Sep 14, 2023

Public workspaceTotal Starch Enzymatic Digestion V.5

This protocol is a draft, published without a DOI.
  • 1Realizing Increased Photosynthetic Efficiency (RIPE)
Lynn Doran
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Protocol Citation: Lynn Doran, Amanda P. De Souza 2023. Total Starch Enzymatic Digestion . protocols.io https://protocols.io/view/total-starch-enzymatic-digestion-czxbx7inVersion created by Lynn Doran
Manuscript citation:
Amaral, LIV do, Gaspar, M., Costa, PMF, Aidar, MPM, & Buckeridge, MS (2007).New fast and sensitive enzymatic method for extracting and measuring starch in plant materials.Hoehnea,34 n.4, 425-431.doi:10.1590/s2236-89062007000400001

Pak S. Chow, Simon M. Landhäusser, A method for routine measurements of total sugar and starch content in woody plant tissues, Tree Physiology, Volume 24, Issue 10, October 2004, Pages 1129–1136, https://doi.org/10.1093/treephys/24.10.1129
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 13, 2023
Last Modified: September 14, 2023
Protocol Integer ID: 87747
Keywords: Starch, Soluble Starch, Enzymatic Digestion, Plant Tissue, GOD-POD
Abstract
Enzymatic digestion of total soluble starch to glucose in plant tissue extracts for preparation for quantification via the GOD-POD Method (NZYtech).
Materials
Reagents
Amount2.09 g of MOPS per Amount1 L of water (Amount0.4598 g for Amount220 mL water), pH adjusted to 6.5 using NaOH.
Store refrigerated for up to 2 months.
  • Acetate Buffer 100mM , pH 4.5
Amount6 mL of acetic acid per Amount1 L of water (Amount1.5 mL per Amount250 mL water), pH adjusted to 4.5 using NaOH.
Store refrigerated for up to 2 months.
  • Ice

Materials
  • Pipette tips

Equipment
  • Graduated cylinder
  • Water bath
  • Floating tube holder
  • Single channel pipette
  • Ice bucket




Before start
  • Prepare fresh daily 120 U/mL α-amylase (Bacillus licheniformis) in MOPS buffer. 1 mL per sample will be needed. Initial concentration of α-amylase is 3000 U/mL. Use C1V1 = C2V2 to calculate the volume of α-amylase and MOPS buffer to use.
Note
α-amylase comes at 3000 U/mL concentration in a 20 mL bottle.

To prepare 250 mL of 120 U/mL α-amylase in MOPS buffer, dilute 10 mL α-amylase (3000 U/mL) into 240 mL MOPS buffer.

  • Prepare fresh daily 30 U/mL amyloglucosidase (Aspergillus niger) in acetate buffer. 1 mL per sample will be needed. Initial concentration of amyloglucosidase is 3260 U/mL. Use C1V1 = C2V2 to calculate the volume of amyloglucosidase and acetate buffer to use.
Note
To prepare 250 mL of 30 U/mL amyloglucosidase in acetate buffer, dilute 2.3 mL amyloclucosidase (3260 U/mL) into 247.7 mL acetate buffer.

Heat the water bath to Temperature75 °C .

Add Amount500 µL 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 Duration00:30:00 at Temperature75 °C in the water bath.

30m
Add another Amount500 µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.
Vortex to suspend all solids.
Incubate for Duration00:30:00 at Temperature75 °C in the water bath.

30m
Cool down the water bath to Temperature50 °C , tubes can be stored at room temp on the counter while water bath cools.
Note
Leaving the lid off, especially if it is a shaking water bath, will help cool. For faster cooling, remove some water from the water bath and refill with cool distilled water. Do not use tap water as mineral buildup or heated chlorine could damage the water bath.

Add Amount500 µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.

Vortex to suspend all solids.
Incubate for Duration00:30:00 at Temperature50 °C in the water bath.

30m
Add another Amount500 µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.

Vortex to suspend all solids.
Incubate for Duration00:30:00 at Temperature50 °C in the water bath.

30m
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.