Total Starch Enzymatic Digestion
Lynn Doran, Amanda P. De Souza
Abstract
Enzymatic digestion of total soluble starch to glucose in plant tissue extracts for preparation for quantification via the GOD-POD Method (NZYtech).
Before start
Extract and dry total starch pellet from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.
Steps
Prepare fresh daily 120 U/mL α-amylase in MOPS buffer. 1 mL per sample will be needed. Initial concentration of α-amylase is 1000 U/mL. Use C1V1 = C2V2to calculate the volume of α-amylase and MOPS buffer to use.
Prepare fresh daily 30 U/mL amyloglucosidase in acetate buffer. 1 mL per sample will be needed. Initial concentration of amyloglucosidase is 3260 U/mL. Use C1V1 = C2V2to calculate the volume of amyloglucosidase and acetate buffer to use.
Heat the water bath to 75°C.
Add 500µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.
Vortex to suspend all solids. Flicking the tube may help dislodge the dried starch pellet.
Incubate for 0h 30m 0s at 75°C in the water bath.
Add another 500µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.
Vortex to suspend all solids.
Incubate for 0h 30m 0s at 75°C in the water bath.
Cool down the water bath to 50°C, tubes can be stored at room temp on the counter while water bath cools.
Add 500µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.
Vortex to suspend all solids.
Incubate for 0h 30m 0s at 50°C in the water bath.
Add another 500µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.
Vortex to suspend all solids.
Place the tubes in ice to stop the reaction, until cool to the touch.
Proceed to total starch (as glucose) quantification by NZYtech GOD-POD method or store the samples at -20°C up to one month.
