TITLE: Amylase activity 
AUTHORS: Bjorn Bartholdy, a.g.henry  

[DESCRIPTION]
This protocol is a scaled-down, modified version of the Enzymatic Assay of α-Amylase (EC 3.2.1.1) found here.

[BEFORE_START]
All instances of dH2O can be replaced with ultrapure water.

[STEPS]
SECTION: Prepare solutions
1. 0.5% Potato starch solution

add a small amount of dH2O to a beaker
add 0.5 Mass Percent 0.5% (w/v) potato starch
Boil for 15 min  while stirring
Take off heat and leave at room temperature
Add dH2O to final volume
Continuous stirring throughout the assay


SECTION: Prepare solutions
2. 0.2% Maltose

 1. add 100 mg maltose to 50 mL dH2O

SECTION: Prepare solutions
3. Buffer

add 250 mL  dH2O to a beaker
add 600 mg of sodium phosphate, monobasic
add 97.5 mg  of sodium chloride
adjust to pH 6.9  with   and   


SECTION: Prepare solutions
4. 2 M NaOH

add 0.8 g   to 10 mL dH2O

SECTION: Prepare solutions
5. 5.3 M potassium sodium tartrate, tetrahydrate solution

add 14.96 mg    to 10 mL  of the 2 M NaOH solution
dissolve solids with heat and stirring

 




SECTION: Prepare solutions
6. 96 mM 3,5-Dinitrosalicylic acid solution

add 0.438 g 3,5-dinitrosalicylic acid to 20 mL dH2O
dissolve solids with heat and stirring

 



SECTION: Prepare solutions
7. Colour reagent

add 12 mL of 50-70 °C dH2O to an appropriate size amber bottle (or something that can protect the solution from light).
add (slowly and with mixing) 8 mL of warm 5.3 Molarity (M)   
add 20 mL  of warm 96 mM 3,5-Dinitrosalicylic acid solution

 


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

Samples should be analysed in duplicates or triplicates.
SECTION: Saliva collection
8. Saliva samples are included as a positive control for amylase activity.

Saliva donors rinse their mouth with water for 30 s 

SECTION: Photometer
23. 
 
Photometer settings:
540 nm filter

Photometer reading 1
Pause
Shake
Pause
Photometer reading 2


 

SECTION: Calculations
25. Prepare the standard curve by regressing (OLS) the ΔA540 of each Standard on mg Maltose (  )

SECTION: Calculations
24. Calculate ΔA540 of each Standard by subtracting the Standard Blank.
  

SECTION: Sample preparation
17. To each well, add 75 µL of sample (or saliva)
 
 

SECTION: Standard curve preparation
12. 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.

 

SECTION: Saliva collection
9. Collect the saliva by spitting into 50 ml plastic centrifuge tubes.

SECTION: Saliva collection
10. Centrifuge the saliva at 1000 x g, 10 min and sample from the supernatant.

SECTION: Standard curve preparation
14. Boil the deep-well microplate(s) for 3 min , then cool on ice to room temperature.

SECTION: Standard curve preparation
15. Add 675 µL of dH2O to each well for a final volume of 900 µL .

SECTION: Standard curve preparation
13.     and repeat the process, but adding artificial saliva instead of dH2O.

SECTION: Sample preparation
18. Then add 75 µL of the  0.5% potato starch solution to each of the wells with samples (and saliva)
 

SECTION: Sample preparation
19. Incubate the plate at 36 °C for 6 min 

SECTION: Sample preparation
20. Remove the plate from the incubator and add 75 µL of colour reagent to the sample wells, 
then boil for 3 min .

 



SECTION: Sample preparation
21. Cool the plate on ice to Room temperature 

then add 675 µL dH2O to each well for a final volume of 900 µL (homogenise the solution with the pipette).


 

SECTION: Sample preparation
22. then transfer 200 µL to a 96 well microplate suitable for the photometer.

SECTION: Standard curve preparation
11. Two standard curves are prepared: one containing dH2O, and one containing artificial saliva.
 

SECTION: Calculations
26. Calculate ΔA540 of each Sample by subtracting the Sample Blank.
  

SECTION: Calculations
27. 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.
SECTION: Calculations
28. 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.  

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