Phytase Analysis, Pitfalls and Interpretation of FTU for

Biological Efficacy

Mike Bedford1 & Ralf Greiner2

1AB Vista Feed Ingredients Ltd, Woodstock Court, Blenheim Rd. Marlborough,

Wilts UK. SN8 1QJ

2Max Rubner-Institute,, Federal Research Institute of Nutrition and Food,,

Department of Food Technology and Bioprocess Engineering, Haid-und-Neu-

Straße 9, 76131 Karlsruhe, Germany
 Phytases

¾ Occurrence
ƒ identified in microorganisms, plants, and some animal tissue

the ability of phytases to hydrolyse phytate is usually

known only from in vitro assays and information on
their in vivo function is rather limited

¾ Definition
ƒ a subgroup of phosphatases that are capable
of initiating the stepwise dephosphorylation
of phytate

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 Phytate-degrading enzymes

¾ acid phytate-degrading enzymes

ƒ histidine acid phosphatases
ƒ cysteine phosphatases
ƒ purple acid phosphatases

¾ alkaline phytate-degrading enzymes

ƒ ß-propeller
p p pphytases,
y Ca2+- dependent
p

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 Determination of Phytase Activity

phytase
InsP6 InsP5, (InsP4, InsP3, InsP2, InsP)

PO43- + [Mo7O24]6-

pH = 5.5 H+/acetone
T = 37°C
[sodium phytate] = 5 mM
[PMo12O40]3-

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 Phytase Assay

linearity with time substrate inhibition

1.4

1.2
1.2
¾ phytate preparation has to be free of
1
1
other phosphorylated compounds
0.8
0.8 ¾ hydrolysis of phytate has to be limited:
ΔE

ΔE
06
0.6 ƒ hydrolysis
0.6y y pproducts could serve as
substrates for the phytases
0.4 0.4
ƒ the released phosphate might act as
an competitive inhibitor
0.2
0.2

0 0
0 10 20 30 0 1 2 3 4
time substrate [mM]

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 Interpretation of FTU for Biological Efficacy
pH activity profiles
¾ phytate hydrolysis in vivo: pH ≠ constant

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 Interpretation of FTU for Biological Efficacy
pH activity profiles
¾ phytate hydrolysis in vivo: pH ≠ constant

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 Interpretation of FTU for Biological Efficacy
pH activity profiles
¾ phytate hydrolysis in vivo: pH ≠ constant

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 Interpretation of FTU for Biological Efficacy
substrate
¾ phytate accessibility limited (feed matrix, precipitated)

¾ phytate concentration low (KM-values range from <10 to 650 µM)

1.2
¾ interaction, inhibitors,
electrostatic environment 1

¾ presence of other 0.8

ΔE
phosphorylated compounds
0.6

0.4

0.2

0
0 1 2 3 4
substrate [mM]

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 Interpretation of FTU for Biological Efficacy
presence of other phosphorylated compounds

broad substrate specificity narrow substrate specificity

relative activity [%]
substrate rel. Activity [%] substrate P1 P2

phytate 100.0 100.0

phytate
p y 100 p-nitrophenyl
p nitrophenyl phosphate 12.3 9.8
p-nitrophenyl phosphate 68 1-naphthyl phosphate 0.7 0.8
1-naphthyl phosphate 49 2-naphthyl phosphate 2.7 2.5
2-naphthyl phosphate 20 2-glycero phosphate 1.9 1.7
2-glycero phosphate 24 , p p
fructose-1,6-diphosphate 8.5 8.5
fructose-1,6-diphosphate 98 fructose-6-phosphate 1.3 1.7
fructose-6-phosphate 5 glucose-6-phosphate 0.4 0.8
glucose-6-phosphate 30
AMP 0 0
AMP 11
ADP 0 0
ADP 87
ATP 0 0
ATP 221
NADP 0 0
GTP 10
Na2H2-pyrophosphate 0 0
Na2H2-pyrophosphate 290
pyridoxal phosphate 0 0
pyridoxalphosphate
id l h h t 14
o-phospho-L-serine 0 0
o-phospho-L-serine 12
GTP 0 0

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 Interpretation of FTU for Biological Efficacy
pH stability and susceptibility to pepsin degradation

¾ pH stability
ƒ residual activity after 24 hrs at pH 2.5
2 5 and 44°C;
C; faba bean phytase: 22%,
22%
P. agglomerans phytase: 31%, Malaysian waste-water bacterium phytase: 95%

¾ susceptibility to pepsin degradation

ƒ in vitro at pH 2.0: Escherichia coli, Klebsiella sp., Malaysian waste-water
bacterium, Yersinia rohdei phytase: more than 80% of initial activity, Aspergillus
niger phytase: 26-42%, Peniophora lycii phytase: 2-20%

ƒ digesta supernatant of the stomach: Bacillus subtilis phytase: 68%,

68% Aspergillus
niger phytase: 60-70%, Peniophora lycii phytase: 59%

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 Interpretation of FTU for Biological Efficacy
phytate degradation pathway

¾ phytate degradation pathway / initiation site of phytate dephosphorylation

ƒ animal feeding studies do not give any clear indication that differences in
bioefficacy are based on the position of initiating phytate dephosphorylation

ƒ InsP6 and InsP5 dephosphorylation

p p y capacity
p y seems to be important
p for bioefficacy;
y;
(a complete transformation of dietary phytate into myo-inositol tetra- and –
trisphosphates in the stomach seems to be much more important for bioefficacy of
supplementary
pp y pphytase
y than a complete
p dephosphorylation
p p y of single
g pphytate
y
molecules)

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Thank You Veryy Much
For Your Attention !

Dr. Ralf Greiner

Department of Food Technology and Bioprocess Engineering
Max Rubner-Institute
Federal Research Institute of Nutrition and Food
Haid-und-Neu-Straße 9 • D-76131 Karlsruhe
Tel.: ++49 (0)721 6625 300 • Fax: ++49 (0)721 6625 303
[email protected]
g @ • www.mri.bund.de

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