
0.05
3
12
17
46
54
261
279
188
Starch
0
0
0
0
0
7
65
96
291
432
Maltose
0
0
5
7
18
140
146
165
240
251
Sucrose
0
0
0
2
48
53
74
132
142
96
Lactose
0
0
2
6
8
12
18
22
39
87
Glucose
0
12
14
16
17
60
85
91
208
158
Galactose
0
0
0
0
0
0
2
6
10
39
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Protein Standard Curve
Absorbance (600nm)
3
2
1
0
0
500
1000
1500
2000
2500
Protein concentration (g/ml)
Fig 1: Standard curve for protein using egg albumin (Lowry et al., 1951 method)
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Acknowledgements
This research was supported by the year 2006 Small Grant Award of the British
Mycological Society with Dr. Adekunle Odunayo Adejuwon as recipient.
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