The document discusses the isolation and characterization of amylase enzyme from fungal strains. It describes isolating fungal strains from soil samples, screening them for amylase production, producing amylase from the best isolate, and characterizing the enzyme. The characterization included determining the effect of pH, temperature, carbon sources and nitrogen sources on amylase activity. The highest amylase activity was observed at pH 5.0 and between 40-60°C. Different carbon and nitrogen sources also affected amylase production levels.

1. Isolation and Characterization of Amylase Enzyme from
Fungal Strains
Persented by:
Ankit Kushwaha (03)
(Prof.) Dr. Vivek Kumar
Srivastava
Head of Department
Department of Biotechnology
RBS ENGINEERING
TECHNICAL
CAMPUS BICHPURI, AGRA

2. TABLE OF CONTENTS
 Introduction
 Materials and Methods
o Sample collection
o Isolation of fungi from soil sample
o Screening of fungal isolates for amylase production
o Inoculums preparation
o Production of amylase from isolate
o Amylase assay
 Result
 Characterization of Enzyme
o Effect of pH on activity of amylase
o Effect of temperature on activity of amylase
o Effect of Carbon sources on amylase production
o Effect of Nitrogen sources on amylase production
 References

3.  Amylases are important hydrolases enzyme which have been widely used since many decades.
 These enzymes randomly cleave internal glycosidic linkages in starch molecules.
 Amylases are the great significance in Biotechnological application ranging from food, fermentation,
detergents, pharmaceutical, etc.
 Because of the importance of amylases, isolation of new microorganisms suitable for amylase production
could provide potential new sources of the enzyme.
 In present day, biotechnology amylase accounts approximately for 25% of the enzyme market.
 There are two types of amylase enzyme

4. •The production of amylase is dependent on the strains, methods of cultivation, cell growth, nutrient
requirement, metal ions, pH, temperature, time of incubation and thermotability.
•Many 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.

5. Materials and Methods
Sample collection
Soil samples were collected in a sterile polyethylene container
The sampling technique were selected randomly from the parking, Boys hostel, Girls Hostel, Playground, and
brought to the laboratory for further processing.

6. Isolation of fungi from soil sample
•Fungal colonies were isolated by diluting 1gm of soil samples in 10 ml of sterile distilled water by serial dilution
method.
•Potato dextrose agar (PDA) (Potato 200 gm, Dextrose 20 gm, Agar 20 gm, Distilled water 1000 ml) media was
prepared
•autoclaved and poured in sterile Petri plates
•The inoculated plates were incubated at ambient temperature for 120 hours (5 days).

7.  Screening of fungal isolates for amylase production
Select the best amylase producing strains.
Isolates were grown in starch agar media comprising the following, in gm/L; yeast extract 1.5, peptone 0.5,
sodium chloride 1.5, starch 10, agar 15, and make the media of the pH similar with pH of soil sample.
Then the plates were incubated at room temperature for 120 hours,

8. Inoculums preparation
Inoculums were prepared by adding sterilized distilled water into a sporulated ( 5 days ) old PDA slant culture.
An inoculums needle was used to displace the spore clusters under sterilized conditions.
then it was shaken thoroughly to prepare homogenized spore suspension.

9. Production of amylase from isolate
Production medium contained
( (g/l) NaNO3- 1.0g; MgSO4.7H2O- 0.5 g; FeSO4-0.01g; soluble starch 20.0 g. 100 ml of medium was taken
in a 250 ml conical flask ),
The flasks were sterilized in autoclave at 121C for 15 min. ,
after cooling the flask was inoculated with fungal cultures,
The inoculated medium was incubated at 30C in shaker incubator,
At the end of the fermentation period,
the culture medium was centrifuged at 5000 rpm for 15 min to obtain the crude extract.

11. Amylase assay
Amylase activity was determined as described by Miller (1959).
The absorbance was measured at 540 nm by spectrophotometer
The concentration of the enzyme produced, and kinetics were evaluated against the
standard amylase enzyme,

12. Result
 The totals of 5 fungal isolates were isolated in the first phase of screening based on colony morphology and
microscopic mount of the isolates. From the total of 5 fungal isolates, 1 isolate with relatively higher clear zones
formation by starch hydrolysis were selected in the second phase of screening and further studied the isolates
belonged to the genera Aspergillus Niger, and the isolate with maximum amylase production

13. Characterization of Enzyme
Effect of pH on activity of amylase
In this study as the pH value increase amylase activity also increases and its highest activity was
observed at pH 5.0
 The maximum amylase activity was found at pH 5
Citation: Negussie F Bussa, Mesfin Moges, Manikandan Muthuswamy, Melese Abdisa. Isolation and Characterization of Amylase Enzyme from Selected Fungal Strains of Wof Washa Forest of
North Shoa, Ethiopia. Sci J Biol & Life Sci. 1(2): 2019. SJBLS.MS.ID.000506. DOI: 10.33552/SJBLS.2019.01.000506

14. Effect of temperature on activity of amylase
As temperature increases amylase activity also increase at a certain temperature.
The highest relative activity of amylase was observed in a range of 40-60°C and reached its maximum activity
at 50°C.
Citation: Negussie F Bussa, Mesfin Moges, Manikandan Muthuswamy, Melese Abdisa. Isolation and Characterization of Amylase Enzyme from Selected Fungal Strains of Wof
Washa Forest of North Shoa, Ethiopia. Sci J Biol & Life Sci. 1(2): 2019. SJBLS.MS.ID.000506. DOI: 10.33552/SJBLS.2019.01.000506

15. Effect of Carbon sources on amylase production
The nature and amount of carbon source in culture media is important energetic source for the production of
extracellular amylase from fungal isolate. Therefore, production of amylase was tested by growing in SSF
containing different carbon source as a substrate.
Citation: Negussie F Bussa, Mesfin Moges, Manikandan Muthuswamy, Melese Abdisa. Isolation and Characterization of Amylase Enzyme from Selected Fungal Strains of Wof Washa
Forest of North Shoa, Ethiopia. Sci J Biol & Life Sci. 1(2): 2019. SJBLS.MS.ID.000506. DOI: 10.33552/SJBLS.2019.01.000506

16. Effect of Nitrogen sources on amylase production
Nitrogen is the secondary energy sources and plays an important role in the growth of the organism and enzyme
production. The nature of the compound and the concentration that we used might stimulate or down reduce the
production of enzymes. In this study the effect of supplementary organic nitrogen sources (Peptone, Tryptone, Urea) and
inorganic nitrogen sources (NH4 )2 SO4 , NH4 NO3 , NH4 Cl) on the production of amylase by fungal strain using SSF
was determined.
Citation: Negussie F Bussa, Mesfin Moges, Manikandan Muthuswamy, Melese Abdisa. Isolation and Characterization of Amylase Enzyme from Selected Fungal Strains of Wof
Washa Forest of North Shoa, Ethiopia. Sci J Biol & Life Sci. 1(2): 2019. SJBLS.MS.ID.000506. DOI: 10.33552/SJBLS.2019.01.000506

17. References
ISSN: 2694-166X DOI: 10.33552/SJBLS.2019.01.000506 Scientific Journal of Biology
& Life Sciences Research Article Copyright © All rights are reserved by Negussie
International Journal of Biotechnology and Food Science Vol. 2(5), pp. 88-93, June
2014 ISSN: 2384-7344 Research Paper
Vol. 9(2) pp. 7-14, March 2018 DOI: 10.5897/IJBMBR2018.0289 Article Number:
9DE922D56542 ISSN 2141-2154 Copyright © 2018 Author(s) retain the copyright of this
article http:// www.academicjournals.org/IJBMBR International Journal of Biotechnology
and Molecular Biology Research.
 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706
Volume 6 Number 4 (2017) pp. 783-788 Journal homepage: http://www.ijcmas.com
Negussie F Bussa, Mesfin Moges, Manikandan Muthuswamy, Melese Abdisa. Isolation
and Characterization of Amylase Enzyme from Selected Fungal Strains of Wof Washa
Forest of North Shoa, Ethiopia. Sci J Biol & Life Sci. 1(2): 2019. SJBLS.MS.ID.000506.
DOI: 10.33552/SJBLS.2019.01.000506