Enzymatic Assay of Amyloglucosidase
(EC 3.2.1.3)
Description
This procedure may be used for the determination of Amyloglucosidase activity using starch as the substrate. The Spectrophotometric Stop Rate Determination [Absorbance at 340 nm (A340), Light path = 1 cm] is based on the following reactions:
Amyloglucosidase
Starch + water ––––––––––––––––> D-Glucose
Hexokinase
D-Glucose + ATP ––––––––––> Glucose-6-Phosphate + ADP
G-6-PDH
Glucose-6-Phosphate + β-NADP ––––––––––> 6-PG + β-NADPH
ADP – Adenosine Diphosphate
ATP – Adenosine Triphosphate
G-6-PDH – Glucose-6-Phosphate Dehydrogenase
β-NADP – β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized form
β-NADPH – β-Nicotinamide Adenine Dinucleotide Phosphate, Reduced form
6-PG – 6-Phospho-D-Gluconate
Unit Definition – One unit of Amyloglucosidase will liberate 1.0 mg of glucose from starch in 3 minutes at pH 4.5 at 55 °C.
Precautions
Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
Reagents and Equipment Required
Sodium acetate, trihydrate (S8625)
Starch from potato (S2004)
6.1 N Trichloroacetic acid solution ~100% (w/v) (T0699]
Glucose Assay Reagent (G3293)
Sodium bicarbonate (S8875)
Preparation Instructions
Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.
Buffer (50 mM Sodium Acetate, pH 4.5 at 55 °C) – Prepare a 6.8 mg/mL solution in ultrapure water using Sodium acetate, trihydrate (S8625). Adjust to pH 4.5 at 55 °C with 1 M HCl.
Starch Solution [1% (w/v)] – Prepare a 10 mg/mL solution in Buffer using soluble Starch from potato (S2004). Facilitate solubilization by heating for ~15 minutes at 60–80 °C. Do not boil. Allow the solution to slowly mix throughout the assay.
Enzyme Solution (Amyloglucosidase) – Immediately before use, prepare a solution (0.40–0.80 mg solid/mL) in cold ultrapure water. Then immediately dilute to 0.3–0.6 units/mL of Amyloglucosidase in 55 °C purified water. The final reaction mixtures must contain 0.15–0.60 units of Amyloglucosidase.
TCA Solution [(50% (w/v) Trichloroacetic Acid Solution] – Prepare a 2-fold dilution of 6.1 N Trichloroacetic acid solution ~100% (w/v) (T0699] in ultrapure water.
Glucose Assay Reagent (HK) – Immediately before use, dissolve the contents of one vial of Glucose Assay Reagent (G3293) with ultrapure water using volume indicated by package size on label.
Procedure
In a 2.00 mL reaction mix, the final concentrations are 25 mM sodium acetate, 0.5% (w/v) starch and 0.15–0.60 unit of amyloglucosidase.
Enzymatic Stop Reaction
1. Pipette the following reagent into suitable containers:
| Reagent | Test 1 (mL) | Test 2 (mL) | Test 3 (mL) | Blank (mL) |
|---|---|---|---|---|
| Starch Solution | 1.00 | 1.00 | 1.00 | 1.00 |
2. Equilibrate to 55 °C. Then add:
| Reagent | Test 1 (mL) | Test 2 (mL) | Test 3 (mL) | Blank (mL) |
|---|---|---|---|---|
| Enzyme Solution | 0.50 | 0.70 | 1.00 | – |
3. Immediately mix by swirling and incubate at 55 °C for exactly 3 minutes. Then add:
| Reagent | Test 1 (mL) | Test 2 (mL) | Test 3 (mL) | Blank (mL) |
|---|---|---|---|---|
| TCA Solution | 0.30 | 0.30 | 0.30 | 0.30 |
| Enzyme Solution | 0.50 | 0.30 | – | 1.00 |
4. Mix by swirling and adjust to pH 7.0 with solid Sodium bicarbonate (S8875).
5. Filter through a 0.1 µM PVDF syringe filter (F7523) and use Filtrate in Enzymatic Activity Determination, step 4.
Enzymatic Activity Determination
1. Pipette the following reagent into suitable cuvettes:
| Reagent | Test 1 (mL) | Test 2 (mL) | Test 3 (mL) | Blank (mL) |
|---|---|---|---|---|
| Glucose Assay Reagent | 2.80 | 2.80 | 2.80 | 2.80 |
2. Equilibrate to 25 °C. Monitor the A340 until constant, using a suitably thermostatted spectrophotometer.
3. Record the initial A340 for both the Tests and the Blank.
4. Then add:
| Reagent | Test 1 (mL) | Test 2 (mL) | Test 3 (mL) | Blank (mL) |
|---|---|---|---|---|
| Appropriate Test Filtrate, Enzymatic Stop Reaction, step 5 | 0.20 | 0.20 | 0.20 | – |
| Blank Filtrate, Enzymatic Stop Reaction, step 5 | – | – | – | 0.20 |
5. Immediately mix by inversion. Monitor the A340 until the DA340/min is <0.0020 and this rate is maintained for at least 5 minutes.
6. Record the final A340 for both the Tests and the Blank.
Results
Calculations
1. ΔA340 = A340 Final – A340 Initial
2.
| Units/mL enzyme = | (ΔA340 Test – ΔA340 Blank) (180) (2.3) (3.0) (df) |
| (6.22) (1,000) (mL Enzyme) (0.20) |
where:
180 = Micrograms of glucose per micromole of glucose
2.3 = Total volume (in milliliters) of Enzymatic Stop Reaction, steps 1–5
3.0 = Total volume (in milliliters) of Enzymatic Activity Determination, steps 1–6
df = Dilution factor
6.22 = Millimolar extinction coefficient of β-NADPH at 340 nm
1,000 = Conversion factor from micrograms to milligrams
ml Enzyme = Volume of Enzyme Solution added in Enzymatic Stop Reaction, step 2
0.20 = Volume (in milliliters) from Enzymatic Stop Reaction used in Enzymatic Activity Determination
3.
| Units/mg solid = | units/mL enzyme |
| mg solid/mL enzyme |
03/14-1
References
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