Enzymatic Assay of Amyloglucosidase (EC 3.2.1.3)
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1. OBJECTIVE
To standardize a procedure for the enzymatic assay of Amyloglucosidase at Sigma-Aldrich St. Louis.
2. SCOPE
This procedure applies to all products that have a specification for Amyloglucosidase activity using starch as a substrate.
3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
3.2. Unit Definition – One unit will liberate 1.0 mg of glucose from starch in 3 min at pH 4.5 at 55°C.
3.3. ADP – Adenosine Diphosphate
3.4. ATP – Adenosine Triphosphate
3.5. G-6-PDH – Glucose-6-Phosphate Dehydrogenase
3.6. β-NADP – β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized form
3.7. β-NADPH – β-Nicotinamide Adenine Dinucleotide Phosphate, Reduced form
3.8. 6-PG – 6-Phospho-D-Gluconate
4. DISCUSSION
Starch + H2O Amyloglucosidase > D - Glucose
D - Glucose + ATP Hexokinase > Glucose 6 - Phosphate + ADP
Glucose 6 - Phosphate + β - NADP G-G-PDH > 6 - PG + β - NADPH
5. RESPONSIBILITIES
It is the responsibility of all Analytical Services personnel to follow this protocol as written.
6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
7.1 CONDITIONS:
T = 55°C, pH = 4.5, A340nm, Light path = 1 cm
7.2 METHOD:
Spectrophotometric Stop Rate Determination
7.3 REAGENTS:
7.3.1 50 mM Sodium Acetate, pH 4.5 at 55ºC (Buffer)
Prepare a 136 mg/ml solution in purified water using Potassium Phosphate, Monobasic, Anhydrous, Sigma-Aldrich Product Number S8625. Adjust to pH 4.5 at 55ºC with 1 M HCl.
7.3.2 1% (w/v) Starch Solution (Starch)
Prepare in Reagent 7.3.1 (Buffer) using Starch, Potato, Soluble, Sigma-Aldrich Product Number Sigma-Aldrich Product Number S2004. Facilitate solubilization by heating for approximately 15 minutes at 60ºC – 80ºC. Do not boil.
7.3.3 Amyloglucosidase Enzyme Solution (Amylogluc)
Immediately before use, prepare a solution at 0.40 to 0.80 mgs solid / ml in cold purified water. Then immediately dilute to 0.3 to 0.6 units / ml of Amyloglucosidase in 55ºC purified water. The final reaction mixtures must contain 0.15 to 0.60 units of Amyloglucosidase.
7.3.4 50% (w/v) Trichloroacetic Acid Solution (TCA)
Prepare in purified water using Trichloroacetic Acid, 6.1 N Solution, approximately 100% (w/v), Sigma-Aldrich Product Number T0699
7.3.5. Glucose (HK) Assay Reagent (HK)
Immediately before use, dissolve the contents of one vial of Glucose (HK) Assay Reagent, Sigma-Aldrich Product Number G3293, per volume on label.
7.4 TEST METHOD
7.4.1 STEP 1 - Enzymatic Stop Reaction (Note that the sample and control should be ran separate due to the short incubation time)
7.4.1.1 Pipette (in milliliters) the following Reagents into suitable containers:
| Test1 | Test2 | Test3 | Blank | |
| Reagent 7.3.2 (Starch) | 1.00 | 1.00 | 1.00 | 1.00 |
7.4.1.2 Equilibrate to 55ºC. Then add:
| Reagent 7.3.3 (Amylogluc) | 0.50 | 0.70 | 1.00 | ---- |
7.4.1.3 Immediately mix by swirling and incubate at 55ºC for exactly 3 minutes. Then add:
| Reagent 7.3.4 (TCA) | 0.30 | 0.30 | 0.30 | 0.30 |
| Reagent 7.3.3 (Amylogluc) | 0.50 | 0.30 | ---- | 1.00 |
7.4.1.4 Mix by swirling and adjust to pH 7.0 with solid Sodium Bicarbonate, Sigma-Aldrich Product Number S8875 .
7.4.1.5 Filter through a 0.1 μm acrodisc syringe filter, Sigma-Aldrich Product Number F7523 and use filtrate in 7.4.2 (Step 2).
7.4.2 Step 2 – Enzymatic Activity Determination
7.4.2.1. Pipette (in milliliters) the following reagents into suitable cuvettes:
| Test 1 | Test 2 | Test 3 | Blank | |
| Reagent 7.3.5 (HK) | 2.80 | 2.80 | 2.80 | 2.80 |
7.4.2.2. Equilibrate to 25ºC. Monitor the A340nm until constant, using a suitably thermostatted spectrophotometer.
7.4.2.3. Record the initial A340nm for both the test and the blank.
7.4.2.4. Then add:
| Reagent 7.4.1.5 (Test) | 0.20 | 0.20 | 0.20 | ---- |
| Reagent 7.4.1.5 (Blank) | ---- | ---- | ---- | 0.20 |
7.4.2.5. Immediately mix by inversion. Monitor the A340nm until the ΔA340nm/min is less than 0.0020, and this rate is maintained for at least 5 minutes.
7.4.2.6. Record the final A340nm for both the test and the blank.
7.5 CALCULATIONS
7.5.1 ΔA340nm = A340nm Final - A340nm Initial
| 7.5.2 | Units/ml enzyme = | (ΔA340nm Test - ΔA340nm Blank) (180) (2.3) (3.0)(df) |
| (6.22)(1000)(1.00)(0.20) |
where:
180 = Micrograms of glucose per micromole of glucose
2.3 = Total volume (in milliliters) of 7.4.1 (Step 1)
3.0 = Total volume (in milliliters) of 7.4.2 (Step 2)
df = Dilution factor
6.22 = Millimolar extinction coefficient of β-NADPH at 340 nm
1000 = Conversion factor from micrograms to milligrams
1.00 = Volume (in milliliters) of enzyme used in 7.4.1 (Step 1)
0.20 = Volume (in milliliters) from 7.4.1 (Step 1) used in 7.4.2 (Step 2)
7.6 FINAL ASSAY CONTENTRATION:
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 units amyloglucosidase.
8.1 Bergmeyer, H. U., Gawehn K,, and Grassl, M. (1974) Methods of Enzymatic Analysis (Bergmeyer, H.U. ed.) Second Edition, Volume I, 434-435.
8.2 Replaces PROC-DEK-OP-003068 (v1).
9. APPROVAL
Review, approvals and signatures for this document will be generated electronically using the EDMS. Print a “For Use” copy if hardcopy with signature verification is required.
