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

1. 1963. Bergmayer, H. U.: Methods of Enzymatic Analysis. Verlag Chemie G.m.b.H., Weinheim/Bergstr. (Germany) and Academic Press, New York and London. 1963, XXIV, 1064 pages with 78 figures, 4 colored diagrams and 41 tables. Clothbound DM 120. ? = $ 30.00. Starch/Stärke. 15(7):272-272. https://doi.org/10.1002/star.19630150713