Content uploaded by Byung-Chul Oh
Author content
All content in this area was uploaded by Byung-Chul Oh
Content may be subject to copyright.
1
2
Springer
Dear Author:
Please find attached the final pdf file of your contribution, which can be viewed using the
Acrobat Reader, version 3.0 or higher. We would kindly like to draw your attention to the
fact that copyright law is also valid for electronic products. This means especially that:
• You may print the file and distribute it amongst your colleagues in the scientific
community for scientific and/or personal use.
• You may make your article published by Springer-Verlag available on your personal
home page provided the source of the published article is cited and Springer-Verlag
and/or other owner is mentioned as copyright holder. You are requested to create a link
to the published article in Springer's internet service. The link must be accompanied by
the following text: "The original publication is available at springerlink.com". Please use
the appropriate DOI for the article. Articles disseminated via SpringerLink are indexed,
abstracted and referenced by many abstracting and information services, bibliographic
networks, subscription agencies, library networks and consortia.
• Without having asked Springer-Verlag for a separate permission your
institute/your company is not allowed to place this file on its homepage.
• You may not alter the pdf file, as changes to the published contribution are
prohibited by copyright law.
• Please address any queries to the production editor of the journal in question, giving
your name, the journal title, volume and first page number.
Yours sincerely,
Springer-Verlag
Appl Microbiol Biotechnol (2004) 63: 362–372
DOI 10.1007/s00253-003-1345-0
MINI-REVIEW
B.-C. Oh
.
W.-C. Choi
.
S. Park
.
Y.-o. Kim
.
T.-K. Oh
Biochemical properties and substrate specificities of alkaline
and histidine acid phytases
Received: 25 November 2002 / Revised: 31 March 2003 / Accepted: 4 April 2003 / Published online: 28 October 2003
# Springer-Verlag 2003
Abstract Phytases are a special class of phosphatase that
catalyze the sequential hydrolysis of phytate to less-
phosphorylated myo-inositol derivatives and inorganic
phosphate. Phytases are added to animal feedstuff to
reduce phosphate pollution in the environment, since
monogastric animals such as pigs, poultry, and fish are
unable to metabolize phytate. Based on biochemical
properties and amino acid sequence alignment, phytases
can be categorized into two major classes, the histidine
acid phytases and the alkaline phytases. The histidine acid
phosphatase class shows broad substrate specificity and
hydrolyzes metal-free phytate at the acidic pH range and
produces myo-inositol monophosphate as the final prod-
uct. In contrast, the alkaline phytase class exhibits strict
substrate specificity for the calcium–phytate complex and
produces myo-inositol trisphosphate as the final product.
This review describes recent findings that present novel
viewpoints concerning the molecular basis of phytase
classification.
Introduction
Phytate (myo-inositol 1,2,3,4,5,6-hexakisphosphate; IP
6
)is
the principal storage form of phosphorus, inositol, and a
variety of minerals in plants, representing approximately
75–80% of the total phosphorus in plant seeds (Cosgrove
1966). In nature, phytate primarily exists as metal–phytate
complexes with nutritionally important cations, such as
Ca
2+
,Zn
2+
, and Fe
2+
(Asada et al. 1969). Monogastric
animals are unable to utilize phytate, since they have no or
low levels of phytase activity in their digestive tracks
(Schroder et al. 1996). Therefore, inorganic phosphate is
frequently added to their feed to facilitate optimal growth
for these animals. This approach could stimulate algal
blooms and eutrophication of surface water, due to the
high content of phytate and inorganic phosphate in the
excretion of the animals. Furthermore, phytate acts as an
anti-nutritional factor by chelating divalent cations and
preventing the absorption of minerals, such as Ca
2+
,Zn
2+
,
and Fe
2+
(Urbano et al. 2000).
Phytases (IP
6
phosphohydrolase) are a special class of
phosphatases that catalyze the sequential hydrolysis of
phytate to less phosphorylated myo-inositol derivatives
and inorganic phosphate (Wyss et al. 1999a). Phytases are
used as an animal feed additive to improve phosphate
bioavailability and to reduce the loss of phosphate and
divalent cations from phytate. Most bacterial, fungal, and
plant phytases belong to the histidine acid phosphatases
(HAP; EC 3.1.3.2; Van Etten et al. 1991). All members of
the HAP class share a conserved active site motif,
RHGXRXP, unique to this enzyme class (Van Etten et
al. 1991).
Alkaline phytases are also widely distributed in nature
(Liu et al. 1998). However, enzymatic characterization of
phytases has been mainly focused on HAPs, since the
most widely used technique to screen phytase activity is
designed to measure the amount of inorganic phosphate
released from metal-free phytate (sodium phytate) as
substrate (Englen et al. 1994). Therefore, the screening of
phytase-producing microorganisms or plants and the
cloning of the phytase-encoding gene according to this
principle led us to find HAPs which react with the metal-
free form of phytate at acidic pH. The International Union
of Pure and Applied Chemistry and the International
Union of Biochemistry (IUPAC–IUB 1975) classified this
B.-C. Oh
.
W.-C. Choi
.
T.-K. Oh (*)
Microbial Genomics Laboratory, Korea Research Institute of
Bioscience and Biotechnology,
P.O. Box 115, 305-600 Yusong, Taejon, Korea
e-mail: otk@kribb.re.kr
Tel.: +82-42-8604374
Fax: +82-42-8604595
S. Park
Comparative Pharmacokinetics and Microbial Pharmacology
College of Veterinary Medicine, Kyungpook National
University,
702-701 Taegu, Korea
Y.-o. Kim
Biotechnology Research Center, National Fisheries Research
and Development Institute,
619-902 Busan, Korea
group of HAPs as 3-phytase (EC 3.1.3.8) or 6-phytase
(EC 3.1.3.26), based on the position-specificity of the
initial hydrolysis from phytate, but no EC number was
given to alkaline phytase.
Despite considerable differences between alkaline
phytases and HAPs, only limited knowledge on the
biochemical and catalytic properties of alkaline phytases
is currently available. In order to expand the knowledge on
phytases, we classified phytases based on both the amino
acid sequences of the phytases and their biochemical
properties. Phylogenetic analysis enabled classification of
the phytases into two major classes (HAPs, alkaline
phytases), which correlated well with the biochemical and
catalytic properties of each class. The present review on
phytases focuses on the differences between well known
HAPs and alkaline phytases from Bacillus and some
plants, based on the physiological nature of their substrate.
The physiological nature of phytate
Phytate can exist in a metal-free form or in metal–phytate
complex, depending on the pH of the solution and the
concentration of metal cations (Fig. 1A). At acidic pH,
protonation of the phosphate groups of phytate generates
the metal-free form. At neutral pH, in contrast, deprotona-
tion of the phosphate groups of phytate enhances the
affinity for divalent metal cations and thus phytate forms
metal–phytate complexes with divalent metal cations,
mostly Mg
2+
and Ca
2+
(Cheryan 1980; Maenz et al. 1999).
In the metal–phytate complex, divalent metal cations
with large ionic radii, such as Ca
2+
(0.99 Å) and
Sr
2+
(1.12 Å), bind two oxianions from the phosphate
groups of phytate in a bidentate fashion (Martin and Evans
1986). However, divalent metal cations with small radii,
such as Mg
2+
(0.65 Å), Fe
2+
(0.74 Å), and Zn
2+
(0.71 Å),
bind in a monodentate fashion within two oxygen atoms
from the phosphate groups of phytate (Fig. 1B). Therefore,
bidentate metal-complex formation prefers divalent metal
cations with large ionic radii (Jonson and Tate 1969;
Cheryan 1980).
During the germination of seed, phytate is degraded by
phytases, providing phosphate and minerals for the
growing seedlings. In addition to its role in phosphate
storage, phytate acts as a strong chelator for divalent metal
cations and exists as a stable metal–phytate complex with
divalent metal cations (mostly K
+
,Mg
2+
,Ca
2+
,orZn
2+
)in
plants (Asada et al. 1969; Reddy et al. 1982).
The biochemical properties of phytases
The biochemical properties of phytases from various
sources are summarized in Table 1. Phytases widely exist
in a variety of microorganisms and plants and in some
animal tissues (Liu et al. 1998).
Phytase activity has been found most frequently in
fungi, such as Aspergillus terreus (Yamada et al. 1968),
Asp. ficuum (Gibson 1987), and Asp. niger (Shieh and
Ware 1968). Phytase is also produced by gram-positive
bacteria, such as Bacillus, and gram-negative bacteria, like
363
Fig. 1A, B Effects of pH and
divalent metal cations on phys-
iological nature of phytate. A
Phytate exists as a metal-free
phytate or a metal–phytate
complex, depending on the pH
and divalent metal cations. The
extent of binding is dependent
upon both pH and divalent
metal cations to phytate ratios.
In addition, at acidic pH and
high cation concentration, a
metal–phytate complex is
formed due to direct electro-
static interaction. B Divalent
metal cations specifically bind
to the phosphate groups of
phytate, depending on the ionic
radii of the metal cations. The
formation of the bidentate metal
complex prefers metal cations
with large ionic radii
364
Aerobacter aerogenes (Greaves et al. 1967), Peudomonas
sp. (Cosgrove 1970), Escherichia coli (Greiner et al.
1993), and Klebsiella (Shah and Parekh 1990; Tambe et al.
1994; Greiner et al. 1997; Jareonkitmongkol et al. 1997).
Generally, phytases from gram-negative bacteria are
intracellular proteins, while phytases from gram-positive
bacteria and fungi are extracellular enzymes (Powar and
Jagannathan 1982; Shimizu 1992; Kerovuo et al. 1998;
Kim et al. 1998a; Choi et al. 2001).
In plants, phytase activity is found in many plant seeds
(Chang 1967; Eskin and Wiebe 1983; Gibson and Ullah
1990; Laboure et al. 1993). However, only wheat, mung
bean, and soybean cotyledon phytases have been purified
and characterized (Mandal et al. 1972; Maiti et al. 1974;
Nagai et al. 1975; Gibson and Ullah 1988).
Most phytases belong to either the acid phytases or the
alkaline phytases, depending on their optimal pH for
catalytic activity. HAPs from E. coli (Greiner et al. 1993),
K. terrigena (Greiner et al. 1997), Asp. niger (Skowronski
1978), Asp. fumigatus (Ullah et al. 2000), canola seeds
(Houde et al. 1990), and spelt (Konietzny et al. 1995) have
an optimal pH range of 4.5–5.5. In contrast, alkaline
phytases from Bacillus (Powar and Jagannathan 1982;
Shimizu 1992; Kerovuo et al. 1998; Kim et al. 1998a;
Choi et al. 2001; Idriss et al. 2002) and some plant seeds,
such as Typha latifolia L. pollen (Hara et al. 1985
) and
Lilium longiflorum pollen (Scott and Loewus 1986), have
an optimal pH range of 6.5–8.0 (Table 1)
All phytases are monomeric proteins, except for phytase
B from Asp. niger, which is a tetramer. The molecular
masses of the enzymes are quite variable, within the
range 38–100 kDa. Glycosylation has no effect on the
specific activity and thermostability of phytases. The
higher molecular weights of fungi and yeast enzymes are
due to glycosylation of the enzymes by the host organism
(Wyss et al. 1999b).
Most phytases have an optimal temperature of 44–60°C.
In contrast, phytases from Asp. fumigatus and B.
amyloliquefaciens have an optimum temperature of
about 70°C (Table 1). To use phytases as animal feed
additives, thermostability of the enzyme is a highly
desirable property during the animal feed-pelleting process
(80–100°C). Alkaline phytases from Bacillus are quite
stable at the high temperature range of 80–95°C (Kim et
Table 1 The biochemical properties of phytases from various organisms. Heat inactivation of each phytase starts at/above the indicated
temperature
Source Origin Molecular
mass
(kDa)
Optimum Heat
inactivation
(°C)
Reference
Temperature
(°C)
pH
Fungi Aspergillus niger (PhyA) 85 58 2.5, 5.0 60 Ullah (1988)
A. niger (PhyB) 85–100 60 2.5 60 Ullah and Phillippy (1994)
A. fumigatus 85–100 58 5.0 60 Ullah et al. (2000)
A. terreus 214 70 4.5 60 Yamamoto et al. (1972), Wyss et al. (1999a)
A. oryzae 120–140 50 5.5 60 Shimizu (1993)
A. nidulans 77.8 55 5.5 60 Wyss et al. (1999b)
Yeast Saccharomyces cerevisiae – 45 4.6 40 Nayini (1984)
Schwanniomyces castellii 490 77 4.4 65 Segueilha et al. (1992)
Bacteria Bacillus subtilis 37 60 7.5 - Powar and Jagannathan (1982)
B. subtilis (natto) 38 60 6.0–6.5 - Shimizu (1992)
B. subtilis 42 55 7.0–7.5 60 Kerovuo et al. (1998)
B. amyloliquefaciens 44 70 7.0–7.5 80 Kim et al. (1998a), Oh et al. (2001)
Escherichia coli 42 55 4.5 60 Greiner et al. (1993)
Klebsiella oxytoca 40 55 5.0–6.0 50 Jareonkitmongkol et al. (1997)
K. terrigena 40 58 5.0 50 Greiner et al. (1997)
Pseudomonas sp. ––5.0 – Irvine and Cosgrove (1971)
Plant Canola seed – 50 5.2 – Houde et al. (1990)
Cucurbita maxima 66.5 48 4.8 – Goel and Sharma (1979)
Lilium longiflorum 36, 88 55–60 8.0 – Scott and Loewus (1986)
Legume seeds ––8.0 – Scott (1991)
Maize 76 55 4.8 – Laboure et al. (1993)
Mung beans 158 57 7.5 – Mandal et al. (1972)
Soybean seeds 119 60 4.5– 4.8 – Sutardi and Buckle (1986),
Gibson and Ullah (1988)
Spelt 60 55 6.0
– Konietzny et al. (1995)
Typa latifolia L. ––8.0 – Hara et al. (1985)
Wheat brane – 47 5.0 – Nelson et al. (1968),
Lim and Tate (1973), Nagai et al. (1975)
al. 1998a; Choi et al. 2001; Tye et al. 2002), while other
phytases are rapidly inactivated above 60°C (Liu et al.
1998; Wyss et al. 1999b; Tomschy et al. 2002). The
phytase from B. amyloliquefaciens is an extremely
thermostable enzyme, based on T
m
values (80°C, the
denaturation temperature) determined by differential scan-
ning calorimetry (Ha et al. 2000).
Mechanism of phytate hydrolysis
Phosphatases are a diverse class of enzymes that catalyze
the hydrolysis of the phosphomonoester bond in biological
systems (Vincent et al. 1992). Phosphatases have been
classified into five different subclasses: alkaline phospha-
tases, purple acid phosphatases, low-molecular-weight
HAPs, high-molecular-weight HAPs, and protein phos-
phatases.
All acid phytases (EC 3.1.3.2) are in a subfamily of the
high-molecular-weight HAPs (Van Etten et al. 1991). The
catalytic mechanism of phosphomonoester hydrolysis by
HAPs was elucidated by site-directed mutagenesis (Osta-
nin et al. 1992; Ostanin and Van Etten 1993) and by the
crystal structure of transition-state analogue complexes
(Lim et al. 2000). On the basis of these results, the
following catalytic mechanism for phosphomonoester
hydrolysis was proposed: the histidine residue in the
conserved motif, RHGXRXP, serves as a nucleophile in
the formation of a covalent phosphohistidine intermediate
(Ostanin et al. 1992; Lindqvist et al. 1994) and the aspartic
acid residue of the C-terminal conserved HD motif serves
as a proton donor to the oxygen atom of the scissile
phosphomonoester bond (Lindqvist et al. 1994; Porvari et
al. 1994).
The catalytic mechanism of phytate hydrolysis by
alkaline phytase was characterized by site-directed muta-
genesis, kinetic analysis, and the crystal structure of the
phytase (Oh et al. 2001; Shin et al. 2001). From these
results, the following catalysis mechanism for phytate
hydrolysis was proposed: a calcium-bound water molecule
in the active-site cleft directly attacks the phosphomono-
ester bond of the calcium-phytate complex in alkaline
phytase.
The catalytic mechanism was confirmed by the
following results. First, the alanine substitution of three
calcium-binding residues at Glu211, Glu260, and Asp314
resulted in the complete loss of catalytic activity (Oh et al.
2001). Second, fluoride (which is used extensively to
probe for a water/hydroxide neucleophile) is able to
replace the calcium-bound hydroxide ion at the enzyme′s
active-site cleft (Shin et al. 2001). Third, there is no other
candidate nucleophile group at the active-site region (Ha et
al. 2000). This kind of nucleophilic attack by metal-bound
water/hydroxide is very common in metalloenzymes, such
as inorganic pyrophosphatase (Baykov et al. 2000), myo-
inositol monophosphatase (Ganzhorn and Chanal 1990),
and superoxide dismutase (Meier et al. 1998).
The biochemical and structural mechanism
of substrate specificity
Substrate specificity and affinity are important properties
of phytases, specifically related to the physiological nature
of the substrate (Fig. 1, Fig. 2). The substrate specificity of
alkaline phytases is far narrower than that of HAPs. The
reciprocal relationship between the substrate specificity
and crystal structure of HAPs or alkaline phytases
provides a new insight into the nature of phytate and
into molecular recognition of phytases. HAPs exhibit a
broad specificity for phytate and various other phosphate
esters. The reaction mechanism of phosphate ester
hydrolysis by HAPs appears to follow nonspecific acid
phosphatases properties (Gibson and Ullah 1988).
Specifically for phytate as a substrate, HAPs can
hydrolyze metal-free phytate at acidic pH, when phytate
exists as a metal-free phytate (Fig. 1A; Maenz et al. 1999;
Wyss et al. 1999a). Recently, the crystal structure of the E.
coli phytase and phytate complex demonstrated that the
positively charged active site of this phytase prefers metal-
free phytate (Fig. 3B, D; Lim et al. 2000). In fact, HAPs
cannot hydrolyze a metal–phytate complex (Maenz et al.
1999; Wyss et al. 1999a). Therefore, chelating agents such
as EDTA and phthalate stimulate the phytase activity by
removing divalent metal cations (Maenz et al. 1999; Wyss
et al. 1999a).
Unlike other HAPs, alkaline phytases exhibit a highly
strict substrate specificity for phytate and have no
enzymatic activity on other phosphate esters (Table 2;
Powar and Jagannathan 1982; Hara et al. 1985; Scott and
Loewus 1986; Scott 1991; Shimizu 1992; Kerovuo et al.
1998; Kim et al. 1998a; Choi et al. 2001; Idriss et al.
2002). More specifically, kinetic studies of B. amyloli-
quefaciens demonstrate that alkaline phytases can hydro-
lyze a calcium–phytate complex as a substrate at pH 7.0–
365
Fig. 2A, B Schematic illustrations of substrate hydrolysis by
histidine acid phosphatases (HAPs) and alkaline phytases. A HAPs
are able to hydrolyze five phosphate groups from phytate at acidic
pH, yielding myo-inositol monophosphate as the final product. B
Alkaline phytases are able to hydrolyze three phosphate groups from
a specific calcium–phytate complex at alkaline pH, producing myo-
inositol trisphosphate as the final product
366
8.0 when phytate exists as a calcium–phytate complex
(Fig. 1B; Oh et al. 2001). Furthermore, the strict substrate
specificity for alkaline phytases is rationalized by the
phosphate bridge formation between Ca
2+
and the two
oxianions from the phosphate groups of phytate (Fig. 1B;
Oh et al. 2001). This kind of strict substrate specificity is
common in the reaction involving phosphorylated inter-
mediates, especially nucleotides. For example, almost all
reactions involving ATP require MgATP as a substrate
(Segel 1975).
Recently, the crystal structure of the phytase from B.
amyloliquefaciens DS11 demonstrated that a negatively
charged active site provides a favorable electrostatic
environment for the positively charged calcium–phytate
complex (Fig. 3A, C; Ha et al. 2000; Shin et al. 2001).
Therefore, EDTA strongly inhibits alkaline phytase activ-
ity (Powar and Jagannathan 1982; Kerovuo et al. 1998;
Kim et al. 1998a; Liu et al. 1998).
In E. coli, the phytase crystal structure clearly shows
that most of the phosphate groups from phytate interact
with the active-site pockets of the phytase (Fig. 3D). The
large active-site region allows HAPs to accommodate
various kind of phosphate esters, such as pNPP, AMP,
ATP, fructose 1,6-bisphosphate, and glucose 6-phosphate
(Table 2). In contrast, the active site region of the phytase
from B. amyloliquefaciens can only bind two phosphate
groups of the calcium–phytate complex, which endows
alkaline phytases with a highly strict substrate specificity
for the calcium–phytate complex (Figs. 2B, 3C).
Molecular classification of phytases
Recently, many alkaline phytase and HAP genes from
various species were cloned and sequenced. Phylogenetic
analysis of the amino acid sequences from various
phytases clearly shows two major classes, which correlates
well with the classification of phytases according to their
biochemical and catalytic properties (Fig. 2, Table 3).
One of the major classes is the family of HAPs sharing a
highly conserved RHGXRXP motif (Van Etten et al.
1991). The HAP class can be further subdivided into three
Fig. 3A–D Electrostatic surface potential of alkaline phytase and
HAP in the active-site region. The surfaces of the substrate-binding
sites of Bacillus amyloliquefaciens (A) and Escherichia coli phytase
(B) are colored according to their local electrostatic potentials,
ranging from −7 kt/e in red to +7kt/e in blue, using GRASPP(Honig
and Nicholls 1995). Stick models of two phosphates (A, C) and
phytate (B, D) are shown in the substrate-binding site. The
positively charged active site of the phytase from E. coli prefers
metal-free phytate. In contrast, a negatively charged active site of the
phytase from B. amyloliquefaciens provides a favorable electrostatic
environment for the positively charged calcium–phytate complex
367
Table 2 Substrate specificity of histidine acid phosphatases (HAPs) and alkaline phytases from various organisms. The hydrolysis rate of sodium phytate was taken as 100% for
comparison. In the case of alkaline phytase, calcium phytate was used, not sodium phytate
Substrate Relative activity (%)
HAPs Alkaline phytases
A. niger (phyA) A. niger (phyB) A. fumigatus Canola seed K. terrigena E. coli Spelt B. subtilis B. subtilis natto B. amyloliquefaciens T. latifolia L.
Sodium phytate 100 100 100 100 100 100 100 100 100 100 100
p-Nitrophenyl phosphate 66 1,125 393 890 24.2 12.3 29 0 0 0 6
Fructose 1,6-bisphosphate 27.3 2,125 393 – 11.9 8.5 103 0 –– –
Fructose 6-phosphate 2 437 43 – 7.2 1.3 121 0 –– –
Glucose 6-phosphate 13 1,062 125 – 2.1 ––0 –– 0
Ribose 5-phosphate 2 562 36 –– ––– – – –
Α-Glycerophosphate 3 562 43 –– ––00 0 –
Β-Glycerophosphate 41 687 157 72 4.9 1.9 38 0 0 0 0
3-Phosphoglycerate 3 750 129 –– ––0 –– –
Na-Pyrophosphate 19.4 – 136 23,190 13.7 – 517 4.2 4.2 2 0
AMP 0 150 14 – 10.3 0.4 11 0 – 00
ADP 8 625 64 – 11 ––0 – 0 –
ATP 48 875 86 17,545 8 0.9 252 0 – 00
Table 3 Molecular and biochemical characteristics of HAPs and alkaline phytases. IP
1
Myo-inositol monophosphate, IP
3
myo-inositol trisphosphate, P
i
inorganic phosphate
Characteristics Histidine acid phytases Alkaline phytase
PhyA PhyB PhyC PhyD
Molecular mass (kDa) 62–128 270 42–45 38–45
Glycosylation Yes Yes No No
Optimum pH 2.5–5.0 2.5 5.0–6.0 7.0–8.0
Optimum temperature(°C) 55–60 55–60 40–60 55–70
Thermal stability Low (60°C) Low (60°C) Low (60°C) High (85–95°C)
Effect of Ca
2+
Inhibition Inhibition Inhibition Stimulation
Effect of EDTA Stimulation Stimulation Stimulation Inhibition
Substrate specificity Broad Broad Broad Specific
Nature of phytate Metal-free phytate Metal-free phytate Metal-free phytate Ca-phytate
Position specificity
D-3 position of phytate D-3 position of phytate D-6 position of phytate D-3 position of phytate
Final product IP
1
+5P
i
IP
1
+5P
i
IP
1
+5P
i
IP
3
+3P
i
Active site (+) Charged amino acid (+) Charged amino acid (+) Charged amino acid (–) Charged amino acid
Crystal structure A large α/β and a small α-domain A large α/β and a small α-domain A large α/β and a small α-domain Six-bladed β-propeller
368
different groups (PhyA–PhyC), based on amino acid
sequence homology and biochemical properties, such as
optimal pH and the position-specificity of phytate hydrol-
ysis (Fig. 2, Table 3 ).
Group I, PhyA, consists of enzymes with 465–
469 amino acids and includes extracellular HAPs from
Asp. niger (Van Hartingsveldt et al. 1993), Asp. niger
(awamori) (Piddington et al. 1993), Asp. fumigatus
(Pasamontes et al. 1997b), Asp. terrus (Mitchell et al.
1997), Emericella nidulans (Pasamontes et al. 1997a),
Myceliophthora thermophila (Mitchell et al. 1997), and
Talaromyces thermophilus (Pasamontes et al. 1997a).
These phytases have two optimal pH values (2.5, 5.5)
and an optimal temperature at 55–60°C (Ullah 1988; Wyss
et al. 1999a).
The molecular mass of the unglycosylated phytases is
predicted to be 48–50 kDa. However, the apparent
molecular mass of glycosylated phytases is determined
to be 62–128 kDa by SDS-PAGE and is ~64–137 kDa by
analytical ultracentrifugation (Wyss et al. 1999b). They
exhibit considerable activity with a broad range of
phosphate compounds, including both phytate and other
phosphate esters (Table 2; Wyss et al. 1999b). Since they
hydrolyze phytate preferentially at the
D-3 position,
generating
D-myo-inositol-(1,2,4,5,6)-pentakisphosphate,
PhyA is classified as a 3-phytase (Cosgrove 1980; Greiner
et al. 1997; Wyss et al. 1999a).
Group II, PhyB, contains phytases that are extracellular
HAPs from Asp. niger (Ethrlich et al. 1993), Saccharo-
myces cerevisiae (Bajwa et al. 1984), and Schizosaccha-
romyces pombe (Elliott et al. 1986). They are composed of
453–479 amino acids and the molecular mass is ~48–
50 kDa. The apparent molecular mass of the glycosylated
proteins is 65 kDa by SDS-PAGE and about 270 kDa by
analytical centrifugation (Kostrewa et al. 1999). The
crystal structure of Asp. niger phytase B clearly shows
the phytases have a tetramer form (Kostrewa et al. 1999).
These phytases have a single optimal pH of 2.5, lack any
activity at pH 5.0 or higher, and have an optimal
temperature of 55–60°C. PhyB also has a broad substrate
specificity (Ullah and Cummins 1987; Wyss et al. 1999a).
PhyB hydrolyzes the same position of metal-free
phytates and is classified as a 3-phytase (Irving and
Cosgrove 1972; Greiner et al. 2001).
Group III, PhyC, contains acid phytases from E. coli
(Dossa and Boquet 1985; Dossa et al. 1990), lysosomal
(Pohlmann et al. 1988; Geier et al. 1991), and prostatic
acid phosphatases (Van Etten et al. 1991) from rat and
human. These phytases are intracellular proteins com-
posed of 354–439 amino acids, with a molecular mass of
~42–45 kDa; and these monomeric proteins are non-
glycosylated enzymes (Wyss et al. 1999b). PhyC have a
single optimal pH (~5.0–6.0) and exhibit an optimal
temperature at 40–60°C.
Despite low sequence homology (14% identity), the
overall crystal structure of E. coli phytase is closely related
to rat prostatic acid phosphatase (Schneider et al. 1993;
Lim et al. 2000). The crystal structure of the E. coli
phytase complex with phytate reveals that the active-site
region contains many positively charged groups, demon-
strating favorable binding for metal-free phytate at acidic
pH (Fig. 3
B, D; Lim et al. 2000). However, HAPs cannot
hydrolyze a metal-phytate complex, due to electrostatic
repulsion between the positively charged active site and
the positively charged metal–phytate complex. Group III
phytases are also capable of hydrolyzing not only metal-
free phytate but also various other phosphate esters, like
other HAPs (Ullah and Cummins 1987; Wyss et al.
1999a). However, these phytases are considered as a 6-
phytase, since they hydrolyze phytate preferentially at the
D-6 (l-4) position (Greiner et al. 1993; Lassen et al. 2001).
In addition, all HAPs are able to hydrolyze five phosphate
groups from metal-free phytate, yielding myo-inositol
monophosphate as the end product (Wyss et al. 1999a;
Fig. 2A).
Another major class (Class II) contains alkaline
phytases, which differ from HAPs in many aspects,
including optimal pH, molecular mass, tertiary structure,
substrate specificity, and calcium ion requirement for
enzymatic catalysis. Based on these biochemical differ-
ences and phylogenetic data, alkaline phytases from
Bacillus and some plant seeds can be classified as another
group: PhyD.
Group IV, PhyD, consists of the phytate-specific
enzymes from Bacillus and some plants, such as T.
lattifolia pollen (Hara et al. 1985), L. longiflorum pollen
(Scott and Loewus 1986; Barrientos et al. 1994), and some
legume seeds (Scott 1991). Comparison of amino acid
sequences among these enzymes is impossible, since no
amino acid sequence data is available for the plant alkaline
phytases. However, these plant enzymes have biochemical
characteristics very similar to those of the phytases from
Bacillus (Oh et al. 2001). Several phytase genes were
cloned from B. amyloliquefaciens (Kim et al. 1998b; Idriss
et al. 2002), B. licheniformis (Tye et al. 2002), and B.
subtilis (Kunst et al. 1997; Kerovuo et al. 1998; Tye et al.
2002). They are composed of 383 amino acids and encode
an extracellular monomeric protein. The molecular mass is
~42 kDa; and SDS-PAGE gives an apparent molecular
mass of ~38–44 kDa.
The amino acid sequences of phytases from Bacillus are
highly homologous to each other, with 64–98% sequence
identity. However, these amino acid sequences do not
align with any other known HAPs or other phosphatases.
Moreover, they do not contain the conserved RHGXRP
active-site motif of HAPs (Van Etten et al. 1991).
Phylogenic analysis clearly shows that alkaline phytases
are not a subfamily of HAPs but are indeed novel phytases
(Fig. 4). Most significantly, this group requires calcium for
catalytic activity and has an optimal pH of ~7.0–8.0
(Powar and Jagannathan 1982; Shimizu 1992; Kerovuo et
al. 1998; Kim et al. 1998a; Choi et al. 2001; Idriss et al.
2002). The optimal temperature of these phytases is ~55–
70°C; and they are quite thermostable at a temperature
range of 80–95°C (Kim et al. 1998a; Choi et al. 2001
;Tye
et al. 2002).
Alkaline phytases are highly specific for the calcium–
phytate complex (Oh et al. 2001), but have no activity on
pNPP and other phosphoseters, the general substrates for
other HAPs (Powar and Jagannathan 1982; Shimizu 1992;
Kim et al. 1998a; Choi et al. 2001). Also, they hydrolyze
the calcium–phytate complex preferentially at the
D-3
position, yielding
D-myo-inositol-(1,2,4,5,6)-pentakispho-
sphate as an initial product (Idriss et al. 2002) and are
classified as 3-phytases. Alkaline phytases are able to
hydrolyze three phosphate groups from the calcium–
phytate complex, producing myo-inositol trisphosphate as
a final product (Fig. 2B; Hara et al. 1985; Barrientos et al.
1994; Kerovuo et al. 2000).
Conclusion
Phytases are added to animal feedstuffs to reduce phos-
phate pollution in the environment, since monogastric
animals are unable to metabolize phytate. Phosphate is a
very important component of DNA, RNA, ATP, and other
biologically active compounds. For an ideal animal feed
additive, it is necessary to consider whether the biochem-
ical and catalytic properties of phytases are optimal not
only for a given physiological reaction but also in the
context of the industrial process in which the phytases are
applied. For example, it is essential that the pH activity/
stability profile for the phytase is sufficiently broad to
accommodate any pH range. Thermostability would be a
highly desirable property during the animal feed-pelleting
process at a temperature of ~65–80°C. Acid and protease
resistance are major concerns in the digestive tract.
Critically, the chemical nature of phytate cannot be
handled in the digestive tract of the animals, where
phytate mostly exists as a calcium–phytate complex when
there is a high content of calcium in the animal feed (30–
40 g/kg diet; Van der Klis et al. 1997).
On the basis of pH profiles, higher thermostability, and
the strict substrate specificity and physiological nature of
phytate in the digestive tract of animals, alkaline phytases
could be ideal candidates for biotechnological applications
as an animal feed additive and in the production of
chemicals such as myo-inositol trisphosphate and myo-
inositol derivatives. Finally, it would be possible to isolate
more alkaline phytase and genes from microorganisms,
plants, and animals if we use another method for
measuring the inorganic phosphate released from calci-
um–phytate complexes (equimolar concentrations of cal-
cium and phytate) at alkaline pH.
Acknowledgements. The authors gratefully acknowledge Drs. M.
Rudolph, S-H Lee, and J-W Kim for critical reading of the
manuscript. This study was supported by the G7 project and in part
by the 21C Frontier R&D Program from the Korean Ministry of
Science and Technology.
References
Asada K, Tanaka K, Kasai Z (1969) Formation of phytic acid in
cereal grains. Ann NY Acad Sci 165:801–814
Bajwa W, Meyhack B, Rudolph H, Schweingruber AM, Hinnen A
(1984) Structural analysis of the two tandemly repeated acid
phosphatase genes in yeast. Nucleic Acids Res 12:7721–7739
369
Fig. 4 Phylogenetic analysis of various HAPs and alkaline
phytases. The phylogenic tree was constructed using the MagAlign
program of Lasergene after the original data set was aligned by the
CLUSTAL method. The length of horizontal lines indicates the
relative evolutionary distance. The amino acid sequence of Asper-
gillus niger NRLL 3153 PhyA (Van Hartingsveldt et al. 1993), Asp.
niger NRLL 3153 PhyB (Ethrlich et al. 1993), Asp. niger (awamori)
ATCC 38854 (Piddington et al. 1993), Asp. fumigatus
ATCC 130703 (Pasamontes et al. 1997b), Asp. terreus 9A1(Mitchell
et al. 1997), rat prostatic acid phosphatase (Roiko et al. 1990),
Myceliophthora thermophilia ATCC 48102 (Mitchell et al. 1997),
Talaromyces thermophilus ATCC 20186 (Pasamontes et al. 1997a),
Emericella nidulans (Pasamontes et al. 1997a), E. coli, Schizosac-
charomyces pombe (Elliott et al. 1986), Saccharomyces cerevisiae
(Bajwa et al. 1984), human lysosomal acid phosphatase (Pohlmann
et al. 1988), mouse lysosomal acid phosphatase (Geier et al. 1991),
human prostatic acid phosphatase (Van Etten et al. 1991), B.
amyloliquefaciens DS11 (Kim et al. 1998b), B. subtilis VTT E-
68013 (Kerovuo et al. 1998), B. subtilis (Kunst et al. 1997), B.
subtilis 168 (Tye et al. 2002), B. licheniformis (Tye et al. 2002), and
B. amyloliquefaciens FZB45 (Idriss et al. 2002) were obtained from
GenBank
370
Barrientos L, Scott JJ, Murthy PP (1994) Specificity of hydrolysis of
phytic acid by alkaline phytase from lily pollen. Plant Physiol
106:1489–1495
Baykov AA, Fabrichniy IP, Pohjanjoki P, Zyryanov AB, Lahti R
(2000) Fluoride effects along the reaction pathway of pyro-
phosphatase: evidence for a second enzyme pyrophosphate
intermediate. Biochemistry 39:11939–11947
Chang CW (1967) Study of phytase and fluoride effects in
germinating corn seed. Cereal Chem 44:129–142
Cheryan M (1980) Phytic acid interactions in food systems. Crit Rev
Food Sci Nutr 13:297–335
Choi YM, Suh HJ, Kim JM (2001) Purification and properties of
extracellular phytase from Bacillus sp. KHU-10. J Protein
Chem 20:287–292
Cosgrove DJ (1966) The chemistry and biochemistry of inositol
polyphosphates. Rev Pure Appl Chem 16:209–215
Cosgrove DJ (1970) Inositol phosphate phosphatases of microbio-
logical origin. Inositol phosphate intermediates in the dephos-
phorylation of the hexaphosphates of myo-inositol, scyllo-
inositol, and
D-chiro-inositol by a bacterial (Pseudomonas sp.)
phytase. Aust J Biol Sci 23:1207–1220
Cosgrove DJ (1980) Phytases and intermediates in the dephosphor-
ylation of P6-inositols by phytase enzymes. In: Cosgrove DJ
(ed)Inositol phosphates: their chemistry, biochemistry, and
physiology. (Studies in organic chemistry 4) Elsevere, Amster-
dam, pp 85–105
Dossa E, Boquet PL (1985) Identification of the gene appA for the
acid phosphatase (pH optimum 2.5) of Escherichia coli. Mol
Gen Genet 200:68–73
Dossa J, Marck C, Boquet PL (1990) The complete nucleotide
sequence of the Escherichia coli gene appA reveals significant
homology between pH 2.5 acid phosphtase and glucose-1-
phosphatase. J Bacteriol 172:5497–5500
Elliott S, Chang CW, Schweingruber ME, Schaller J, Rickli EE,
Carbon J (1986) Isolation and characterization of the structural
gene for secreted acid phosphatase from Schizosaccharomyces
pombe. J Biol Chem 261:2936–2941
Englen AJ, Heeft FC van der, Randsdrop PH, Smit EL (1994)
Simple and rapid determination of phytase activity. J AOAC Int
77:760–764
Eskin NAM, Wiebe S (1983) Changes in phytase activity and
phytate during germination of two fababean cultivars. J Food
Sci 48:270–271
Ethrlich KC, Montalbano BG, Mullaney EJ, Dischinger HC, Ullah
AHJ (1993) Identification and cloning of a second phytase gene
(phyB) from Aspergillus niger (ficcum). Biochem Biophys Res
Commun 195:53–57
Ganzhorn AJ, Chanal MC (1990) Kinetic studies with myo-inositol
monophosphatase from bovine brain. Biochemistry 29:6065–
6071
Geier C, Figura K von, Pohlmann R (1991) Molecular cloning of the
mouse lysosomal acid phosphatase. Biol Chem 372:301–304
Gibson D (1987) Production of extracellular phytase from Asper-
gillus ficuum on starch media. Biotechnol Lett 9:305–310
Gibson DM, Ullah AH (1988) Purification and characterization of
phytase from cotyledons of germinating soybean seeds. Arch
Biochem Biophys 260:503–513
Gibson DM, Ullah, AB (1990) Phytase and their action on phytic
acid in inositol metabolism in plants. Arch Biochem Biophys
262:77–92
Goel M, Sharma CB (1979) Multiple forms of phytase in
germinating cotyledons of Cucurbita maxima. Phytochemistry
18:1939–1942
Greaves MP, Anderson G, Webley DM (1967) The hydrolysis of
inositol phosphates by Aerobacter aerogenes. Biochim Biophys
Acta 132:412–
418
Greiner R, Konietzny U, Jany KD (1993) Purification and
characterization of two phytases from Escherichia coli. Arch
Biochem Biophys 303:107–113
Greiner R, Haller E, Konietzny U, Jany KD (1997) Purification and
characterization of a phytase from Klebsiella terrigena. Arch
Biochem Biophys 341:201–206
Greiner R, Alminger ML, Carlsson NG (2001) Stereospecificity of
myo-inositol hexakisphosphate dephosphorylation by a phytate-
degrading enzyme of baker′s yeast. J Agric Food Chem
49:2228–2233
Ha NC, Oh BC, Shin S, Kim HJ, Oh TK, Kim YO, Choi KY, Oh BH
(2000) Crystal structures of a novel, thermostable phytase in
partially and fully calcium-loaded states. Nat Struct Biol 7:147–
153
Hara A, Ebina S, Kondo A, Funagua T (1985) A new type of
phytase from Typha latifolia L. Agric Biol Chem 49:3539–
3544
Honig B, Nicholls A (1995) Classical electrostatics in biology and
chemistry. Science 268:1144–1149
Houde RL, Alli I, Kermasha S (1990) Purification and character-
isation of canola seed (Brassica sp.) phytase. J Food Biochem
14:331–351
Idriss EE, Makarewicz O, Farouk A, Rosner K, Greiner R, Bochow
H, Richter T, Borriss R (2002) Extracellular phytase activity of
Bacillus amyloliquefaciens FZB45 contributes to its plant-
growth-promoting effect. Microbiology 148:2097–2109
Irvine GCJ, Cosgrove, DJ (1971) Inositol phosphate phosphatases of
microbiological origin. J Biol Sci 24:547
Irving GC, Cosgrove DJ (1972) Inositol phosphate phosphatases of
microbiological origin: the inositol pentaphosphate products of
Aspergillus ficuum phytases. J Bacteriol 112:434–438
IUPAC–IUB (1975) Enzyme nomenclature recommendation
supplement 1: correction and additions. Biochem Biophys
Acta 429:1
Jareonkitmongkol S, Ohya M, Watanabe R, Takagi H, Nakamori S
(1997) Partial purification of phytase from a soil isolate
bacterium, Klebsiella oxytoca MO-3. J Ferment Bioeng
83:393–394
Jonson LF, Tate ME (1969) The conformational analysis of phytic
acid based on NMR spectra. J Chem 47:63–73
Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N, Apajalahti J
(1998) Isolation, characterization, molecular gene cloning, and
sequencing of a novel phytase from Bacillus subtilis. Appl
Environ Microbiol 64:2079–2085
Kerovuo J, Rouvinen J, Hatzack F (2000) Analysis of myo-inositol
hexakisphosphate hydrolysis by Bacillus phytase: indication of
a novel reaction mechanism. Biochem J 352 Pt 3:623–628
Kim YO, Kim HK, Bae KS, Yu JH, Oh TK (1998a) Purification and
properties of a thermostable phytase from Bacillus sp. DS11.
Enzyme Microb Technol 22:2–
7
Kim YO, Lee JK, Kim HK, Yu JH, Oh TK (1998b) Cloning of the
thermostable phytase gene (phy) from Bacillus sp. DS11 and its
overexpression in Escherichia coli. FEMS Microbiol Lett
162:185–191
Konietzny U, Greiner R, Jany K-D (1995) Purification and
characterisation of a phytase from spelt. J Food Biochem
18:165–183
Kostrewa D, Wyss M, D′Arcy A, Loon AP van (1999) Crystal
structure of Aspergillus niger pH 2.5 acid phosphatase at 2.4 Å
resolution. J Mol Biol 288:965–974
Kunst F, et al (1997) The complete genome sequence of the gram-
positive bacterium Bacillus subtilis. Nature 390:249 –256
Laboure AM, Gagnon J, Lescure AM (1993) Purification and
characterization of a phytase (myo-inositol- hexakisphosphate
phosphohydrolase) accumulated in maize (Zea mays) seedlings
during germination. Biochem J 295:413–419
Lassen SF, et al (2001) Expression, gene cloning, and characteriza-
tion of five novel phytases from four basidiomycete fungi:
Peniophora lycii, Agrocybe pediades,aCeriporia sp., and
Trametes pubescens. Appl Environ Microbiol 67:4701–4707
Lim D, Golovan S, Forsberg CW, Jia Z (2000) Crystal structures of
Escherichia coli phytase and its complex with phytate. Nat
Struct Biol 7:108–113
Lim PE, Tate ME (1973) The phytases. II. Properties of phytase
fractions F1 and F2 from wheat bran and the myo-inositol
phosphates produced by fraction F2. Biochim Biophys Acta
302:316–328
Lindqvist Y, Schneider G, Vihko P (1994) Crystal structures of rat
acid phosphatase complexed with the transition-state analogs
vanadate and molybdate. Implications for the reaction mech-
anism. Eur J Biochem 221:139–142
Liu BL, Rafiq A, Tzeng YM, Rob A (1998) The induction and
characterization of phytase and beyond. Enzyme Microb
Technol 22:415–424
Maenz DD, Engele-Schaan CM, Newkirk RW, Classen HL (1999)
The effect of minerals and mineral chelators on the formation of
phytase-resistant and phytase-susceptible forms of phytic acid
in solution and in a slurry of canola meal. Anim Feed Sci
Technol 81:177–192
Maiti IB, Majumder, AL, Biswas BB (1974) Purification and mode
of action of phytase from Phaseolus aureus. Phytochemistry
13:1047–1051
Mandal NC, Burnman S, Biswas, BB (1972) Isolation, purification
and characterization of phytase from germinating mung beans.
Phytochemistry 11:495–502
Martin CJ, Evans WJ (1986) Phytic acid–metal ion interactions II.
The effect of pH on Ca(II) binding. J Inorg Biochem 27:17–30
Meier B, Scherk C, Schmidt M, Parak F (1998) pH-dependent
inhibition by azide and fluoride of the iron superoxide
dismutase from Propionibacterium shermanii. Biochem J
331:403–407
Mitchell DB, Vogel K, Weimann BJ, Pasamontes L, Loon AP van
(1997) The phytase subfamily of histidine acid phosphatases:
isolation of genes for two novel phytases from the fungi
Aspergillus terreus and Myceliophthora thermophila. Microbi-
ology 143:245–252
Nagai M, Nishibu M, Sugita Y, Yoneyama Y (1975) The effects of
inositol hexaphosphate on the allosteric properties of two beta-
99-substituted abnormal hemoglobins, hemoglobin Yakima and
hemoglobin Kempsey. J Biol Chem 250:3169–3173
Nayini NRP (1984) The phytase of yeast. Lebensm Wiss Technol
17:24–26
Nelson TS, Shieh TR, Wodzinski RJ, Ware JH (1968) The
availability of phytate phosphorus in soybean meal before
and after treatment with a mold phytase. Poult Sci 47:1842–
1848
Oh BC, Chang BS, Park KH, Ha NC, Kim HK, Oh BH, Oh TK
(2001) Calcium-dependent catalytic activity of a novel phytase
from Bacillus amyloliquefaciens DS11. Biochemistry 40:9669–
9676
Ostanin K, Van Etten RL (1993) Asp304 of Escherichia coli acid
phosphatase is involved in leaving group protonation. J Biol
Chem 268:20778–20784
Ostanin K, Harms EH, Stevis PE, Kuciel R, Zhou MM, Van Etten
RL (1992) Overexpression, site-directed mutagenesis, and
mechanism of Escherichia coli acid phosphatase. J Biol
Chem 267:22830–22836
Pasamontes L, Haiker M, Henriquez-Huecas M, Mitchell DB, Loon
AP van (1997a) Cloning of the phytases from Emericella
nidulans and the thermophilic fungus Talaromyces thermo-
philus. Biochim Biophys Acta 1353:217–223
Pasamontes L, Haiker M, Wyss M, Tessier M, Loon AP van (1997b)
Gene cloning, purification, and characterization of a heat-stable
phytase from the fungus Aspergillus fumigatus. Appl Environ
Microbiol 63:1696–1700
Piddington CS, et al (1993) The cloning and sequencing of the genes
encoding phytase (phy) and pH 2.5 optimum acid phosphatase
(aph) from Aspergillus niger var awamori. Gene 133:55–62
Pohlmann R, Houston CS, Paloheimo M, Cantrell M, Miettinen-
Oinonen A, Nevalainen H, Rambosek J (1988) Human
lysosomal acid phosphatase: cloning, expression and chromo-
somal assignment. EMBO J 7:2343–2350
Porvari KS, Herrala AM, Kurkela RM, Taavitsainen PA, Lindqvist
Y, Schneider G, Vihko PT (1994) Site-directed mutagenesis of
prostatic acid phosphatase. Catalytically important aspartic
acid 258, substrate specificity, and oligomerization. J Biol
Chem 269:22642–22646
Powar VK, Jagannathan V (1982) Purification and properties of
phytate-specific phosphatase from Bacillus subtilis. J Bacteriol
151:1102–1108
Reddy NR, Sathe SK, Salunkhe DK (1982) Phytates in legumes and
cereals. Adv Food Res 28:1–92
Roiko K, Janne OA, Vihko P (1990) Primary structure of rat
secretory acid phosphatase and comparison to other acid
phosphatases. Gene 89:223–229
Schneider G, Lindqvist Y, Vihko P (1993) Three-dimensional
structure of rat acid phosphatase. EMBO J 12:2609–2615
Schroder B, Breves G, Rodehutscord M (1996) Mechanisms of
intestinal phosphorus absorption and availability of dietary
phosphorus in pigs. Dtsch Tieraerztl Wochenschr 103:209–214
Scott JJ (1991) Alkaline phytase activity in nonionic detergent
extracts of legume seeds. Plant Physiol 95:1298–1301
Scott JJ, Loewus FA (1986) A calcium-activated phytase from
pollen of Lilium longiflorum. Plant Physiol 82:333–335
Segel IH (1975) In: Segel IH (ed) Enzyme kinetics: substrate–
activator complex is the true substrate. Wiley, New York,
pp 242–272
Segueilha L, Lambrechts C, Boze H, Moulin G, Galzy P (1992)
Purification and properties of the phytase from Schwannio-
myces castellii. J Ferment Bioeng 74:7–11
Shah V, Parekh LJ (1990) Phytase from Klebsiella sp. No. PG-2:
purification and properties. Indian J Biochem Biophys 27:98–
102
Shieh TR, Ware JH (1968) Survey of microorganisms for the
production of extracellular phytase. Appl Microbiol 16:1348–
1351
Shimizu M (1992) Purification and characterization of phytase from
Bacillus subtilis (natto) N-77. Biosci Biotechnol Biochem
56:1266–1269
Shimizu M (1993) Purification and characterization of phytase and
acid phosphatase produced by Aspergillus oryzae K1. Biosci
Biotechnol Biochem 57:1364–1365
Shin S, Ha NC, Oh BC, Oh TK, Oh BH (2001) Enzyme mechanism
and catalytic property of beta propeller phytase. Structure
9:851–858
Skowronski T (1978) Some properties of partially purified phytase
from Aspergillus niger. Acta Microbiol Pol 27:41–48
Sutardi, Buckle KA (1986) The characteristics of soybean phytase. J
Food Biochem 10:197–216
Tambe SM, Kaklij GS, Kelkar SM, Parekh LJ (1994) Two distinct
molecular forms of phytase from Klebsiella aerogenes: evi-
dence for unusually small active enzyme peptide. J Ferment
Bioeng 77:23–27
Tomschy A, et al (2002) Engineering of phytase for improved
activity at low pH. Appl Environ Microbiol 68:1907–1913
371
372
Tye AJ, Siu FK, Leung TY, Lim BL (2002) Molecular cloning and
the biochemical characterization of two novel phytases from B.
subtilis 168 and B. licheniformis. Appl Microbiol Biotechnol
59:190–197
Ullah AH (1988) Aspergillus ficuum phytase: partial primary
structure, substrate selectivity, and kinetic characterization.
Prep Biochem 18:459–471
Ullah AH, Cummins BJ (1987) Purification, N-terminal amino acid
sequence and characterization of pH 2.5 optimum acid
phosphatase (EC 3.1.3.2) from Aspergillus ficuum. Prep
Biochem 17:397–422
Ullah AH, Phillippy BQ (1994) Substrate selectivity in Aspergillus
ficcum phytase and acid phosphtase using myo-inositol
phosphates. J Agric Food Chem 42:423–425
Ullah AH, Sethumadhavan K, Lei XG, Mullaney EJ (2000)
Biochemical characterization of cloned Aspergillus fumigatus
phytase (phyA). Biochem Biophys Res Commun 275:279–285
Urbano G, Lopez-Jurado M, Aranda P, Vidal-Valverde C, Tenorio E,
Porres J (2000) The role of phytic acid in legumes: antinutrient
or beneficial function? J Physiol Biochem 56:283–294
Van der Klis JD, Versteegh HA, Simons PC, Kies AK (1997) The
efficacy of phytase in corn-soybean meal-based diets for laying
hens. Poult Sci 76:1535–1542
Van Etten RL, Davidson R, Stevis PE, MacArthur H, Moore DL
(1991) Covalent structure, disulfide bonding, and identification
of reactive surface and active site residues of human prostatic
acid phosphatase. J Biol Chem 266:2313–2319
Van Hartingsveldt W, et al (1993) Cloning, characterization and
overexpression of the phytase-encoding gene (phyA)ofAsper-
gillus niger. Gene 127:87–94
Vincent JB, Crowder MW, Averill BA (1992) Hydrolysis of
phosphate monoesters: a biological problem with multiple
chemical solutions. Trends Biochem Sci 17:105–110
Wyss M, Brugger R, Kronenberger A, Remy R, Fimbel R,
Oesterhelt G, Lehmann M, Loon AP van (1999a) Biochemical
characterization of fungal phytases (myo-inositol hexakispho-
sphate phosphohydrolases): catalytic properties. Appl Environ
Microbiol 65:367–373
Wyss M, Pasamontes L, Friedlein A, Remy R, Tessier M,
Kronenberger A, Middendorf A, Lehmann M, Schnoebelen
L, Rothlisberger U, Kusznir E, Wahl G, Muller F, Lahm HW,
Vogel K, Loon AP van (1999b) Biophysical characterization of
fungal phytases (myo-inositol hexakisphosphate phosphohy-
drolases): molecular size, glycosylation pattern, and engineer-
ing of proteolytic resistance. Appl Environ Microbiol 65:359–
366
Yamada K, Minoda, Y, Yamada K (1968) Phytase from Aspergillus
terreus. Part 1. Production, purification, and some general
properties of the enzyme. Agric Biol Chem 32:1275–
1283
Yamamoto S, Minoda Y, Yamada K (1972) Chemical and physi-
cochemical properties of phytase from Aspergillus terreus.
Agric Biol Chem 36:2097–2103
