Title: Characterization of Alpha Amylase and Invertase Using Polyacrylamide Gel Electrophoresis (PAGE)
Aim: To characterize alpha amylase and invertase enzymes using Polyacrylamide Gel Electrophoresis (PAGE) for assessing their molecular weight and purity.
Introduction:
Alpha amylase and invertase are industrially significant enzymes involved in starch and sucrose hydrolysis, respectively. Their characterization using PAGE helps determine their molecular weight, purity, and electrophoretic mobility. This technique is essential for enzyme quality control, purification assessment, and structural studies.
Theory:
Polyacrylamide Gel Electrophoresis (PAGE) is a technique used to separate proteins based on their size and charge. Native PAGE maintains the enzyme’s activity and structure, whereas SDS-PAGE denatures proteins, allowing for molecular weight determination. Staining techniques like Coomassie Brilliant Blue (CBB) or specific enzyme activity staining can be used for visualization.
Principle:
Proteins migrate through a polyacrylamide gel matrix under an electric field based on their size and charge. In native PAGE, enzymes retain their structure and activity, enabling in-gel activity assays. In SDS-PAGE, proteins are denatured and separated based on molecular weight.
Requirements:
Alpha amylase and invertase enzyme samples
Polyacrylamide Gel Electrophoresis (PAGE) apparatus
Tris-glycine buffer (pH 8.3)
Acrylamide and bisacrylamide solution
Ammonium persulfate (APS) and TEMED
SDS (for SDS-PAGE)
Sample loading dye (with or without SDS)
Molecular weight markers
Coomassie Brilliant Blue staining solution
Gel destaining solution (methanol-acetic acid-water)
Starch and sucrose for enzyme activity staining
Lugol’s iodine (for starch staining)
Nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS) for invertase staining
Electrophoresis power supply
Gel documentation system
Procedure:
Preparation of Polyacrylamide Gel: a. Prepare resolving gel (10-12% acrylamide for SDS-PAGE, 8% for native PAGE) and pour into the gel casting apparatus. b. Allow the gel to polymerize, then overlay with stacking gel (5% acrylamide) and insert combs. c. After polymerization, remove the comb and set the gel in the electrophoresis chamber with Tris-glycine buffer.
Sample Preparation: a. For SDS-PAGE: Mix enzyme samples with SDS loading dye and boil for 5 minutes. b. For Native PAGE: Mix samples with non-denaturing loading buffer. c. Load molecular weight markers in one well.
Electrophoresis Run: a. Load 10-20 µL of enzyme samples into wells. b. Run the gel at 100-150V until the dye front reaches the bottom.
Protein Staining and Visualization: a. Stain the gel with Coomassie Brilliant Blue for 1 hour and destain with methanol-acetic acid-water solution. b. Observe bands under a gel documentation system.
Enzyme Activity Staining (Optional): a. For alpha amylase: Incubate the gel in 1% starch solution, wash, and stain with Lugol’s iodine. Clear zones indicate enzyme activity. b. For invertase: Incubate the gel in sucrose solution with NBT and PMS. Dark bands indicate enzyme activity.
Observation:
Distinct protein bands indicate enzyme presence and purity.
Clear zones in activity staining confirm enzyme functionality.
Band intensity and mobility help estimate molecular weight and homogeneity.
Result: The characterization of alpha amylase and invertase was performed using PAGE, confirming their molecular weight, purity, and activity.
Conclusion: Polyacrylamide Gel Electrophoresis effectively characterizes alpha amylase and invertase, providing insights into their purity and molecular properties. The study aids in enzyme quality control and further biochemical analysis.