Title: Production of Bacterial or Fungal Alpha Amylase
Aim: To study the production of alpha amylase by bacterial or fungal cultures through fermentation.
Introduction:
Alpha amylase is an important hydrolytic enzyme that breaks down starch into simpler sugars. It is widely used in industries such as food, textile, and pharmaceuticals. Bacteria (e.g., Bacillus subtilis) and fungi (e.g., Aspergillus niger) are common microbial sources of alpha amylase, and their production is optimized through fermentation techniques.
Theory:
The production of alpha amylase by microorganisms involves fermentation, where the microbes utilize starch or other carbon sources as substrates. The enzyme is secreted into the medium and hydrolyzes starch into maltose and glucose. The efficiency of production depends on factors such as pH, temperature, aeration, and substrate concentration.
Principle: Microbial fermentation is carried out in a nutrient-rich medium that supports the growth of the organism and stimulates enzyme production. The enzyme activity can be detected by iodine staining or quantified using a spectrophotometric assay.
Requirements:
Bacterial culture (Bacillus subtilis) or fungal culture (Aspergillus niger)
Starch-containing fermentation medium
Inoculation loop
Conical flasks
Shaker/incubator (30-37°C)
Autoclave
Spectrophotometer
DNS reagent (for enzyme assay)
Iodine solution (for qualitative starch hydrolysis test)
Procedure:
Prepare a starch-containing medium (e.g., nutrient broth with 1% starch).
Sterilize the medium by autoclaving at 121°C for 15 minutes.
Inoculate the sterilized medium with a loopful of bacterial or fungal culture. Incubate at 30-37°C for 48-72 hours under shaking conditions.
After incubation, centrifuge the culture broth at 10,000 rpm for 10 minutes. Collect the supernatant, which contains the extracellular alpha amylase.
Qualitative Analysis (Iodine Test):
- a. Take 1 mL of culture supernatant and mix with 1 mL of 1% starch solution.
- b. Incubate at 37°C for 10 minutes.
- c. Add iodine solution and observe color change. A clear zone indicates starch hydrolysis.
- a. Mix 1 mL of enzyme extract with 1 mL of 1% starch solution.
- b. Incubate at 37°C for 10 minutes.
- c. Add 1 mL of DNS reagent and boil for 5 minutes.
- d. Cool the tubes and measure absorbance at 540 nm using a spectrophotometer.
Observation:
A clear zone around colonies in the iodine test confirms starch hydrolysis.
Increased absorbance at 540 nm indicates higher enzyme activity.
Result: The bacterial or fungal strain produces alpha amylase, which hydrolyzes starch efficiently. The enzyme activity can be quantified using spectrophotometry.
Conclusion: Alpha amylase production by microorganisms is a key process in biotechnology. Optimization of growth conditions can enhance enzyme yield for industrial applications.
