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 (Catalog No. S8625)
Starch from potato (Catalog No. S2004)
6.1 N Trichloroacetic acid solution [~100% (w/v), Catalog No. T0699]
Glucose Assay Reagent (Catalog No. G3293)
Sodium bicarbonate (Catalog No. 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 (Catalog No. 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 (Catalog No. 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), Catalog No. T0699] in ultrapure water.

Glucose Assay Reagent (HK) – Immediately before use, dissolve the contents of one vial of Glucose Assay Reagent (Catalog No. 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 (Catalog No. S8875).

5. Filter through a 0.1 µM PVDF syringe filter (Catalog No. 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