Amylase, Alpha AssayMethod: That of (1951) wherein the reducing groups released from starch are measured by the reduction of 3,5-dinitrosalicylic acid. One unit releases from soluble starch one micromole of reducing groups (calculated as maltose) per minute at 25°C and pH 6.9 under the specified conditions.Reagents

0.02 M Sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride

2 N Sodium hydroxide

Dinitrosalicylic acid color reagent. Prepare by dissolving 1.0 gm of 3,5-dinitrosalicylic acid in 50 ml of reagent grade water. Add slowly 30.0 gms sodium potassium tartrate tetrahydrate. Add 20 ml of 2 N NaOH. Dilute to a final volume of 100 ml with reagent grade water. Protect from carbon dioxide and store no longer than 2 weeks.

1% Starch. Prepare by dissolving 1.0 gm soluble starch, (Merck) in 100 ml 0.02 M sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride. Bring to a gentle boil to dissolve. Cool and bring volume to 100 ml, with water, if necessary. Incubate at 25°C for 4-5 minutes prior to assay.

Maltose Stock Solution. Prepare by dissolving 180 mg maltose (MW 360.3) in 100 ml reagent grade water in a volumetric flask.

Enzyme

Dilute to a concentration of 1-10 micrograms/ml. A minimum of three different concentrations in this range should be run.

Procedure

Adjust spectrophotometer to 540 nm and 25°C.

Using the maltose stock solution prepare a maltose standard curve as follows: In numbered tubes, prepare 10 maltose dilutions ranging from 0.3 to 5.0 micromoles per ml. Include two blank tubes with reagent grade water only. Into a series of corresponding numbered tubes pipette 1 ml of each dilution of maltose. Add 1 ml of dinitrosalicylic acid color reagent. Incubate in boiling water bath for 5 minutes and cool to room temperature. Add 10 ml distilled water to each tube and mix well. Read A540 versus micromoles maltose.

Enzyme assay: Pipette 0.5 ml of respective enzyme dilutions into a series of numbered test tubes. Include a blank with 0.5 ml reagent grade water. Incubate tubes at 25°C for 3-4 minutes to achieve temperature equilibration. At timed intervals, add 0.5 ml starch solution (at 25°C). Incubate exactly 3 minutes and at timed intervals add 1 ml dinitrosalicylic acid color reagent to each tube. Incubate all tubes in a boiling water bath for 5 minutes. Cool to room temperature and add 10 ml reagent grade water. Mix well and read A540 versus blank. Determine micromoles maltose released from standard curve.

Calculation

Up: Worthington Enzyme Manual

Micro Assay for Flour Alpha Amylase Activity

Micro Assay for Flour Alpha Amylase Activity

Adapted by Mary Guttieri for the Soft Wheat Quality Laboratory

The new method adapts the AACC Method 22-02 using the Ceralpha K-CERA (Megazyme) alpha-amylase assay procedure for higher throughput to determine flour alpha-amylase activity in a microwell plate. All reagents, controls and precautions are as described in the Megazyme manual.

Required Materials

Ceralpha Alpha-Amylase Kit (AACC Method 22-02) 50 mL conical centrifuge tubes Centrifuge with rotor to spin 50 mL conical tubes at 1000 xg Analytical balance Microplate reader and plate (510 nm) Vortex mixer Water bath at 40o C Multichannel repeating pipette

Ceralpha Substrate and Stopping Reagent

Ceralpha substrate is prepared as described and stored frozen (-20oC) in 1 mL aliquots in microcentrifuge tubes.

Additional Stopping Reagent is prepared using 1% w/v sodium phosphate tribasic dodecahydrate in distilled water adjusted to pH11.

Enzyme Extraction

1. Accurately weigh 3.0 g of ground grain or flour into a 50 mL conical centrifuge tube.

2. Add 20.0 mL of 1X Extraction Buffer solution (pH 5.4) to each tube and mix vigorously.

3. Allow enzyme to extract over 20 minutes in a 40°C water bath, with occasional mixing.

4. Centrifuge 1,000 x g for ten minutes.5. Assay enzyme activity within two hours.

Reaction Blank

A single set of triplicate Reaction Blanks (non-enzymatic control) is prepared as follows for each batch of samples being analyzed.

1. 0.3 mL of stopping reagent2. 20 μL of substrate solution at the start of the reaction time3. 20 μL of any enzyme preparation in the sample set

The mean absorbance of the non-enzymatic control is subtracted from all assays conducted during that day to establish the background or blank absorbance.

Assay Procedure

1. Dispense 20 μL aliquots of Ceralpha Reagent Solution into a microtiter plate and pre-incubate the tubes and contents at 40°C for 5 min. Dispense 3 aliquots for each enzyme extract (assay each extract in triplicate).

2. To each well containing Ceralpha Reagent solution (20 μL), add 20 μL of wheat α-

amylase extract directly to the bottom of the well at 30 second intervals.3. Incubate at 40°C for exactly twenty min from time of addition.4. Following the 20 min incubation period, add exactly 0.3 mL of Stopping Reagent.5. Read the absorbance of the solutions and the reaction blank at 400 nm against

340 μL distilled water.