Enzyme Kinetics: Action of Salivary Amylase on Starch
What you will learn
Investigate the digestive enzymatic action of human salivary amylase (ptyalin) on starch and determine the achromic point (time required for complete hydrolysis) at 37°C.
Materials needed
- 1% Soluble Starch solution (freshly prepared) mLWhat is it?1% Soluble Starch solution (freshly prepared) mL₹50
Soluble Potato Starch (Amylum solubile) analytical reagent grade.
Specifications- Purity
- High purity soluble indicator starch
- Solubility
- Dissolves clear in boiling water
- Concentration
- 1.0% w/v freshly prepared
- Dilute Salivary Amylase solution (1:10 filtered human saliva in distilled water) mLWhat is it?Dilute Salivary Amylase solution (1:10 filtered human saliva in distilled water) mL₹95
White porcelain 12-cavity testing spot plate for sequential achromic point timing.
Specifications- Wells
- 12 numbered circular depressions
- Glaze
- Acid-proof white glazed ceramic
- Contrast
- Pure white background for iodine color transition
- Lugol's Dilute Iodine Solution dropper bottleWhat is it?Lugol's Dilute Iodine Solution dropper bottle₹140
Thermostatic 37°C laboratory biological water bath incubator.
Specifications- Temperature
- Pre-set to 37.0 °C physiological body temperature
- Stability
- ± 0.2 °C microprocessor control
- Porcelain Spotting Tile with multiple depression cavitiesWhat is it?Porcelain Spotting Tile with multiple depression cavities₹185
Standard laboratory apparatus used in Enzyme Kinetics: Action of Salivary Amylase on Starch.
Specifications- Category
- Biology Apparatus
- Grade
- CBSE / NEP 2020 Laboratory Standard
- Water Bath maintained at 37°C (human body temperature)What is it?Water Bath maintained at 37°C (human body temperature)₹230
Standard laboratory apparatus used in Enzyme Kinetics: Action of Salivary Amylase on Starch.
Specifications- Category
- Biology Apparatus
- Grade
- CBSE / NEP 2020 Laboratory Standard
- Digital Stopwatch and Pipettes (1 mL and 5 mL) setWhat is it?Digital Stopwatch and Pipettes (1 mL and 5 mL) set₹275
Standard laboratory apparatus used in Enzyme Kinetics: Action of Salivary Amylase on Starch.
Specifications- Category
- Biology Apparatus
- Grade
- CBSE / NEP 2020 Laboratory Standard
- Test tubes and test tube rack setWhat is it?Test tubes and test tube rack set₹320
Standard laboratory apparatus used in Enzyme Kinetics: Action of Salivary Amylase on Starch.
Specifications- Category
- Biology Apparatus
- Grade
- CBSE / NEP 2020 Laboratory Standard
Tick items as you collect them. Tap + to see the product, specifications and buy options.
Stay safe
- Maintain the water bath temperature strictly around 37°C (higher temperatures denature the amylase enzyme).
- Ensure starch solution is completely free from reducing sugars before the test.
- Rinse the sampling dropper between minute intervals to prevent early transfer of enzyme.
- Dilute saliva appropriately so the reaction takes between 5 to 10 minutes for measurable tracking.
Steps
- 1Rinse mouth with lukewarm water. Collect 2 mL saliva in a beaker and dilute with 18 mL distilled water to obtain 1:10 enzyme solution.Observation
Rinse mouth with warm water; collect 2 mL clean saliva in beaker; dilute with 18 mL distilled water to make 10% salivary enzyme solution.
SafetyOnly use student's own saliva; wear gloves and dispose of saliva tubes in disinfectant.
- 2Place 1 drop of dilute iodine solution in each depression well of the porcelain spotting tile.Observation
Prepare 1% soluble starch solution by dissolving 1 g starch in 100 mL boiling distilled water; cool to 37°C.
SafetyStir boiling starch solution continuously to prevent bottom scorching.
- 3In a test tube, take 5 mL of 1% starch solution and incubate in a 37°C water bath for 5 minutes.Observation
Place drops of dilute Lugol's iodine in all cavities of a white glazed porcelain spot plate.
SafetyArrange spot plate cavities in a numbered grid corresponding to 1-minute intervals.
- 4Add 1 mL of dilute salivary amylase solution to the starch tube, mix quickly, and start the stopwatch immediately.Observation
Mix 5 mL starch solution with 1 mL diluted saliva in 37°C water bath; start stopwatch immediately.
SafetyMaintain 37°C physiological temperature using water bath incubator.
- 5At 1-minute intervals, withdraw 1 drop from the reaction mixture using a clean dropper and add to an iodine depression well.Observation
Every 60 seconds, transfer 1 drop of reaction mixture into an iodine cavity; note progressive color shift.
SafetyColor progresses: Blue-Black -> Purple -> Red-Brown -> Yellow-Brown -> Yellow (Achromic Point).
- 6Observe the color progression: initially dark blue-black (starch present), fading to violet (erythrodextrin), brownish-yellow (achroodextrin), and finally no color change (maltose).Observation
Record achromic point at 8.0 minutes at 37°C; compare with boiled enzyme (remains blue-black permanently).
SafetyDecontaminate all glassware with 1% sodium hypochlorite bleach after practical.
- 7Record the exact time when no blue color develops; this is the achromic point (complete starch digestion).
Tap + on a step to see what you will observe and any safety tips.
Record your observations
| Time Interval (Minutes) | Tile Spot Color (Room Temp 37°C) | Tile Spot Color (Boiled Amylase 100°C) | Tile Spot Color (Ice Cold 0°C) | Starch Digestion State |
|---|---|---|---|---|
| 0 Minutes (Initial) | Deep Blue-Black | Deep Blue-Black | Deep Blue-Black | 100% Undigested starch present |
| 2 Minutes | Blue-Violet | Deep Blue-Black | Deep Blue-Black | Partial hydrolysis to amylodextrin |
| 4 Minutes | Reddish-Brown (Erythrodextrin) | Deep Blue-Black | Deep Blue-Black | Erythrodextrins formed |
| 6 Minutes | Yellowish-Brown (Achroodextrin) | Deep Blue-Black | Deep Blue-Black | Achroodextrins formed |
| 8 Minutes (Achromic Point) | Pale Yellow (Iodine color unchanged) | Deep Blue-Black | Blue-Violet | Complete hydrolysis to Maltose; Achromic Point |
| 10 Minutes | Pale Yellow (No starch) | Deep Blue-Black (Permanent) | Blue-Violet | Enzyme fully active at 37°C; denatured at 100°C |
Tap a highlighted cell to write your own observation.
Result and conclusion
Blue-black iodine response gradually lightens through violet and red-brown to reach the achromic point (colorless) at 6.5 minutes at body temperature (37°C), proving complete enzyme breakdown to maltose.
| Observation | What it means |
|---|---|
| Enzymatic Starch Breakdown | Salivary α-amylase hydrolyzes α(1->4) glycosidic bonds in starch, degrading it step-wise into maltose disaccharides. |
| Achromic Point Concept | The time taken (8.0 min at 37°C) for starch to stop giving blue/violet/brown iodine color is termed the achromic point. |
| Thermal Denaturation at 100°C | Boiling salivary amylase permanently denatures its tertiary protein structure, completely abolishing catalytic activity. |
| Kinetic Inactivation at 0°C | At 0°C ice temperature, enzyme is kinetically dormant due to low molecular kinetic energy, delaying the achromic point beyond 40 mins. |
Sample readings. Replace with your own results.