Oct 27, 2021

Public workspaceAmylase activity V.1

This protocol is a draft, published without a DOI.
  • 1Leiden University
  • BYOC
Bjørn Peare Bartholdy
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Bjorn Bartholdy, a.g.henry 2021. Amylase activity . protocols.io https://protocols.io/view/amylase-activity-bw8jphun
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: August 09, 2021
Last Modified: October 27, 2021
Protocol Integer ID: 52203
Abstract
This protocol is a scaled-down, modified version of the Enzymatic Assay of α-Amylase (EC 3.2.1.1) found here.
Materials
Chemicals
Potato starch
D-(+)-maltose monohydrate
Sodium phosphate monobasic
Sodium chloride
potassium sodium tartrate, tetrahydrate
Sodium Hydroxide
3,5 Dinitrosalicylic acid

Equipment
Pipettes
  • 2 -- 20 μL
  • 20 -- 200 μL
  • 100 -- 1000 μL
8-channel pipette (ca. 20 - 300 μL) [OPTIONAL, but recommended]
96 well microplate
Spectrophotometer (with 540 nm filter)
Protocol materials
Reagent1 M NaOH
Reagent1 M HCl
ReagentNaOH
ReagentPotassium sodium tartrate tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2377
ReagentPotassium sodium tartrate tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2377
Before start
All instances of dH2O can be replaced with ultrapure water.
Prepare solutions
Prepare solutions
15m
15m
0.5% Potato starch solution

  1. add a small amount of dH2O to a beaker
  2. add Concentration0.5 Mass Percent 0.5% (w/v) potato starch
  3. Boil for Duration00:15:00 while stirring
  4. Take off heat and leave at room temperature
  5. Add dH2O to final volume
  6. Continuous stirring throughout the assay


15m
0.2% Maltose

1. add Amount100 mg maltose to Amount50 mL dH2O

Buffer

  1. add Amount250 mL dH2O to a beaker
  2. add Amount600 mg of sodium phosphate, monobasic
  3. add Amount97.5 mg of sodium chloride
  4. adjust to Ph6.9 with Reagent1 M NaOHContributed by users and Reagent1 M HCl


2 M NaOH

  1. add Amount0.8 g ReagentNaOHContributed by users to Amount10 mL dH2O

5.3 M potassium sodium tartrate, tetrahydrate solution

  1. add Amount14.96 mg ReagentPotassium sodium tartrate tetrahydrateSigma AldrichCatalog #S2377 to Amount10 mL of the 2 M NaOH solution
  2. dissolve solids with heat and stirring

Safety information
DO NOT heat to a boil




96 mM 3,5-Dinitrosalicylic acid solution

  1. add Amount0.438 g 3,5-dinitrosalicylic acid to Amount20 mL dH2O
  2. dissolve solids with heat and stirring

Safety information
DO NOT heat to a boil



Colour reagent

  1. add Amount12 mL of Temperature50-70 °C dH2O to an appropriate size amber bottle (or something that can protect the solution from light).
  2. add (slowly and with mixing) Amount8 mL of warm Concentration5.3 Molarity (M) ReagentPotassium sodium tartrate tetrahydrateSigma AldrichCatalog #S2377
  3. add Amount20 mL of warm 96 mM 3,5-Dinitrosalicylic acid solution

Note
The colour reagent is stable for 6 months in a dark place at ambient temperature


Saliva collection
Saliva collection
30s
30s
Saliva samples are included as a positive control for amylase activity.

Saliva donors rinse their mouth with water for Duration00:00:30

30s
Collect the saliva by spitting into 50 ml plastic centrifuge tubes.

Centrifuge the saliva at Centrifigation1000 x g, 00:10:00 and sample from the supernatant.

10m
Standard curve preparation
Standard curve preparation
Two standard curves are prepared: one containing dH2O, and one containing artificial saliva.
Protocol
Artificial saliva
NAME

Artificial saliva

CREATED BY
Bjørn Peare Bartholdy

Add Amount300 mL distilled (or deionized) dH2O to a Amount1000 mL beaker, with stirring and heat Temperature60 °C .

Add:

- Amount2.5 g ReagentMucin from porcine stomach (Type III)Becton Dickinson (BD)Catalog #M1778
- Amount5 g ReagentTrypticase™ PeptoneBecton Dickinson (BD)Catalog #211921
- Amount10 g ReagentOxoid™ Proteose PeptoneBecton Dickinson (BD)Catalog #LP0085B
- Amount5 g ReagentBacto Yeast ExtractBecton Dickinson (BD)

Let the reagents completely dissolve before continuing to the next step

Add:

- Amount2.5 g ReagentKClBecton Dickinson (BD)
- Amount0.35 g ReagentNaClBecton Dickinson (BD)
- Amount0.2 g ReagentCaCl2Becton Dickinson (BD)
- Amount0.74 g ReagentSodium phosphate dibasicBecton Dickinson (BD)Catalog #7558-79-4
- Amount0.54 g ReagentNaHCO3Becton Dickinson (BD)
- Amount2.5 mg ReagentHeminBecton Dickinson (BD)

Add the remaining Amount700 mL distilled (or deionized) dH2O

Adjust to Ph7 with ReagentNaOHBecton Dickinson (BD) and stirring

Transfer to two 1000 ml bottles, so half of each bottle is filled.

Autoclave at Temperature121 °C , Pressure1 Bar for Duration00:15:00 minutes
Safety information
Do NOT screw bottle caps on tightly.

Loosely screw the caps on the bottles or cover the tops with foil

15m
Once the solution has cooled, add:

  • Amount1 mg ReagentMenadioneBecton Dickinson (BD)
  • Amount0.3 g ReagentUreaBecton Dickinson (BD)
  • Amount0.17 g ReagentL-ArginineBecton Dickinson (BD)Catalog #A5006


Store in fridge at ca. Temperature4 °C

Occasionally test the pH to ensure it stays around Ph7

Add the following reagents to the wells of a deep-well microplate so each well contains a total volume of 225 μL. Each column in the table represents a single well.

Add the Maltose to each well first, then dH2O, then colour reagent.

ReagentSTD1STD2STD3STD4STD5STD6STD7STD Blank
0.2% Maltose3.7515304560751500
dH2O146.2513512010590750150
Colour reagent7575757575757575
All quantities are in μL

Go togo to step #12 and repeat the process, but adding artificial saliva instead of dH2O.

Boil the deep-well microplate(s) for Duration00:03:00 , then cool on ice to room temperature.

3m
Add Amount675 µL of dH2O to each well for a final volume of Amount900 µL .

Sample preparation
Sample preparation
Samples are prepared in a 96 deepwell plate with approx. 1 mL volume per well.

Samples should be analysed in duplicates or triplicates.
To each well, add Amount75 µL of sample (or saliva)
Note
It is best to make duplicates or triplicates of the samples

Then add Amount75 µL of the 0.5% potato starch solution to each of the wells with samples (and saliva)
Safety information
Once the starch has been added to the sample, the reaction will begin. The starch should be added quickly; ideally with a multichannel pipette .

Incubate the plate at Temperature36 °C for Duration00:06:00

6m
Remove the plate from the incubator and add Amount75 µL of colour reagent to the sample wells,
then boil for Duration00:03:00 .

Safety information
The colour reagent will stop the reaction, so should be added with a multichannel pipette.



3m
Cool the plate on ice to TemperatureRoom temperature

then add Amount675 µL dH2O to each well for a final volume of Amount900 µL (homogenise the solution with the pipette).


Note
Use a new pipette tip for each sample.

then transfer Amount200 µL to a 96 well microplate suitable for the photometer.

Photometer
Photometer

Equipment
Multiskan FC
NAME
Microplate Photometer
TYPE
Thermo Scientific
BRAND
51119000
SKU
Photometer settings:
540 nm filter

  1. Photometer reading 1
  2. Pause
  3. Shake
  4. Pause
  5. Photometer reading 2


Note
Make sure to include a sample blank (with dH2O for saliva positive controls, and stock artificial saliva for samples)

Calculations
Calculations
Calculate ΔA540 of each Standard by subtracting the Standard Blank.

Prepare the standard curve by regressing (OLS) the ΔA540 of each Standard on mg Maltose (Go togo to step #12 )

Calculate ΔA540 of each Sample by subtracting the Sample Blank.

Then calculate the mg of Maltose released (x) using the regression coefficients:


Where y is the , b is the intercept, and a is the slope.
Units per mL enzyme can be calculated as:


where dilution factor is how much the sample was diluted (if at all), and mL enzyme is how much of the sample was added in step 17.Go togo to step #17

A Unit is defined as mg of maltose released from starch in 6 minutes.