Screening of Endophytic Bacteria Isolated from Selected Plants in Hawassa for the Production and Characterization of Amylase
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Date
2024-06
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Addis Ababa University
Abstract
Amylases are crucial enzymes in the global industry, constituting 25% of enzyme production. Endophytes, known for producing bioactive compounds and enzymes, offer promise for industrial applications. The aim of the study was to screen amylase producing endophytic bacteria, to produce amylase under controlled laboratory conditions and characterize the enzyme. Purposive sampling was used to select the study areas as well as the plants screened for amylase
producing endophytic bacteria. After collection the plant samples were washed and surface sterilized before isolation of the endophytic bacteria. The endophytic bacteria were isolated then screened for amylase production on starch agar media and purified. The pure isolates were characterized using morphological and biochemical test. Amylase was produced through submerged fermentation, and its production was optimized using different carbon and nitrogen sources, pH and temperatures. Ammonium sulphate precipitation and dialysis were used for partial purification and the partially purified enzyme was assayed for its activity using the dinitrosalicylic acid (DNS) method. Out of 60 endophytic bacteria isolated from the plants, 32 demonstrated amylase production. From these, 16 pure isolates were found to be the most efficient amylase producers and 7 best amylase producing isolates based on submerged fermentation. Among these top 16 producers, 12 were Gram-positive bacteria and 4 were Gramnegative bacteria.The 7 isolates were identified to species level by rRNA gene sequence analysis.
Then 3 isolates were selected for further purification and characterization. Diameter (Mean ± SD) of clear zone on starch agar ranged from 4.48 ± 0.54 for isolate I10L2 from Enset to 11.53 ± 0.34 for isolate D10F2 from Datura. The highest (p < 0.05) amylase production was recorded for isolate D10L2 when glucose or starch was used as carbon source. The highest amylase production was recorded for all isolates in the presence of ammonium sulfate and the lowest in the presence of tryptone as nitrogen source. The optimum temperature for production of amylase was 30 OC, but differences in the optimum pH ranging from pH 5 to pH 8 were recorded based on the isolates preferences. After partial purification the highest enzyme activity was recorded at pH 7 and pH 9, 50 OC, and 0.5% and 1% calcium ion (Ca 2+) concentrations. The maximum activity in the presence of 1mM Ca2+ was about 16.5 U/mL. These findings highlight the potential of endophytic bacteria for amylase production. With further optimization and scale up, the amylase can be produced in large quantities and for specific applications.
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Keywords
Amylase, Endophyte, Enzyme Activity, Starch, Submerged Fermentation