Title: Partial Purification of Alpha Amylase by Salt Precipitation Method
Aim: To partially purify alpha amylase enzyme using salt precipitation method.
Introduction:
Alpha amylase is an industrially important enzyme that hydrolyzes starch into smaller sugar units. Purification of this enzyme is crucial for its characterization and industrial applications. One of the commonly used methods for enzyme purification is salt precipitation, particularly using ammonium sulfate, which selectively precipitates proteins based on their solubility.
Theory:
Salt precipitation relies on the principle of differential solubility of proteins in high salt concentrations. Ammonium sulfate is commonly used as it stabilizes proteins and removes unwanted contaminants. As the salt concentration increases, the solubility of proteins decreases, leading to their precipitation. The precipitated enzyme can then be collected by centrifugation and re-dissolved in buffer for further analysis.
Principle:
At high salt concentrations, water molecules become less available for protein solvation, causing proteins to aggregate and precipitate. Different proteins precipitate at different salt concentrations, allowing for selective purification of the target enzyme.
Requirements:
Crude enzyme extract (alpha amylase from bacterial or fungal culture)
Ammonium sulfate (solid or saturated solution)
Magnetic stirrer or shaker
Centrifuge
Measuring cylinders
pH meter
Phosphate buffer (pH 7.0)
Ice bath
Spectrophotometer
DNS reagent for enzyme assay
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.
Filter the supernatant to remove any cell debris.
Place the crude extract in an ice bath.
Slowly add ammonium sulfate to achieve 40% saturation while stirring continuously.
Allow the mixture to stir for 30 minutes at 4°C.
Centrifuge at 10,000 rpm for 15 minutes at 4°C and discard the pellet.
To the supernatant, add more ammonium sulfate to achieve 80% saturation.
Stir the solution for another 30 minutes at 4°C.
Centrifuge again at 10,000 rpm for 15 minutes at 4°C and collect the precipitate.
Dissolve the precipitate in phosphate buffer (pH 7.0), and Dialyze the enzyme solution against the same buffer overnight to remove excess salt.
Perform a DNS assay to determine the enzyme activity before and after purification.
Measure absorbance at 540 nm using a spectrophotometer.
DNS Assay Method:
- 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:
The formation of a white precipitate indicates enzyme precipitation.
Increased specific activity of the enzyme after purification suggests successful removal of contaminants.
Result: The enzyme was partially purified using ammonium sulfate precipitation, with improved specific activity compared to the crude extract.
Conclusion: Partial purification of alpha-amylase by salt precipitation is an efficient and cost-effective method. This step enhances enzyme purity and stability, which is essential for industrial and research applications.