Update on Characteristics of Commercial Phytases

Ralf Greiner
Max Rubner-Institut, Federal Research Institute of Nutrition and Food,
Department of Food Technology and Bioprocess Engineering,
Haid-und-Neu-Strae 9, 76131 Karlsruhe, Germany
 Phytases

Occurrence
identified in microorganisms, plants, and some animal tissue

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

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 Enzymatic Phytate Degradation
20

16
InsP6
g inositol phosphates

InsP4
12

InsP3
InsP5

0
0 120 240 360 480
time [min]
Greiner et al., J. Agric. Food Chem. 50, 6858-6864 (2002)
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 Phytases

Occurrence
the ability of phytases
identified in microorganisms, plants, andtosome
hydrolyse phytate is usually
animal tissue
known only from in vitro assays and information on
their in vivo function is rather limited

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

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 Phytate-degrading Enzymes

acid phytate-degrading enzymes

histidine acid phytate-degrading enzymes
cysteine phytate-degrading enzymes
purple acid phytate-degrading enzymes

alkaline phytate-degrading enzymes

-propeller phytases, Ca2+- dependent

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 Enzymatic Phytate Degradation

Ins(1,3,4,5,6)P5 Ins(1,2,3,4,6)P5
lily

A. niger

D-Ins(1,2,4,5,6)P5 Ins(1,2,3,4,5,6)P6 D-Ins(2,3,4,5,6)P5

E. coli
rye

D-Ins(1,2,3,5,6)P5 D-Ins(1,2,3,4,5)P5
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 Enzymatic Phytate Degradation

Ins(1,2,3,4,5,6)P6

Ins(2)P D-Ins(1,2,4,5,6)P5 Ins(2,4,6)P3

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 Phytases

the first commercial phytase products were launched into market in 1991

meanwhile phytase is present in around 62% of monogastric feed

its market volume exceeds US$250 million and is growing at around

10% per year

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 Phytases for Feed Application

commercially available phytase products can be classified according to:

their origin (fungal or bacterial)

the carbon in the myo-inositol ring of phytate at which dephosphorylation

is initiated (3 or 6-phytase)

on the presence of any protection against the high temperature present in

feed production (coated or non-coated)

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 Phytases for Feed Application

phytases from four different sources can be found in the marketplace today:

Peniophora lycii 6-phytase of fungal origin, HAP

Aspergillus niger 3-phytase of fungal origin, HAP

Escherichia coli 6-phytase of bacterial origin, HAP

Penicillium funiculosum 3-phytase of fungal origin, HAP

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 Phytases for Feed Application

phytases from four different sources can be found in the marketplace today:

Peniophora lycii 6-phytase of fungal origin, HAP

Aspergillus niger 3-phytase of fungal origin, HAP

Escherichia coli 6-phytase of bacterial origin, HAP

furthermore, variants of the above mentioned phytases with significant

improvements over the parent enzymes through selective molecular
modification have found its way to the marketplace

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 Phytases for Feed Application

no indication that fungal or bacterial phytases are more efficient in animals

this statement is even more true with improvement of enzyme properties through
molecular modifications

the preference of the third or sixth phosphate group is not known to affect
phytase efficacy
no synergistic (P. lycii + E. coli, A. niger + E. coli) effect was observed on plasma
inorganic phosphate and growth performance (Stahl et al. 2004)

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 Phytases for Feed Application

ideal phytases for animal feed applications should fulfil a series of quality
criteria:

effective release of phytate phosphate in the digestive tract

feed processing stability

storage stability

economical production

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 Phytases for Feed Application

ideal phytases for animal feed applications should fulfil a series of quality
criteria:

effective release of phytate phosphate in the digestive tract

pH optimum and pH profile
kinetic constants (KM, vmax, kcat) under acidic conditions
pH stability under acidic conditions
substrate specificity
resistance to endogenous protease activity in the stomach

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 Characteristics of Commercial Phytases
pH optimum, pH profile, kinetic constants

characteristics P. lycii A. niger E. coli

pH optimum 4.0 - 5.0 2.2 + 5.5 3.5 - 4.5
pH profile - activity at pH 3.0 (activity 18 - 25% 38 - 50% 86 - 149%
at pH 5.5 = 100%)
kinetic constants at pH optimum:
KM [M] 33 27 130 - 370
kcat [s-1] 2200 350 1401 - 2378
kcat/KM [106 s-1 M-1] 66 13 10 - 13
kinetic constants at pH 3.0:
KM [M] 73 - 81 113 - 135 174 - 386
kcat [s-1] 1216 - 1325 166 - 198 823 - 1076
kcat/KM [106 s-1 M-1] 15 - 18 1.3 - 1.7 3.5 - 6.2

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 Characteristics of Commercial Phytases
effect on phytate-degrading activity

characteristics P. lycii A. niger E. coli

phytate concentration 4.5 4.0 2.0
in phytase assay [mM]
phytase activity at 5 mM phytate / test 0.97 0.98 0.76
concentration
phytase activity (acetate = 100%):
+ BSA - - + 5%
+ triton - - + 25%
+ 1 mM Ca2+ + 10% + 20% -
+ 500 mM NaCl + 10% + 10% - 10%
(KM doubled) (KM doubled) (KM doubled)
+ citrate - - - 50%
+ succcinate - - - 75%
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 Characteristics of Commercial Phytases
pH stability, pepsin resistance

characteristics P. Lycii A. niger E. coli

pH stability
pH 2.0, 37C, 1 hr 42 - 49% 69 - 83% 88 - 93%
+ BSA (1 mg ml-1) 45 - 50% 74 - 84% 94 - 100%
protease resistance
pH 2.0, 37C, 1 hr, 2000 U pepsin 14 - 25% 40 - 50% 92 - 98%
+ BSA (1 mg ml-1) 15 - 25% 39 - 51% 93 - 97%
37C, 1 hr
+ stomach digesta 53 - 60% 60 - 70% 83 - 93%
+ crop digesta 92 - 97% 93 - 98% 93 - 96%

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 Characteristics of Commercial Phytases
substrate specificity

characteristics P. lycii A. niger E. coli

substrate specificity intermediate- intermediate- narrow
broad broad

InsP6-dephosphorylation rapid rapid rapid

InsP5-dephosphorylation rapid rapid rapid
InsP4-dephosphorylation rather slow rather slow slow
InsP3-dephosphorylation slow slow slow

final phytate degradation product Ins(2)P Ins(2)P Ins(2)P

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 Phytases for Feed Application

phytatesoluble in equilibrium with phytateinsoluble, equilibrium is pH-dependent

pH optimum of the phytase might be different to that determined with highly
soluble sodium phytate

change on diet composition and quality of ingredients will interfere on the final
concentration of phytate in feed
phytase needs to keep the same high activity even on low phytate concentrations

effects of feed components on phytase activity, not in all phytases in a parallel

and synchronous manner

studies of pepsin effect on P- lycii phytase in the presence of soy/corn meal

pH 3.0, 30 min, 80% residual activity = tolerance effect

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 Phytases for Feed Application

ideal phytases for animal feed applications should fulfil a series of quality
criteria:

effective release of phytate phosphate in the digestive tract

feed processing stability

heat stability / thermo-tolerance

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 Characteristics of Commercial Phytases
temperature optimum, temperature stability

characteristics P. lycii A. niger E. coli

temperature optimum [C] 50 - 55 50 - 58 55 - 60
refolding capacity (after heat denatur.) high high low
80C, 10 min, 0.1 Naac pH 5.5, 32 - 38% 52 - 60% 72 - 85%
[residual activity]
feed pelleting [residual activity]:
70C 100% 75 - 92% 80 - 96%
80C 86 - 90% 68 - 85% 86 - 90%
90C 78 - 82% 52 - 69% 78 - 82%

MRI Department of Food Technology and Bioprocess Engineering 03.01.2013 21

 Phytases for Feed Application

ideal phytases for animal feed applications should fulfil a series of quality
criteria:

effective release of phytate phosphate in the digestive tract

feed processing stability

storage stability
stability of the pure phytase preparations
stability in the presence of pre-mixes, feed

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 Characteristics of Commercial Phytases
temperature optimum, temperature stability

characteristics OptiPhos Phyzyme Ronozyme

storage stability, pure enzyme, 23C
30 days > 91% > 91% > 91%
60 days > 85% > 85% > 85%
90 days > 78% > 78% > 78%
120 days > 71% > 71% > 71%
180 days > 85% > 85% > 85% (uncoated
only 50%)
storage stability, pure enzyme, 37C,
30 days
coated 91 - 93% 69 - 74% 69%
uncoated 91 - 93% 69 - 74% 36%

Sulabo et al., J. Anim. Sci. 89, 4262-4271 (2011)

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 Characteristics of Commercial Phytases
temperature optimum, temperature stability

characteristics OptiPhos Phyzyme Ronozyme

storage stability, 23C, 180 days
pure enzyme > 85% > 85% > 85% (uncoated
only 50%)
in mineral pre-mixes > 73% > 73% (uncoated > 73%
only 67%)
in vitamin-mineral pre-mixes > 60% (uncoated > 60% > 60%
only 43%)
storage stability, 23C, 360 days
in mineral pre-mixes > 83% > 75% > 63%
in vitamin-mineral pre-mixes > 83% > 75% > 63%

Sulabo et al., J. Anim. Sci. 89, 4262-4271 (2011)

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 Phytases for Food Application

most stable at <=23C

pure phytases more stable than in pre-mixes
reduced activity in pre-mixes when stored longer and at higher temperatures
OptiPhos is the most stable product in pre-mixes
coated more stable than non-coated

post-fermentation activities such as product formulation and optimization can

influence the properties of the final phytase preparation
especially efficacy and storage stability of the phytase are dependent on product
formulation

MRI Department of Food Technology and Bioprocess Engineering 03.01.2013 25

 Phytases for Feed Application

ideal phytases for animal feed applications should fulfil a series of quality
criteria:

effective release of phytate phosphate in the digestive tract

feed processing stability

storage stability

economical production
specific activity
production system / expression system / phytase-encoding gene

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 Characteristics of Commercial Phytases
molecular mass, production system, specific activity

characteristics P. lycii A. niger E. coli

monomer monomer monomer
number of amino acids 409 448 433
molecular mass (non-glycosylated) 44 49 42
molecular mass (glycosylated) 70 - 72 66 - 120 47 - 56
production organism A. oryzae A. niger P. pastoris
S. pombe
spec. activity at pH optimum [U mg-1] 1080 103 751 811

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 Phytases for Food Application
Conclusion

no two phytases perform the same

an understanding of the differences between phytases is necessary to secure

optimal animal performance

biochemical characterization of enzymes is essential, but is solely inaccurate to

predict enzyme performance in swine and poultry
properties could be guidelines on potential functionality in animal feeds and
digestive systems

there is no unique point responsible for the better performance in vivo but a
conjunction of all factors

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Thank You Very Much
For Your Attention !

Prof. Dr. Ralf Greiner

Department of Food Technology and Bioprocess Engineering
Max Rubner-Institut
Federal Research Institute of Nutrition and Food
Haid-und-Neu-Strae 9 D-76131 Karlsruhe
Tel.: ++49 (0)721 6625 300 Fax: ++49 (0)721 6625 303
ralf.greiner@mri.bund.de www.mri.bund.de

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