WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Sandhu et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue 9, 1448-1458. Research Article ISSN 2278 – 4357

STUDY OF EFFECT OF TEMPERATURE ON AMYLASE

PRODUCTION BY SOIL MYCOTIC FLORA OF JABALPUR REGION

Sonal Sareen Pathak1, Suneel Kumar3, R. C. Rajak2, Sardul Singh Sandhu*3

1
Department of Biotechnology, Mata Gujri Mahila Mahavidyalaya (Autonomous), Jabalpur-
482001, M.P. India
2
SGH Center for Rural Biotechnology and Management, Jabalpur- 482001, M.P. India
3
Fungal Biotechnology and Invertebrate Pathology Laboratory, Department of Biological
Sciences, Rani Durgawati University, Jabalpur- 482001, M.P. India.

ABSTRACT
Article Received on
12 July 2014, The effect of temperature on enzyme activity shows that enzyme from
Revised on August
2014, different fungal isolates have their optimum temperature. The
Accepted on 28 August 2014
nutritional and cultural conditions are required for the optimum growth
and production of amylase from the mycotic flora. The present study
*Correspondence for deals with the isolation of amylolytic fungi from soil samples. The
Author fungal isolates showing amlolytic activity are used to extract the crude
Sardul Singh Sandhu
amylase to study the effect of temperature for optimum production of
Fungal Biotechnology and
extracellular amylase. In the present research work, Aspergillus niger,
Invertebrate Pathology
Laboratory, Department of Aspergillus flavus, Rhizopus sp. and Fusarium sp. were isolated from
Biological Sciences, Rani the soil sample. A part from these isolates, Absidia species, Alternaria
Durgawati University, alternata, Curvularia lunata and Pythium species were also reported in
Jabalpur, M.P. India
the samples. Among them, the maximum amylolytic activity was
showed by Aspergillus niger and Aspergillus flavus. The enzyme
activity was observed for the isolates at different temperature range of 28°C, 42°C and 60°C
and it was inferred that the activity varied for different isolate at different temperature. The
production of amylase is influenced by temperature which is related to the growth of
organism.

Key Words: Amylase, temperature, optimum, extracellular, amylolytic.

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INTRODUCTION
Current development in biotechnology is yielding new application for enzymes. Many soil
dwelling bacteria and fungi produce amylase, the ‘starch degrading enzyme’. Amylases are
produced by a variety of living organism ranging from microorganism to plants and humans.
Bacteria and fungi secrete amylases to the outside of their cells to carry out extracellular
digestion when they have broken down the insoluble starch and the soluble end products such
as glucose or maltose when they are absorbed into their cells. Although, they can be derived
from several sources, such as plants, animal and microorganism, the enzyme from microbial
sources are generally used for industrial applications.[1] Amylolytic enzyme are commonly
produced by filamentous fungi preferred strains belong to the Aspergillus species and
Rhizopus species. Aspergillus niger is used for commercial production of α-amylase.[2]

α-amylase enzymes are important enzymes employed in starch processing industries for
hydrolysis of polysaccharides such as starch into simple sugar constituents.[3] Starch
degrading enzymes like amylase have received great deal of attention because of their
perceived technological significance and economic benefit.[4] Amylase is also used for
commercial production of glucose. Nowadays the new potential of using microorganism as
biotechnological sources of industrially relevant enzymes has stimulated interest in
exploration of extra cellular enzymatic activities in several microorganisms.[3] These enzymes
are found in animals (saliva, pancreas), plants (malt), bacteria and molds. Amylase of fungal
origin was found to be more stable than the bacterial enzymes on a commercial scale; many
attempts have been made to optimize culture conditions and suitable strains of fungi. The
enzyme cellulase also extracted from the fungi which have very important role in hydrolyzing
the components into small glucose units.[5] Few attempts have been made to elucidate the
control mechanisms involved in formation and secretion of extracellular enzymes.[6]

The influence of temperature on amylase production is related to the growth of organism.

Among fungi, most amylase production studies have been done with mesophillic fungi within
the temperature range of 25-37ºC. Optimum yields of amylase were achieved at 30-37ºC for
Aspergillus oryzae.[7] Temperature optima for enzyme activity is one of the most important
parameters in starch processing industry, as starch granules are kept at high temperatures
during the initial process of starch hydrolysis, gelatinization which is followed by the process
of enzymatic liquefaction where the use of thermostable amylolytic enzyme is
indispensable.[8] Molds are capable of producing high amount of amylase; Aspergillus niger

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is used for the commercial production of amylases. Studies on fungal amylases, especially in
developing countries have focused on Aspergillus sp. because of their ubiquitous nature and
non fastidious nutritional requirements of these organisms.[6]

MATERIALS AND METHODS

Collection of soil sample
In the present study, different soil samples were collected from garden, play ground and
garbage heap of Jabalpur region for isolating amylolytic fungi. The sample was brought to
the laboratory in the sterile polyethene bags for further processing.

Isolation of fungi
The isolation of fungi was done by serial dilution method. The soil samples were serially
diluted and spreaded on potato dextrose agar media plate containing antibiotic
chloramphenicol to avoid bacterial contamination and incubated at 28 ±1oC for 5-7 days.
After the proper growth, the pure colony was transferred to PDA slant and preserved at 4-5oC
temperature.

Identification of fungi
All the isolated soil fungal strains were identified on the basis of morphological
characteristics according to Domasch.[9] The colonies appearing on petri-plates were sub-
cultured into the tube containing potato dextrose agar medium for identification. All the
plates were observed for macroscopic characters of fungi that is for color, hyphae and texture
and microscopic character by preparing the slides from different fungal colony.

Screening of soil fungi for Starch Hydrolysis

Fungi isolated from different soil sources were grown on solidified starch agar media. They
were incubated at 28 ±1°C for 72 hours. The total number of colonies of different fungi
appearing on the plates was counted and recorded. The fungal colonies were then examined
for amylase production through Starch Hydrolysis Test.[10]

Enzyme Production
The production of amylase from selected fungal was done in the fermentation media.[11] The
selected isolates were screened quantitatively in amylase production medium comprising of
KH2PO4 (1.4 gm/L), NH4NO3 (10 gm/L), KCl (0.5gm/L), MgSO4.7H2O (0.1gm/L),
FeSO4.7H2O (0.01gm/L), Soluble starch (20gm/L)} and pH adjusted to 6.5. The inoculum

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(showing maximum hydrolysis during screening) was inoculated in the media and incubated
for 72 hrs at 28 ±1ºC on a shaker at 150 rpm.

Crude Enzyme extraction and Protein estimation

Crude enzyme was extracted from fermented media by adding Tris-buffer (pH 6.5), agitating
the flask in shaker at 180 rpm for 1 hour, the mixture was filtered through cheese cloth and
centrifuged at 8000 rpm at 4ºC for 5 minutes. The supernatant was collected and treated as
crude enzyme. The concentration of protein in crude enzyme (extracted from flask) was
determined by Lowry’s methods of protein estimation using Bovine serum albumin as a
standard.[12]

Enzyme Assay in Crude Enzyme

0.5 ml of crude enzyme extract was taken into a test tube to which 0.5 ml of 1% soluble
starch was added. The test tube was then incubated in a water bath at 100ºC for 15 minutes.
For blank, 0.5 ml of enzyme extract (that has been boiled for 15 minutes in order to inactivate
the enzyme) was added to starch solution. The reaction was ceased by adding 1 ml of DNS
[13]
Reagent. The test tube was boiled for 15 minutes and cooled immediately. 10 ml of
distilled water was added and color intensity at 540 nm was determined. Amount of maltose
released was determined by comparing the absorbance reading of the test enzyme at 540 nm
with the standard graph of maltose. The graph was plotted between the known concentrations
of maltose ranging from 0.05-0.5 mg/ml.

Effect of temperature and thermo-stability studies

The crude enzyme extracted from different fungal isolates was allowed to undergo the
enzymatic reaction at different temperature 28ºC, 42ºC and 60ºC. Reaction mixtures were
incubated at above respective temperature and enzyme activity was determined in order to
find out the optimum temperature for amylase production. Thermostability test was
performed by pre-incubating the crude enzyme in a range of temperature of 28 ºC to 60ºC for
3 hours.

RESULTS
Isolation of soil fungi
In the present research work, fungi were isolated from the different soil sample of play
ground, garbage soil and garden. A total 26 fungi were isolated which belong to different
genera as shown in Table 1.

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Table 1. Number of different fungi isolated from various soil samples

Fungi from Fungi from play Fungi from Total number
Fungi
garden soil ground soil garbage soil of fungi
Absidia sp. --- 2 --- 2
Aspergillus flavus 2 --- 1 3
Aspergillus fumigatus --- 3 --- 3
Aspergillus niger 2 1 2 5
Aspergillus sp. 1 1 --- 2
Cladosporium sp. --- --- 1 1
Curvularia lunata --- 2 --- 2
Fusarium sp. --- 2 --- 2
Pythium sp. --- --- 3 3
Rhizopus sp. 3 --- --- 3
Total 8 11 7 26

Identification of fungi
Identification of soil fungi was done according the protocol of Aggarwal and Hasija[15] and
Domasch[9]. The isolated species were described according to their macroscopic features such
as colour, shape and growth of cultured colonies, as well as microscopic characteristics like
structure of hyphae, conidia and conidiophores. Obtained data was then compared with the
descriptions of fungi species present in the literature. 10 species were successfully identified
as Absidia sp., Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus sp.,
Cladosporium sp., Curvularia lunata, Fusarium sp., Pythium sp., Rhizopus sp. as depicted in
Table 2.

Table 2. Frequency of fungi isolated from different soil samples

S.
Name of Fungus Isolation site % Frequency
No.
1. Absidia sp. Play ground soil 07.69
2. Aspergillus flavus Parden soil & garbage soil 15.53
3. Aspergillus fumigatus Play ground soil 11.53
Play ground soil garden soil &
4. Aspergillus niger 19.23
garbage soil
5. Aspergillus sp. Garden soil & play ground soil 07.69
6. Cladosporium sp. Garbage soil 03.84
7. Curvularia lunata Play ground soil 07.69
8. Fusarium sp. Play ground soil 12.53
9. Pythium sp. Garbage soil 12.53
10. Rhizopus sp. Garden soil 11.53

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Fig 1. Frequency of fungal isolates from different soil samples

Screenig of Amylase producing soil fungi

The fungal isolates were screened for amylase production by Starch Hydrolysis Test. The
four fungal isolates i.e. Aspergillus flavus, Aspergillus niger, Fusarium sp. and Rhizopus sp.
were found to give positive results (Table 3). Among these fungal isolates, Aspergillus flavus,
Aspergillus niger and Rhizopus sp. were found to be potential amylase producer and capable
of producing excessive amylase. Though the amount of amylase produced was different in
each case. But, other species Absidia sp., Cladosporium sp., Pythium sp., Curvularia lunata,
Aspergillus fumigatus showed negative results.

Table 3. Starch Hydrolysis test

S.No. Fungi Observation Inference
1. Aspergillus flavus +++ Abundant
2. Aspergillus niger +++ Abundant
3. Fusarium sp. ++ Average
4. Rhizopus sp. +++ Abundant

Amylase Production & Protein estimation

The culture showing maximum zone during screening were inoculated in the flask containing
fermentation media. The amount of protein in the crude enzymes extracted from fermented
flasks was determined by Lowry’s method.

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Table 4. Protein estimation by Lowry’s Method

28ºC 42ºC 60ºC
Name of the Protein Protein Protein
S.No. OD at OD at OD at
Species concentration concentration concentration
650 nm 650 nm 650 nm
(mg/ ml) (mg/ ml) (mg/ ml)
Aspergillus
1 0.126 0.15 0.224 0.21 0.014 0.02
flavus
Aspergillus
2 0.059 0.06 0.405 0.40 0.028 0.03
niger
3 Fusarium sp. 0.030 0.03 0.145 0.15 0.012 0.02
4 Rhizopus sp. 0.058 0.06 0.267 0.29 0.017 0.02

Enzyme assay at different temperature

The crude enzyme extracted from different fungal isolates was incubated at different
temperature 28ºC, 42ºC and 60ºC. The different fungal isolates from various soil samples
showed different enzyme activity.[1] The amylolytic activity at temperature 28ºC of
Aspergillus niger was 16.33 U/ml/min, Rhizopus sp. was 12 U/ml/min, Aspergillus flavus was
22.66 U/ml/min, and Fusarium sp. was 7 U/ml/min, at temperature 42ºC of Aspergillus niger
was 15.33 U/ml/min, Rhizopus sp. was 9.7 U/ml/min, Aspergillus flavus was 8.4 U/ml/min
and Fusarium sp. was 6 U/ml/min and at temperature 60°C of Aspergillus niger was 9.66
U/ml/min, Rhizopus sp. was 10.6 U/ml/min, Aspergillus flavus was 11.66 U/ml/min and
Fusarium sp. was 6 U/ml/min (Table 5). From this data, it was inferred that Aspergillus
flavus and Aspergillus niger were best amylase producers.

Table 5. Enzyme assay at different temperature

Enzyme activity (U/ml/min)
S.No. Name of organism
28ºC 42ºC 60ºC
1. Aspergillus flavus 22.66 8.4 11.66
2. Aspergillus niger 16.33 15.3 9.66
3. Rhizopus sp. 12 9.7 10.6
4. Fusarium sp. 7 6 6

From the above studies, the crude enzyme extracted from fungal isolates was used further for
thermostability studies. It was found that the highest activity was at 28ºC but there was
decrease in enzyme activity with increase in temperature as shown in Fig 3. This suggests
that the enzyme is more thermostable at 28ºC - 30ºC and can be used in various
biotechnological processes and industrial applications. This is in agreement with studies
reported earlier.[1]

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Fig 2. Enzyme Activity at different temperature

Fig 3. Thermo-stability studies

DISCUSSION
Amylase is a very significant enzyme, having biological, clinical, biochemical and industrial
importance. Several fungi are able to produce amylases. In this present work, a total of 26
fungi were isolated from soil samples of Jabalpur region. Similarly, Mishra et al [14] studied
the isolation and production of amylase and xylanase from soil fungi of Rajasthan.

In the present study, 10 different fungi Absidia sp. (7.69%), Aspergillus flavus (15.53%),
Aspergillus fumigatus (11.53%), Aspergillus niger (19.23%), Aspergillus sp. (7.69%),
Cladosporium sp. (3.84%), Curvularia lunata (7.69%), Fusarium sp. (12.53%), Pythium sp.

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(12.53%), Rhizopus sp. (11.53%) were isolated from soil samples. Similarly, Sohail et al [8]
studied the survey of amylolytic fungi and bacteria from native soil samples.

Morphological investigations using both macroscopic and microscopic features, have resulted
in the identification of ten fungal species: Absidia sp. Aspergillus flavus, Aspergillus
fumigatus, Aspergillus niger, Aspergillus sp., Cladosporium sp., Curvularia lunata, Fusarium
[11]
sp., Pythium sp., Rhizopus sp. Similarly, Ominyi et al isolated and identified amylase
producing fungi based on morphological characters.

In the present study, the isolated soil fungi were then screened for amylase production by
starch hydrolysis test. The four fungal isolates i.e. Aspergillus flavus, Aspergillus niger,
Fusarium sp. and Rhizopus sp. were found to be maximum amylase producers. The study of
effect of temperature was performed on the amylase activity of these fungal isolates. The
results showed that the enzyme production is greatly affected by temperature. The activity
varied for different isolate at different temperature. The inactivation at high temperature can
be possible because of amino acid destruction or hydrolysis of the peptide chain. The isolates
showed a decrease in enzyme activity when temperature range fell outside the mesophillic
range. This study of effect of temperature on enzyme activity showed that enzyme from
[16]
different fungal isolates have their optimum temperature. Similarly, Alva et al also
studied the production and characterization of enzyme isolated from Aspergillus sp. JGI 12 in
solid state cultures in which the enzyme was found to have highest stability at 25°C and least
stability at 60°C.

CONCLUSION
Soil appears to be an important biotope for searching and exploration of industrially
important enzymes. Amylases have potential application in the food fermentation, textile,
paper and pharmaceutical industries. Amylases have been most widely reported to occur in
microorganisms, although they are also found in plants and animals. Currently, they comprise
about 30% of the world enzyme production. Low yield of enzymes has always been a
problem in the commercial production of amylases. Moreover, thermal stability is a desirable
feature for economic viability of enzymatic processes. There are several processes in the
medical and clinical areas that involve the application of amylase. The present work deals
with the study of effect of temperature on the amylase production of different fungal isolates.
The main object of the work was to focus on the effect of temperature on amylase production
which includes isolation of the amylase producing fungi from soil, their characterization and

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crude enzyme assay. The probable genera with maximum amylase production were
Aspergillus niger and Aspergillus flavus. These fungal isolates showed varied enzyme
activity at different temperature range. These were then screened at different temperature
range for amylase production and it was found that the fungal isolates showed maximum
activity at 28°C and pH 6.5. These can be used further to study the effect of different cultural
parameters like pH, buffer, substrate concentration, nitrogen source, carbon source,
incubation temperature etc. The strain improvement studies and recombinant DNA
technology can be carried out further for the maximum amylase production and for industrial
applications.

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