Enzymatic Assay of Phytase (EC 3.1.3.8)

Document History

Derived from procedure SSPHYT02. Includes template updates to current SOP specifications and incorporation of notes into the procedure. Refer to SOP-DEK-ENZ18.

1. Objective

The objective of this procedure is to determine the activity of phytase, Sigma-Aldrich Product Number P9792.

2. Scope

The scope of this procedure includes the following reaction:
Phytic Acid + H2O    Phytase   > p-myo-inositol 1,2,4,5,6-PKP + Pi

3. Definitions

3.1.    Purified Water – Water from a deionizing system, resistivity > or = 18MΩcm @ 25°C.

3.2.    p-myo-inositol 1,2,4,5,6-PKP = p-myo-inositol 1,2,4,5,6-Pentakisphosphate

3.3.    Phytic Acid = Myo-Inositol Hexakis(dihydrogen phosphate)

3.4.    Pi = Inorganic Phosphate

3.5.    Unit definition of Phytase = One unit will liberate 1.0 μmole of inorganic phosphorus from 4.2 X 10-2 M phytate per minute at pH 2.5 at 37°C.

4. Discussion

4.1.    Phytic Acid + H2O    Phytase   > p-myo-inositol 1,2,4,5,6-PKP + Pi

4.2.    Method = Colorimetric Determination, Temperature (T) = 37°C, pH = 2.5, A400nm , Light path = 1cm

5. Responsibilities

It is the responsibility of all Sigma-Aldrich personnel to follow this procedure as written.

6. Safety

Refer to the MSDS of each chemical used before beginning this assay.

7. Procedure

7.1    Reagents

7.1.1    200 mM Glycine Buffer, pH 2.8 at 37°C (Buffer)

7.1.1.1.    Prepare 100 ml in purified water using Glycine, Hydrochloride, Sigma-Aldrich Product Number G7126.

7.1.1.2.    Adjust to pH 2.5 at 37°C with 1M NaOH or 1M HCl.

7.1.2.     44.1 mM Phytic Acid Solution, pH 2.5 at 37°C (Phytic Acid)

7.1.2.1.    Prepare 10 ml in Reagent 7.1.1 using Phytic Acid, Dipotassium, Sigma-Aldrich Product Number P5681.

7.1.2.2.    Adjust to pH 2.5 at 37°C with 1M HCl.

7.1.3.    5% (w/v) Ammonium Molybdate Solution (Amm Moly)
Prepare 100 ml in purified water using Molybdic Acid, Ammonium Tetrahydrate Salt, Aldrich-Product Number M0878.

7.1.4    5N Sulfuric Acid Solution
Prepare 100 ml in purified water using Sulfuric Acid, 95-98%, A.C.S. Reagent, Aldrich Product Number 258105.

7.1.5    Acetone
Use Acetone, A.C.S. Reagent Grade, Aldrich Product Number 179124.

7.1.6    Color Reagent Solution (CRS)

7.1.6.1    Prepare 100 ml by adding 25 ml of Reagent 7.1.3 to 25 ml of Reagent 7.1.4 and 50 ml of Reagent 7.1.5.

7.1.6.2    Mix and use immediately.

7.1.6.3    PREPARE FRESH.

7.1.7    50 mM Potassium Phosphate Solution (P Std.)
Prepare 10 ml in deionized water using Potassium Phosphate, Monobasic,, Anhydrous, Sigma-Aldrich Product Number P5379.

7.1.8    Phytase Enzyme Solution (Enzyme)
Immediately before use, prepare a solution containing 0.5 – 2.0 units/ml of Phytase in cold Reagent 7.1.1.

7.2.     Procedure – Blank/Test Preparation

7.2.1.     Pipette (in milliliters) the following reagents into a 4 dram vial:

  Test Blank
Reagent 7.1.2 (Phytic Acid) 0.50 0.50

 

7.2.2.     Equilibrate to 37°C, then add:

Reagent 7.1.8 (Enzyme) 0.025 ----
Reagent 7.1.1 (Buffer) ---- 0.025

 

7.2.3.    Immediately mix by inversion and incubate at 37°C for exactly 30 minutes. Then add:

Reagent 7.1.6 (CRS) 4.00 4.00
Purified Water 0.025 0.025

 

7.2.4.    Mix by swirling, then transfer to suitable cuvettes and record the A400nm for both the test and blank using a suitable spectrophtometer.

7.3.    Procedure – Standard Curve

7.3.1    Prepare by pipetting (in milliliters) the follow reagents into suitable containers:

  Std 1 Std 2 Std 3 Std 4 Std 5 Std. Blk.
Purified Water 0.04 0.03 0.02 0.01 ---- 0.05
Reagent 7.1.7 (P Std.) 0.01 0.02 0.03 0.04 0.05 ------
Reagent 7.1.2 (Phytic Acid) 0.50 0.50 0.50 0.50 0.50 0.50

 

7.3.2    Mix by swirling and incubate at 37°C for 30 minutes. Then add:

Reagent 7.1.6 (CRS) 4.00 4.00 4.00 4.00 4.00 4.00


7.3.3    Mix by swirling, then transfer to suitable cuvettes and record the A400nm for both the standards and the standard blank using a suitable spectrophtometer.

7.4.    Calculations

7.4.1    Standard Curve

7.4.1.1    ΔA400nm Standard = A400nm Standard - A400nm Standard Blank

7.4.1.2    Prepare a standard curve by plotting ΔA400nm versus μmoles of phosphate.

7.4.2    Sample Determination

7.4.2.1    ΔA400nm Test = A400nm Test - A400nm Test Blank

7.4.2.2    Determine the μmoles of phosphate liberated using the standard curve.

7.5.2.3 Units/ml enzyme = (µmoles of phosphate released) (df)
(30)(0.025)

 

where:
    df = dilution factor
    30 = Time (in minutes) of assay per the Unit Definition
    0.025 = volume (in milliliters) of enzyme used

7.4.2.4 Units/mg solid = units/ml enzyme
Mg solid/ml enzyme

8. References & Attachments

Heinonen, J.K. and R.J. Lahti (1981) Analytical Biochemistry. 113, 313-317.

9. Approval

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