Phytase

Application and Principle of Phytase

This procedure is used to determine the activity of enzymes releasing phosphate from phytate. The assay is based on enzymatic hydrolysis of sodium phytate under controlled conditions by measurement of the amount of ortho phosphate released.

 

Reagents and Solutions     
[Note: All glassware must be acid washed, rinsed, and scrupulously cleaned to ensure the absence of phosphate.]
Acetate Buffer (pH 5.5) Dissolve 1.76 g of 100% acetic acid (C2H4O2), 30.02 g of sodium acetate trihydrate (C2H3O2Na·3H2O), and 0.147 g of calcium chloride dihydrate in about 900 mL of water. Transfer the solution into a 1000-mL volumetric flask, dilute to volume with water, and mix. The pH should be 5.50 ± 0.05.
Substrate Solution Dissolve 8.40 g of sodium phytate decahydrate (C6H6O24P6Na12·10H2O) (Sigma Chemical Co.) in 900 mL of Acetate Buffer. Adjust the pH to 5.50 ± 0.05 at 37.0° ± 0.1° by adding 4 M acetic acid. Cool to ambient temperature. Quantitatively transfer the mixture to a 1000-mL volumetric flask, dilute to volume with Acetate Buffer, and mix. Prepare fresh daily.
Nitric Acid Solution (27%) While stirring, slowly add 70 mL of 65% nitric acid to 130 mL of water.
Ammonium Heptamolybdate Solution Dissolve 100 g of ammonium heptamolybdate tetrahydrate [(NH4)6Mo7O24·4H2O] in 900 mL of water in a 1000-mL volumetric flask. Add 10 mL of 25% ammonia solution, dilute to volume with water, and mix. This solution is stable for 4 weeks when stored at ambient temperature and shielded from light.
Ammonium Vanadate Solution Dissolve 2.35 g of ammonium monovanadate (NH4VO3) in 400 mL of warm (60°) water. While stirring, slowly add 20 mL of Nitric Acid Solution (27%). Cool to ambient temperature. Quantitatively transfer the mixture to a 1000-mL volumetric flask, dilute to volume with water, and mix. This solution is stable for 4 weeks when stored at ambient temperature and shielded from light.
Color/Stop Solution While stirring, add 250 mL of Ammonium Vanadate Solution to 250 mL of Ammonium Heptamolybdate Solution. Slowly add 165 mL of 65% nitric acid. Cool to ambient temperature. Quantitatively transfer the mixture to a 1000-mL volumetric flask, dilute to volume with water, and mix. Prepare fresh daily.
Potassium Dihydrogen Phosphate Solution Dry a sufficient amount of potassium dihydrogen phosphate (KH2PO4) in a vacuum oven at 100° to 104° for 2 h. Cool to ambient temperature in a desiccator over dried silica gel.
In duplicate (solutions A and B), weigh approximately 0.245 g of dried potassium dihydrogen phosphate accurately to within 1 mg and dilute with Acetate Buffer to 1 L to obtain solutions containing 1.80 mmol/L of potassium dihydrogen phosphate.
Phytase Reference Preparation (Highly concentrated phytase preparation) This preparation can be obtained from Gist-Brocades, Delft, The Netherlands, with an assigned activity (by collaborative assay), or the activity of the reference preparation can be determined according to Procedure 2.
Phytase Reference Solutions, Procedure 1 Weigh an amount of Phytase Reference Preparation corresponding with 20,000 phytase units accurately to within 1 mg in duplicate in 200-mL volumetric flasks. Dissolve in and dilute to volume with Acetate Buffer, and mix. Dilute with Acetate Buffer to obtain dilutions containing approximately 0.01, 0.02, 0.04, 0.06, and 0.08 phytase units per 2.0 mL of the final dilution.
Sample Preparation, Procedure 1 Suspend or dissolve and dilute accurately weighed amounts of sample in Acetate Buffer so that 2.0 mL of the final dilution will contain between 0.02 and 0.08 phytase units.

Sample Preparation, Procedure 2 In duplicate, accurately weigh amounts of Phytase Reference Preparation and dissolve and dilute in Acetate Buffer to obtain dilutions containing 0.06 ± 0.006 phytase units per 2.0 mL of the final dilution.

 

Procedures
Procedure 1 (Determination of the phytase activity) Transfer 2.00 mL of the Sample Preparation, Procedure 1, and the Phytase Reference Solutions, Procedure 1, into separate 20- × 150-mm glass test tubes. Using a stopwatch and starting at time equals zero, in the order of the series and within regular time intervals, place the tubes into a 37.0° ± 0.1° water bath and allow their contents to equilibrate for 5 min. At time equals 5 min, in the same order of the series and with the same time intervals, add 4.0 mL of Substrate Solution (previously equilibrated to 37.00 ± 0.10) to each test tube. Mix, and replace in the 37.0° ± 0.1° water bath. At time equals 65 min, in the same order and within the same time intervals, terminate the incubation by adding 4.0 mL of Color/Stop Solution. Mix, and cool to ambient temperature.
Prepare blanks by transferring 2.00 mL of the Sample Preparation, Procedure 1, and the Phytase Reference Solutions, Procedure 1, into separate 20- × 150-mm glass test tubes. Using a stopwatch and starting at time equals zero, in the order of the series and within regular time intervals, place the tubes into a 37.0° ± 0.1° water bath and allow them to equilibrate for 5 min. At time equal 5 min, in the same order of the series and within the same time intervals, add 4.0 mL of Color/Stop Solution. Mix, and cool to ambient temperature. Next add 4.00 mL of Substrate Solution to the blank tubes, and mix.
Centrifuge all test tubes for 5 min at 3000 × g. Determine the absorbance of each solution at 415 nm in a 1-cm path-length cell with a suitable spectrophotometer, using water to zero the instrument.
Procedure 2 (Determination of the phytase activity of the Phytase Reference Preparation) Transfer 2.00 mL of Sample Preparation, Procedure 2, and 2.00 mL (three times from Potassium Dihydrogen Phosphate Solution A and two times from B) of Potassium Dihydrogen Phosphate Solutions into separate 20- × 150-mm glass test tubes. Using a stopwatch and starting at time equals zero, in the order of the series and within regular time intervals, place the tubes into a 37.0° ± 0.1° water bath and allow their contents to equilibrate for 5 min. At time equals 5 min, in the same order of the series and within the same time intervals, add 4.0 mL of Substrate Solution (previously equilibrated to 37.0° ± 0.1°) to the test tubes. Mix, and replace in the 37.0° ± 0.1° water bath. At time equals 35 min, in the same order and within the same time intervals, terminate the incubation by adding 4.0 mL of Color/Stop Solution. Mix, and cool to ambient temperature.

Prepare blanks by transferring 2.00 mL of Sample Preparation, Procedure 2, into separate 20- × 150-mm glass test tubes. Prepare Reagent Blanks by transferring 2.00 mL of water into a series of five separate 20- × 150-mm glass test tubes. Add 4.0 mL of Color/Stop Solution to all blank tubes and mix. Next add 4.0 mL of Substrate Solution, and mix. Determine the absorbance of each solution at 415 nm in a 1-cm path-length cell with a suitable spectrophotometer, using water to zero the instrument.

 

Calculations
Calculation, Procedure 1 One Phytase (fytase) unit (FTU) is the amount of enzyme that liberates inorganic phosphate at 1 µmol/min from sodium phytate 0.0051 mol/L at 37.00 at pH 5.50 under the conditions of the test. Calculate the corrected absorbance (sample minus blank) for each Sample Preparation and Phytase Reference Solution. Plot the accurately calculated phytase activity (FTU per 2 mL) of each Phytase Reference Solution against the corresponding absorbance. From the curve, determine the phytase activity in each Sample Preparation (FTU per 2 mL):

Activity (FTU/g) = (FTU per 2 mL × dilution)/sample weight (g).


Calculation, Procedure 2 Calculate the corrected absorbances AR for each Sample Preparation (absorbance Phytase Reference Solution minus corresponding absorbance blank) and for each Potassium Dihydrogen Phosphate Solution, Ap (absorbance Potassium Dihydrogen Phosphate Solution minus average absorbance reagent blank). Calculate C, the phosphate concentration of each Potassium Dihydrogen Phosphate Solution:

(W × 1000 × 2)/MW = C (mmol/2 mL).

Calculate the absorbances D for each Potassium Dihydrogen Phosphate Solution after correction for the amount of potassium dihydrogen phosphate weighed:

Ap/C = D(absorbance units/mmol of phosphate per 2 mL).

Calculate the average of results D, giving E (maximum allowable difference, 5%).
Calculate the activity for each Phytase Reference Preparation:

(AR × f)/(30 × R × E) = FTU/g,

in which AR equals the corrected absorbance of the Phytase Standard Solution; f equals the total dilution factor of the reference preparation; 30 equals the incubation time, in min; R equals sample weight, in g; E equals average of D factors; W equals the weight of potassium dihydrogen phosphate, in g; and MW equals the molecular weight of potassium dihydrogen phosphate, 136.09 (g/mol).

 

CERTIFICATE OF ANALYSIS

 

Packaging and storage

Solid food packaging bag, 25 kg / barrel.

 

Phytase should be stored in dry, cool and well ventilated places away from the sun, heat.

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