Pathway of dephosphorylation of myo-inositol hexakisphosphate by phytases of legume seeds

Article abstract of DOI:10.1021/jf025620t

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytases purified from faba bean and lupine seeds, respectively, was established. The data demonstrate that the legume seed phytases under investigation dephosphorylate myo-inositol hexakisphosphate in a stereospecific way. The phytase from faba bean seeds and the phytase LP2 from lupine seeds degrade phytate by sequential removal of phosphate groups via D-Ins(1,2,3,5,6)P₅, D-Ins(1,2,5,6)P₄, D-Ins(1,2,6)P₃, and D-Ins(1,2)P₂ to finally Ins(2)P, whereas the phytases LP11 and LP12 from lupine seeds generate the final degradation product Ins(2)P via D-Ins(1,2,4,5,6)P₅, D-Ins(1,2,5,6)P₄, D-Ins(1,2,6)P₃, and D-Ins(1,2)P₂.

Full text of DOI:10.1021/jf025620t

J. Agric. Food Chem. 2002, 50, 6865−6870 6865
Pathway of Dephosphorylation of myo-Inositol
Hexakisphosphate by Phytases of Legume Seeds
,
†
‡
‡
RALF GREINER,* MARIE LARSSON ALMINGER, NILS-GUNNAR CARLSSON,
§
§
§
MERCEDES MUZQUIZ, CARMEN BURBANO, CARMEN CUADRADO,
MERCEDES M. PEDROSA,§ AND CARMEN GOYOAGA§
Centre for Molecular Biology, Federal Research Centre for Nutrition, Haid-und-Neu-Strasse 9,
D-76131 Karlsruhe, Germany; Department of Food Science, Chalmers University of Technology,
c/o SIK, Box 5401, SE-402 29 G o¨ teborg, Sweden; and SGIT-INIA, Area de Tecnologia de Alimentos,
Apartado 811, 28080 Madrid, Spain
Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway
of dephosphorylation of myo-inositol hexakisphosphate by the phytases purified from faba bean and
lupine seeds, respectively, was established. The data demonstrate that the legume seed phytases
under investigation dephosphorylate myo-inositol hexakisphosphate in a stereospecific way. The
phytase from faba bean seeds and the phytase LP2 from lupine seeds degrade phytate by sequential
removal of phosphate groups via D-Ins(1,2,3,5,6)P
to finally Ins(2)P, whereas the phytases LP11 and LP12 from lupine seeds generate the final
2
degradation product Ins(2)P via D-Ins(1,2,4,5,6)P , D-Ins(1,2,5,6)P , D-Ins(1,2,6)P , and D-Ins(1,2)P .
5 4 3 2
, D-Ins(1,2,5,6)P , D-Ins(1,2,6)P , and D-Ins(1,2)P
5
4
3
KEYWORDS: Faba bean; legume phytase; lupine; myo-inositol phosphate isomers; phytate degradation
INTRODUCTION
by controlling hypercholesterolemia and atherosclerosis (12, 13),
prevention of renal stone formation (14, 15), and reduced risk
of colon cancer (16, 21). Because different structural isomers
may result in different physiological functions, identification
of the myo-inositol phosphates generated during enzymatic
phytate degradation is of great importance to exploit the full
potential of the naturally occurring phytases. The complete
phytate degradation pathway was elucidated for only a few
phytases, focusing on those of microbial (1-3, 11, 22) or cereal
origin (4-7). There is only one paper describing the phytate
degradation pathway of a legume phytase (8).
Because the bioactive myo-inositol phosphate isomers gener-
ated by legume phytases may be different from those generated
by phytases from microbial or cereal origin and because it was
shown recently that legume phytases may be an alternative to
the corresponding enzymes of microorganisms and cereal seeds
for a biotechnological application to reduce phytate content
during food and feed processing (23), the objective of this study
was to elucidate the complete hydrolysis pathway of phytate
by phytases purified from faba bean and lupine seeds, respec-
tively.
Phytases [myo-inositol(1,2,3,4,5,6)hexakisphosphate phos-
phohydrolases] belong to a special group of phosphatases that
are capable of sequentially hydrolyzing phytate [myo-inositol-
(1,2,3,4,5,6)hexakisphosphate] to a series of lower myo-inositol
phosphates and orthophosphate. In vitro it was shown that a
single phytase can catalyze the sequential breakdown from myo-
inositol hexakisphosphate to myo-inositol monophosphate or
even myo-inositol (1-8). With respect to the final phytate
degradation product, phytases can be divided into two groups.
Enzymes exhibiting an acidic pH optimum release five or even
all six phosphate groups of phytate and myo-inositol mono-
phosphate and myo-inositol, respectively, have been detected
as their final degradation products. On the other hand, the
alkaline phytases (9-11) are not capable of accepting a myo-
inositol phosphate with three or fewer phosphate residues as a
substrate. Thus, myo-inositol trisphosphate was shown to be the
end product of phytate degradation by these alkaline phytases.
Depending on the phytase used, different phosphate residues
of phytate may be released at different rates and in different
order, resulting in the generation of different phosphate-
containing intermediates.
MATERIALS AND METHODS
Certain myo-inositol phosphates have been suggested to have
beneficial health effects, such as amelioration of heart disease
Chemicals. Vicia faba var. Alameda was obtained from CIDA of
Cordoba (Spain), Lupinus albus var. Amiga from S u¨ dwestsaat GbR
(Rastatt, Germany), and Aspergillus niger phytase from Novo Nordisk
*
Author to whom correspondence should be addressed [telephone
+
49 (0) 721/6625 479; fax +49 (0) 721/6625 457; e-mail ralf.greiner@
(Copenhagen, Denmark). Phytic acid dodecasodium salt was purchased
bfe.uni-karlsruhe.de].
from Aldrich (Steinheim, Germany). Ultrasep ES 100 RP18 was
obtained from Bischoff (Leonberg, Germany), and a high-performance
ion chromatography (HPIC) column Carbo-Pac PA-100 from Dionex
†
Federal Research Centre for Nutrition.
‡
Chalmers University of Technology.
§
SGIT-Inia.
1
0.1021/jf025620t CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/11/2002

6866 J. Agric. Food Chem., Vol. 50, No. 23, 2002
Greiner et al.
(
Sunnyvale, CA). AG1 X-4, 100-200 mesh, resin was purchased from
formate. After lyophilization, the residues were dissolved in 500 µL
of a solution of pyridine/bis(trimethylsilyl)trifluoroacetamide (1:1 v/v)
and incubated at room temperature for 24 h. The silylated products
were injected at 270 °C into a gas chromatograph coupled with a mass
spectrometer. The stationary phase was methylsilicon in a fused silica
column (0.25 mm × 15 m). Helium was used as the carrier gas at a
Bio-Rad (M u¨ nchen, Germany).
Purification of the Phytases. Purification of the phytases of
Aspergillus niger (3), Escherichia coli (24), rye (25), faba bean seeds
(26), and lupine seeds (23) was performed as described previously.
All phytases were purified to apparent homogeneity according to
denaturing and non-denaturing polyacrylamide gel electrophoresis.
Assay of Phytase Activity. Phytase activity measurements were
carried out at 35 °C. The enzymatic reactions were started by the
addition of 10 µL of enzyme to the assay mixtures. The incubation
mixture for phytase activity determination consisted of 350 µL of 0.1
M sodium acetate, pH 4.5 (Escherichia coli and Aspergillus niger),
-
1
flow rate of 0.5 m s . The following heating program was used for
-1
the column: increase from 100 to 340 °C at 4 °C min . Ionization
was performed by electron impact at 70 eV and 250 °C.
Statistical Methods. For statistical comparison Student’s t test was
used.
0
(
.1 M sodium acetate, pH 6.0 (rye), and 0.1 M sodium acetate, pH 5.0
faba bean and lupine), respectively, containing 500 nmol of sodium
phytate.
To determine the kinetic parameters for enzymatic dephosphorylation
RESULTS
Intermediates of Enzymatic myo-Inositol Hexakisphos-
phate Dephosphorylation. The identification of the hydrolysis
products of myo-inositol hexakisphosphate generated by the
phytase purified from faba bean seeds was performed by isomer-
specific HPIC analysis (Figure 1). The chromatographic profile
of the zero-time control indicated only the myo-inositol hexa-
kisphosphate peak (peak 1). After 15 min of incubation, the
quantity of peak 1 (myo-inositol hexakisphosphate) had de-
creased and peak 4 [D/L-Ins(1,2,3,4,5)P5] appeared as the major
degradation product, accompanied by small amounts of peak 3
[D/L-Ins(1,2,4,5,6)P5]. After 30 min of incubation, a further
decrease in peak 1 (myo-inositol hexakisphosphate) was ob-
served. Both myo-inositol pentakisphosphates peak 4 [D/L-
Ins(1,2,3,4,5)P5] and peak 3 [D/L-Ins(1,2,4,5,6)P5] had also
decreased, whereas peak 8 (D/L-Ins(1,2,5,6)P4) had increased.
Furthermore, small amounts of peak 12 [D/L-Ins(1,2,3,4)P4] and
peak 18 [D/L-Ins(1,2,6)P3 and/or Ins(1,2,3)P3] were found. After
of individual myo-inositol phosphates by the phytase from faba bean
and lupine seeds, 10 milliunits of the phytase were added to sequentially
diluted solutions of the purified myo-inositol phosphate isomer (2.0,
1
.0, 0.5, 0.25, 0.125, 0.06, 0.03, and 0.015 mM) in 400 µL of 0.1 M
sodium acetate buffer, pH 5.0, at 35 °C. The following myo-inositol
phosphate isomers were used: D-Ins(1,2,4,5,6)P , D-Ins(1,2,3,4,5)P
D-Ins(1,2,3,5,6)P , and the myo-inositol pentakisphosphate produced
5
5
,
5
by the legume phytase under investigation.
After an incubation period of 30 min, the liberated phosphate was
quantified by using the ammonium molybdate method (27) with some
modification. The rate of reaction was linear for the 30 min incubation
time (data not shown); 1.5 mL of a freshly prepared solution of
acetone/5 N sulfuric acid/10 mM ammonium molybdate (2:1:1 v/v)
and thereafter 100 µL of 1.0 M citric acid were added. Any cloudiness
was removed by centrifugation prior to the measurement of absorbance
at 355 nm. To quantify the released phosphate, a calibration curve was
produced over the range of 5-600 nmol phosphate. Activity (units)
was expressed as micromoles of phosphate liberated per minute. Blanks
were run by addition of the ammonium molybdate solution prior to
addition of the enzyme solution to the assay mixture. The kinetic
6
0 min of incubation, a further decrease in peaks 1, 3, and 4
(myo-inositol hexakisphosphate and pentakisphosphates) and a
further increase in peak 8 [D/L-Ins(1,2,5,6)P4], peak 12 [D/L-
Ins(1,2,3,4)P4], and peak 18 [D/L-Ins(1,2,6)P3 and/or Ins(1,2,3)P3]
was observed. In addition, small amounts of peak 22 [D/L-
Ins(1,2)P , Ins(2,5)P , and/or D/L-Ins(4,5)P ] appeared. After
constants (K and Vmax) were calculated from Lineweaver-Burk plots
M
of the data. For calculation of kcat the following molecular masses were
used: faba bean, 66 kDa (26); lupine LP11, 57 kDa; lupine LP12, 57
kDa; and lupine LP2, 64 kDa (23).
2
2
2
9
0 min of incubation, myo-inositol hexakisphosphate (peak 1)
Preparation of Lower myo-Inositol Phosphates. The phytases from
Aspergillus niger, Escherichia coli, and rye were used to generate
D-Ins(1,2,4,5,6)P , D-Ins(1,2,3,4,5)P , and D-Ins(1,2,3,5,6)P . myo-
5 5 5
and the myo-inositol pentakisphosphates (peaks 3 and 4) were
completely degraded to lower myo-inositol phosphates, mainly
peak 8 [D/L-Ins(1,2,5,6)P4], peak 18 [D/L-Ins(1,2,6)P3 and/or
Ins(1,2,3)P3], and peak 22 [D/L-Ins(1,2)P2, Ins(2,5)P2, and/or
D/L-Ins(3,5)P2]. In addition, traces of peak 12 [D/L-Ins(1,2,3,4)P4]
were still present. After 120 min of incubation, only small
amounts of peak 8 [D/L-Ins(1,2,5,6)P4] remained within the
myo-inositol tetrakisphosphates. They were nearly completely
degraded to peak 18 [D/L-Ins(1,2,6)P and/or Ins(1,2,3)P ] and
Inositol hexakisphosphate (500 µmol) was incubated at 35 °C in a
mixture containing 50 mM ammonium acetate, pH 4.5 (Escherichia
coli and Aspergillus niger), 50 mM ammonium acetate, pH 6.0 (rye),
and 50 mM ammonium acetate, pH 5.0 (faba bean and lupine),
respectively, and 10 units of the phytases in a final volume of 200
mL. After an incubation period of 30 min, the reactions were stopped
by heat treatment (95 °C for 10 min). The myo-inositol pentakis-
phosphates were purified as described previously (3).
3
3
peak 22 [D/L-Ins(1,2)P2, Ins(2,5)P2, and/or D/L-Ins(4,5)P2].
Incubation of the faba bean phytase with a limiting amount of
phytate resulted in the generation of one myo-inositol mono-
phosphate, which was identified as Ins(2)P by gas chromatog-
raphy-mass spectrometry (data not shown).
Production of Enzymatically Formed Hydrolysis Products. The
enzymatic reaction was started at 35 °C by the addition of 50 µL of
the suitably diluted solution of the different legume phytases to the
-
1
incubation mixtures (100 milliunits mL ). The incubation mixture
consisted of 1250 µL of 0.1 M sodium acetate buffer, pH 5.0, containing
A detailed characterization of the hydrolysis pathway of myo-
inositol hexakisphosphate by the phytases from lupine seeds
(Figure 2) revealed that LP11 and LP12 degrade phytate via
D/L-Ins(1,2,4,5,6)P5 (peak 3), D/L-Ins(1,2,5,6)P4 (peak 8),
Ins(1,2,3)P or D/L-Ins(1,2,6)P (peak 18), and D/L-Ins(1,2)P
2
.5 µmol of sodium phytate. From the incubation mixture, 100 µL
samples were removed periodically, and the reaction was stopped by
heat treatment (90 °C for 5 min).
Identification of Enzymatically Formed Hydrolysis Products
(
InsP
chromatographed on an HPIC system using a Carbo Pac PA-100 (4 ×
50 mm) analytical column and a gradient of 5-98% HCl (0.5 M, 0.8
6
)InsP
2
). Fifty microliters of the heat-treated samples was
3
3
2
or Ins(2,5)P2 or D/L-Ins(4,5)P2 (peak 22) to finally Ins(2)P,
whereas LP2 hydrolyzes phytate, as the phytase from faba bean
seeds, via D/L-Ins(1,2,3,4,5)P5 (peak 4), D/L-Ins(1,2,5,6)P4
2
-1
mL min ) as described by Skoglund et al. (28). The eluants were mixed
in a postcolumn reactor with 0.1% Fe(NO ‚9H O in a 2% HClO
solution (0.4 mL min-1) according to the method of Phillippy and Bland
29). The combined flow rate was 1.2 mL min-1
Identification of the myo-Inositol Monophosphate Isomer. myo-
3
)
3
2
4
(peak 8), Ins(1,2,3)P3 or D/L-Ins(1,2,6)P3 (peak 18), and D/L-
Ins(1,2)P2 or Ins(2,5)P2 or D/L-Ins(4,5)P2 (peak 22) to finally
Ins(2)P.
(
.
Kinetic Studies. To determine the kinetic parameters for the
hydrolysis of some myo-inositol phosphate isomers at pH 6.0
and 35 °C by the legume phytase under investigation, the
Inositol monophosphate was produced by incubation of 1.0 unit of the
legume phytase with a limiting amount of myo-inositol hexakisphos-
phate (0.1 µmol) in a final volume of 500 µL of 50 mM ammonium

Enzymatic Phytate Degradation
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6867
Figure 1. HPIC analysis of the enzymatically formed hydrolysis products of myo-inositol hexakisphosphate (InsP
faba bean seeds reference sample (A). The source of the reference myo-inositol phosphates is as indicated in Skoglund et al. (28), phytase from faba
bean seeds. Peaks: (1) Ins(1,2,3,4,5,6)P ; (2) Ins(1,3,4,5,6)P ; (3) D/L-Ins(1,2,4,5,6)P ; (4) D/L-Ins(1,2,3,4,5)P ; (5) Ins(1,2,3,4,6)P ; (6) D/L-Ins(1,4,5,6)P
7) Ins(2,4,5,6)P ; (8) D/L-Ins(1,2,5,6)P ; (9) D/L-Ins(1,3,4,5)P ; (10) D/L-Ins(1,2,4,5)P ; (11) Ins(1,3,4,6)P ; (12) D/L-Ins(1,2,3,4)P ; (13) D/L-Ins(1,2,4,6)P
; (15) Ins(4,5,6)P ; (16) D/L-Ins(1,5,6)P ; (17) D/L-Ins(1,4,5)P ; (18) D/L-Ins(1,2,6)P , Ins(1,2,3)P ; (19) D/L-Ins(1,3,4)P ; (20) D/L-
, D/L-Ins(2,4,5)P ; (21) D/L-Ins(2,4)P ; (22) D/L-Ins(1,2)P , Ins(2,5)P , D/L-Ins(4,5)P ; (23) D/L-Ins(1,4)P , D/L-Ins(1,6)P
6 2
−InsP ) by the phytase purified from
6
5
5
5
5
4
;
(
4
4
4
4
4
4
4
;
(
14) Ins(1,2,3,5)P
4
3
3
3
3
3
3
Ins(1,2,4)P
3
3
2
2
2
2
2
2
.
enzymes were added to sequentially diluted solutions
of Ins(1,2,3,4,5,6)P6, D-Ins(1,2,4,5,6)P5, D-Ins(1,2,3,5,6)P5,
D-Ins(1,2,3,4,5)P5, and the major myo-inositol pentakis-
phosphate generated by the legume phytase under investigation.
The kinetic parameters (KM and kcat) were calculated from
Lineweaver-Burk plots of the data (Table 1). KM and kcat for
the enzymatic hydrolysis of Ins(1,2,3,4,5,6)P6 were determined
the myo-inositol pentakisphosphates included in the study for
every single enzyme, it is obvious that the kinetic parameters
for the myo-inositol pentakisphosphate generated by the enzyme
under investigation are not statistically significant different
(P < 0.05) from the kinetic parameters for the hydrolysis
of D-Ins(1,2,3,5,6)P5 (faba bean and lupine LP2) and
D-Ins(1,2,4,5,6)P5 (lupine LP11 and LP12), respectively.
-
1
-1
to be KM ) 148 µmol L (faba bean), 80 µmol L (lupine
-
1
-1
LP11), 300 µmol L (lupine LP12), and 130 µmol L (lupine
DISCUSSION
-
1
-1
LP2) and kcat ) 704 s (faba bean), 523 s (lupine LP11),
89 s (lupine LP12), and 533 s (lupine LP2), respectively.
-
1
-1
5
Several studies have been performed to elucidate the hy-
drolysis pathway of phytate by phytases from bacterial (1, 2,
11), fungal (3, 22), and cereal (4-7) origins, but only one study
on the phytate hydrolysis pathway of a legume phytase has been
reported (8). The phytase from mung beans seeds was reported
In comparison to myo-inositol hexakisphosphate, the affinity
of the myo-inositol pentakisphosphates included in the study
for the legume phytases under investigation and their maximal
rates of hydrolysis were higher. Comparing the hydrolysis of

6868 J. Agric. Food Chem., Vol. 50, No. 23, 2002
Greiner et al.
Figure 2. HPIC analysis of the enzymatically formed hydrolysis products of myo-inositol hexakisphosphate (InsP
lupine seeds reference sample (A). The source of the reference myo-inositol phosphates is as indicated in Skoglund et al. (28). Reference sample: see
Figure 1, incubation time ) 60 min. (B) Phytase LP11; (C) phytase LP12; (D) phytase LP2. Peaks: (1) Ins(1,2,3,4,5,6)P ; (2) Ins(1,3,4,5,6)P ; (3)
D/L-Ins(1,2,4,5,6)P ; (4) D/L-Ins(1,2,3,4,5)P ; (5) Ins(1,2,3,4,6)P ; (6) D/L-Ins(1,4,5,6)P ; (7) Ins(2,4,5,6)P ; (8) D/L-Ins(1,2,5,6)P ; (9) D/L-Ins(1,3,4,5)P
10) D/L-Ins(1,2,4,5)P ; (11) Ins(1,3,4,6)P ; (12) D/L-Ins(1,2,3,4)P ; (13) D/L-Ins(1,2,4,6)P ; (14) Ins(1,2,3,5)P ; (15) Ins(4,5,6)P ; (16) D/L-Ins(1,5,6)P
17) D/L-Ins(1,4,5)P ; (18) D/L-Ins(1,2,6)P , Ins(1,2,3)P ; (19) D/L-Ins(1,3,4)P ; (20) D/L-Ins(1,2,4)P , D/L-Ins(2,4,5)P ; (21) D/L-Ins(2,4)P ; (22) D/L-
Ins(1,2)P , Ins(2,5)P , D/L-Ins(4,5)P ; (23) D/L-Ins(1,4)P , D/L-Ins(1,6)P
6 2
−InsP ) by the phytase purified from
6
5
5
5
5
4
4
4
4
;
(
4
4
4
4
4
3
3
;
(
3
3
3
3
3
3
2
2
2
2
2
2
.
to degrade phytate via two independent routes: The routes pro-
ceed via D-Ins(1,2,3,5,6)P5, D-Ins(1,2,3,6)P4, D-Ins(1,2,6)P3, and
D-Ins(2,6)P2 to finally Ins(2)P and via D-Ins(1,2,3,5,6)P5,
D-Ins(1,2,3,6)P4, D-Ins(1,2,3)P3, and D-Ins(1,2)P2 to finally
Ins(2)P.
It was concluded that the faba bean phytase and the phytase
LP2 from lupine seeds generate D-Ins(1,2,3,5,6)P5 as the major
myo-inositol pentakisphosphate intermediate, whereas the major
myo-inositol pentakisphosphate intermediate generated by the
phytases LP11 and LP12 from lupine seeds is D-Ins(1,2,4,5,6)P5,
because the kinetic parameters for the myo-inositol pentakis-
phosphate generated by the individual phytase under investiga-
tion are not statistically significantly different (P < 0.05) from
the kinetic parameters for the hydrolysis of D-Ins(1,2,3,5,6)P5
rice, Saccharomyces cereVisiae, Pseudomonas, Klebsiella ter-
rigena, and Aspergillus niger (1, 2, 4-8, 30, 31) fit into this
general consideration. However, this is not a general rule, as
exemplified by indications of 3-phytase activity in lupine (LP11
and LP12) and soybean seeds and 6-phytase activity in
Paramecium (22) and Escherichia coli (2).
The myo-inositol phosphate intermediates generated by the
legume phytases under investigation are consistent with the
degradation pathways shown in Figure 3. All enzymes remove
phosphate stepwise from the phytate molecule, whereby each
myo-inositol intermediate is released from the enzyme and may
become a substrate for further hydrolysis. The major phytate
degradation pathway of the phytases LP11 and LP12 from
lupine seeds proceeds via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4,
D-Ins(1,2,6)P3, and D-Ins(1,2)P2 to finally Ins(2)P, whereas the
faba bean phytase and the phytase LP2 from lupine seeds
degrade phytate via D-Ins(1,2,3,5,6)P5, D-Ins(1,2,5,6)P4,
D-Ins(1,2,6)P3, and D-Ins(1,2)P2 to finally Ins(2)P. The phytase
purified from faba bean seeds exhibited to a small extent also
3-phytase activity. Thereby, D/L-Ins(1,2,4,5,6)P5 is generated,
which may be linked to the major degradation pathway by
dephosphorylation to D/L-Ins(1,2,5,6)P4. A further minor
pathway seems to proceed from D-Ins(1,2,3,5,6)P5 via
D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, and/or D-Ins(1,2,6)P3 and
D-Ins(1,2)P2 to finally Ins(2)P.
(faba bean and lupine LP2) and D-Ins(1,2,4,5,6)P5 (lupine LP11
and LP12), respectively. Because the phytases under investiga-
tion generate D-Ins(1,2,4,5,6)P5 (lupine LP11 and LP12) and
D-Ins(1,2,3,5,6)P5 (faba bean and lupine LP2), respectively, as
the major myo-inositol pentakisphosphate intermediate, the
lupine phytases LP11 and LP12 are 3-phytases (EC 3.1.3.8),
whereas the phytase purified from faba bean seeds and the
phytase LP2 from lupine seeds are 6-phytases (EC 3.1.3.26).
Phytases from microorganisms are considered to be 3-phytases
(EC 3.1.3.8), whereas seeds of higher plants are said to contain
6-phytases (EC 3.1.3.26). Thus, the phytases from faba bean
seeds and the phytase LP2 from lupine seeds as well as the
phytases from mung bean, rye, barley, spelt, oat, wheat bran,
All theoretically existing myo-inositol pentakis- and tetra-
kisphosphate isomers are well resolved on the HPIC system

Enzymatic Phytate Degradation
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6869
Table 1. Kinetic Constants for Enzymatic myo-Inositol Phosphate
Dephosphorylation
a
-1
kcat (s-1)
a
substrate
InsP6
enzyme
KM (µmol L )
faba bean
148 ± 9
80 ± 5
300 ± 17
130 ± 7
704 ± 31
523 ± 21
589 ± 26
533 ± 18
lupine LP11
lupine LP12
lupine LP2
InsP5b
faba bean
99 ± 5a
65 ± 3b
152 ± 12c
84 ± 4d
921 ± 54a
671 ± 19b
684 ± 42b
691 ± 38b
lupine LP11
lupine LP12
lupine LP2
D-Ins(1,2,4,5,6)P5
D-Ins(1,2,3,5,6)P5
D-Ins(1,2,3,4,5)P5
faba bean
nd
nd
lupine LP11
lupine LP12
lupine LP2
68 ± 5b
146 ± 9c
nd
664 ± 23b
696 ± 31b
nd
faba bean
104 ± 6a
nd
nd
936 ± 42a
nd
nd
lupine LP11
lupine LP12
lupine LP2
87 ± 3d
679 ± 19b
faba bean
411 ± 27e
nd
nd
271 ± 18c
nd
nd
lupine LP11
lupine LP12
lupine LP2
521 ± 35f
196 ± 13d
a
Entries followed by different letters in a column differ (P < 0.05). nd, not
determined. The data are mean values ± standard deviation of five independent
b
experiments. Generated by the legume phytase under investigation.
used (Figure 1), so the identities of the myo-inositol pentakis-
and tetrakisphosphate isomers generated by the phytases under
investigation is well established. A clear identification of the
formed myo-inositol trisphosphate isomers by HPIC was not
possible until now, because not all theoretically existing isomers
are available. Theoretically D-Ins(1,2,5)P3, D-Ins(1,2,6)P3, and
D-Ins(2,5,6)P3 as well as D-Ins(1,2)P2, D-Ins(2,5)P2, and
D-Ins(2,6)P2 may occur during degradation of D-Ins(1,2,5,6)P4
to Ins(2)P. According to HPIC, D-Ins(2,6)P2 (peak 21) has to
be excluded as an intermediate, because this myo-inositol
bisphosphate elutes well resolved from the InsP2 peak (peak
Figure 3. Suggested degradation pathways of phytate by the phytase
under investigation: (solid arrow) Major pathway of myo-inositol hexakis-
phosphate dephosphorylation by the legume phytase under investigation;
(
broken arrow) minor pathway of myo-inositol hexakisphosphate dephos-
phorylation by the faba bean phytase. The faba bean enzyme generates
also small amounts of D/L-Ins(1,2,4,5,6)P
5
.
analysis and kinetic studies (4), gave identical results, the
suitability of the latter to reveal the stereospecificity of
enzymatic myo-inositol hexakisphosphate degradation has al-
ready been demonstrated.
2
2) generated during myo-inositol hexakisphosphate dephos-
phorylation by the phytases under investigation (Figures 1 and
). In addition, only a myo-inositol trisphosphate intermediate
2
coeluting with D-Ins(1,2,6)P3 (peak 18) could serve as a
precursor of Ins(2)P. Thus, Ins(2)P is generated from
D-Ins(1,2,5,6)P4 via D-Ins(1,2,6)P3, and D-Ins(1,2)P2.
Inositol phosphates containing the 1,2,3-trisphosphate cluster
are iron-binding antioxidants (32, 33), and D-Ins(1,2,3,6)P4 has
a structure that is moderately effective in opening calcium
channels (34, 35). In addition, D-Ins(1,2,6)P3 has anti-inflam-
matory and antisecretory properties (36). Whether myo-inositol
phosphate isomers have nutritional significance in food is for
the most part speculative, even if an in vivo antioxidative effect
of dietary myo-inositol phosphates has been reported (37, 38).
Until now, the diversity and practical unavailability of the
individual myo-inositol phosphate intermediates precluded their
being tested as bioactive. Studies are now under way to make
individual myo-inositol phosphates available in pure form and
in sufficient quantities for physiological studies.
Thus, the phytate degradation pathways elucidated for the
legume phytases under investigation are identical either to the
phytate degradation pathway determined for the phytases from
Saccharomyces cereVisiae (3) and Pseudomonas (1) (lupine
LP11 and LP12) or to the phytate degradation pathway obtained
for the phytases from rye, barley, spelt, oat (4), wheat bran (7),
and rice (5) (faba bean and lupine LP2). Only the minor phytate
degradation pathway of the phytase from faba bean seeds shows
some similarities to that of the phytase from mung bean (8).
The most striking difference is the appearance of only
D-Ins(1,2,3,6)P4 as the myo-inositol tetrakisphosphate intermedi-
ate during phytate hydrolysis by the phytase from mung bean
seeds, whereas the phytase from faba bean seeds generates
mainly D-Ins(1,2,5,6)P4. The difference may be due to not only
the different specificities of the two enzymes but also the
different analytical approaches to elucidate the enzymatic
phytate degradation pathways. However, because myo-inositol
hexakisphosphate dephosphorylation by the wheat phytase
deduced using an NMR technique (7) and by the phytase D21
from spelt, an old wheat variety, using a combination of HPIC
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