ralf greiner and mike bedford - phytase analysis
TRANSCRIPT
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
Occurrence
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
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
Definition
their in vivo function is rather limited their in vivo function is rather limited
a subgroup of phosphatases that are capableof initiating the stepwise dephosphorylation of phytateof phytate
20.10.2010 2MRI – Department of Food Technology and Bioprocess Engineering
Phytate-degrading enzymes
acid phytate-degrading enzymes
histidine acid phosphatases
cysteine phosphatases
purple acid phosphatases
alkaline phytate-degrading enzymes
ß-propeller phytases, Ca2+- dependentp p p y p
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Determination of Phytase Activity
InsP InsP (InsP InsP InsP InsP)phytase
InsP6InsP5, (InsP4, InsP3, InsP2, InsP)
PO43- + [Mo7O24]6-
H+/acetonepH = 5.5T 37°C
[PMo12O40]3-
T = 37°C[sodium phytate] = 5 mM
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Phytase Assay
1.4
linearity with time substrate inhibition
1
1.2
1
1.2
phytate preparation has to be free of other phosphorylated compounds
0 6
0.8ΔE
0.6
0.8
ΔE
other phosphorylated compounds
hydrolysis of phytate has to be limited:hydrolysis products could serve as
0.4
0.6
0.4
0.6y y psubstrates for the phytasesthe released phosphate might act as an competitive inhibitor
0
0.2
0 10 20 300
0.2
0 1 2 3 4
an competitive inhibitor
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time substrate [mM]
Interpretation of FTU for Biological EfficacypH activity profiles
phytate hydrolysis in vivo: pH ≠ constant
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Interpretation of FTU for Biological EfficacypH activity profiles
phytate hydrolysis in vivo: pH ≠ constant
20.10.2010 7MRI – Department of Food Technology and Bioprocess Engineering
Interpretation of FTU for Biological EfficacypH activity profiles
phytate hydrolysis in vivo: pH ≠ constant
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Interpretation of FTU for Biological Efficacysubstrate
(KM-values range from <10 to 650 µM)
phytate accessibility limited (feed matrix, precipitated)
phytate concentration low
interaction, inhibitors, electrostatic environment 1
1.2
presence of other phosphorylated compounds
0.6
0.8
ΔE
0.4
0
0.2
0 1 2 3 4
20.10.2010 9MRI – Department of Food Technology and Bioprocess Engineering
substrate [mM]
Interpretation of FTU for Biological Efficacypresence of other phosphorylated compounds
relative activity [%]
broad substrate specificity narrow substrate specificity
substrate
rel. Activity [%]
phytate
100
substrate P1
P2
phytate
p-nitrophenyl phosphate
100.0
12.3
100.0
9.8p y
p-nitrophenyl phosphate
1-naphthyl phosphate
2-naphthyl phosphate
2-glycero phosphate
68
49
20
24
p nitrophenyl phosphate
1-naphthyl phosphate
2-naphthyl phosphate
2-glycero phosphate
fructose-1,6-diphosphate
12.3
0.7
2.7
1.9
8.5
9.8
0.8
2.5
1.7
8.5 fructose-1,6-diphosphate
fructose-6-phosphate
glucose-6-phosphate
AMP
98
5
30
11
, p p
fructose-6-phosphate
glucose-6-phosphate
AMP
ADP
1.3
0.4
0
0
1.7
0.8
0
0 ADP
ATP
GTP
Na2H2-pyrophosphate
id l h h t
87
221
10
290
14
ATP
NADP
Na2H2-pyrophosphate
pyridoxal phosphate
0
0
0
0
0
0
0
0
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pyridoxalphosphate
o-phospho-L-serine
14
12
o-phospho-L-serine
GTP
0
0
0
0
Interpretation of FTU for Biological EfficacypH stability and susceptibility to pepsin degradation
pH stabilityresidual activity after 24 hrs at pH 2 5 and 4°C; faba bean phytase: 22%residual activity after 24 hrs at pH 2.5 and 4 C; faba bean phytase: 22%, P. agglomerans phytase: 31%, Malaysian waste-water bacterium phytase: 95%
susceptibility to pepsin degradationin 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%
digesta supernatant of the stomach: Bacillus subtilis phytase: 68% Aspergillusdigesta supernatant of the stomach: Bacillus subtilis phytase: 68%, Aspergillus niger phytase: 60-70%, Peniophora lycii phytase: 59%
20.10.2010 11MRI – Department of Food Technology and Bioprocess Engineering
Interpretation of FTU for Biological Efficacyphytate degradation pathway
phytate degradation pathway / initiation site of phytate dephosphorylationanimal feeding studies do not give any clear indication that differences inanimal feeding studies do not give any clear indication that differences in bioefficacy are based on the position of initiating phytate dephosphorylation
InsP6 and InsP5 dephosphorylation capacity seems to be important for bioefficacy; 6 5 p p y p y p 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 phytase than a complete dephosphorylation of single phytate pp y p y p p p y g p ymolecules)
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Thank You Very MuchThank You Very MuchyyFor Your Attention !For Your Attention !
Dr. Ralf Greiner
Department of Food Technology and Bioprocess EngineeringMax Rubner-InstituteFederal Research Institute of Nutrition and FoodFederal Research Institute of Nutrition and FoodHaid-und-Neu-Straße 9 • D-76131 KarlsruheTel.: ++49 (0)721 6625 300 • Fax: ++49 (0)721 6625 [email protected] • www.mri.bund.deg @
20.10.2010 13MRI – Department of Food Technology and Bioprocess Engineering