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Title: Detection of Enzyme Production by Bacteria & Fungi – Alpha-Amylase

Aim: To detect the production of alpha-amylase enzyme by bacteria and fungi using the starch agar plate method.

Introduction:

Enzymes are biological catalysts produced by microorganisms playing a crucial role in various industrial and biochemical processes. Alpha-amylase is an extracellular enzyme that breaks down starch into smaller sugar molecules such as maltose and glucose. Microorganisms such as Bacillus subtilis (bacteria) and Aspergillus oryzae (fungi) are prolific producers of alpha-amylase. The detection of alpha-amylase production by bacteria and fungi is important in fields such as biotechnology, food processing, and fermentation industries.

Theory:

Alpha-amylase breaks the α-1,4 glycosidic bonds in starch, yielding dextrins, maltose, and glucose. Microorganisms capable of producing this enzyme secrete it into the surrounding medium, breaking down the starch. The presence of alpha-amylase can be detected using iodine staining, as iodine forms a blue-black complex with starch. If the starch is hydrolyzed, a clear zone appears around the microbial growth, indicating enzyme activity.

Principle:

When iodine solution is added to a starch-containing medium, it forms a dark blue-black complex. If alpha-amylase is present, it hydrolyzes starch, preventing the formation of the complex and creating a clear halo around the microbial colony. The size of the clear zone is proportional to the enzyme activity.

Requirements:

1. Microbial cultures (bacterial and fungal strains)
2. Starch agar plates (Nutrient agar/Sabouraud’s agar + 1% starch)
3. Inoculation loop
4. Sterile cotton swabs
5. Incubator (35–37°C for bacteria, 25–28°C for fungi)
6. Iodine solution (Gram’s iodine or Lugol’s iodine)
7. Petri plates
8. Sterile water

Procedure:

1. Prepare nutrient agar for bacterial cultures or Sabouraud’s agar for fungal cultures.
2. Add 1% soluble starch to the medium.
3. Sterilize the medium by autoclaving at 121°C for 15 minutes.
4. Pour the medium into sterile Petri plates and allow it to solidify.
5. Using a sterile inoculation loop, pick a bacterial or fungal colony and streak it onto the starch agar plate.
6. Alternatively, use a sterile cotton swab to evenly spread liquid culture over the plate surface, and Label the plates properly.
7. Incubate bacterial plates at 35–37°C for 24–48 hours, and fungal plates at 25–28°C for 3–5 days.
8. After incubation, flood the starch agar plates with iodine solution.
9. Allow the iodine to react for 2–3 minutes.
10. Observe the plates for the formation of a clear zone around the microbial growth.

Observation:

• A clear zone around the bacterial or fungal colony indicates the hydrolysis of starch due to alpha-amylase production.
• No clear zone suggests the absence of enzyme production.
• The intensity and size of the clear zone correlate with the enzyme activity level.

Fig: Colony showing zone of clearance

Result:

The presence of a clear zone around microbial growth confirms the production of alpha-amylase by the tested bacterial or fungal strain.

Conclusion:

The starch agar plate method effectively detects alpha-amylase production by microorganisms. The presence of a clear zone after iodine staining confirms enzymatic starch hydrolysis. This experiment demonstrates the industrial importance of amylase-producing microorganisms in biotechnology applications such as food processing, biofuel production, and pharmaceutical industries.