Journal of Applied Microbiology ISSN 1364-5072

REVIEW ARTICLE

Phytases: crystal structures, protein engineering and

potential biotechnological applications
M.-Z. Yao, Y.-H. Zhang, W.-L. Lu, M.-Q. Hu, W. Wang and A.-H. Liang
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan,
China

Keywords
biotechnological applications, crystal structure,
heterologous gene expression, phytase,
phytate, protein engineering, transgenics.
Correspondence
Aihua Liang, Key Laboratory of Chemical
Biology and Molecular Engineering of Ministry
of Education, Institute of Biotechnology,
Shanxi University, Taiyuan 030006, China.
E-mail: aliang@sxu.edu.cn

Summary
Phytases are a group of enzymes capable of releasing phosphates from phytates,
one of the major forms of phosphorus (P) in animal feeds of plant origin.
These enzymes have been widely used in animal feed to improve phosphorus
nutrition and to reduce phosphorus pollution in animal waste. This review
covers the basic nomenclature and crystal structures of phytases and emphasizes both the protein engineering strategies used for the development of new,
effective phytases with improved properties and the potential biotechnological
applications of phytases.

2011 0699: received 25 April 2011, revised

19 September 2011 and accepted 7 October
2011
doi:10.1111/j.1365-2672.2011.05181.x

Introduction
Phytases (myo-inositol hexakisphosphate phosphohydrolases) catalyse the partial or complete hydrolytic removal
of orthophosphates from phytates (myo-inositol hexakisphosphates). Phytate is the principal form in which phosphorus and inositol are stored in cereals, legumes used in
commercial animal feeds and oilseeds; phytates constitute
c. 6090% of the total phosphorus content in plants
(Reddy et al. 1982). Phytate is hydrolysed by phytase into
one molecule of inositol and six molecules of inorganic
phosphate (Fig. 1). Unfortunately, phytates are not hydrolysed in the monogastric gut, and the phytate-associated phosphates remain unabsorbed, requiring the
exogenous addition of phosphate to avoid phosphorus
deficiency. In addition to increasing basic food costs, the
phosphates in the excreted phytates are available to
microbial soil consortia, which break them down to create phosphate run-off and serious environmental pollution (Greiner and Konietzny 2006). Furthermore, phytates
are a strong chelator of cations and bind minerals such as
Ca2+, Zn2+ and Fe2+ (Lopez et al. 2002; Vats and Banerjee

2004), making them unavailable for absorption in the

intestine of the monogastric animal. In addition, phytates
are known to form complexes with proteins under both
acidic and alkaline pH conditions. These interactions were
found to affect the proteins structure, thus decreasing the
enzymatic activity, protein solubility and proteolytic
digestibility (Kies et al. 2006) (Fig. 1). Supplemental
microbial phytases in cornsoybean meal diets for monogastric animals can reduce these problems, improve the
animals utilization of the phytate phosphorus and reduce
their faecal phosphorus excretion by up to 50% (Leytem
et al. 2008; Kim et al. 2010).
So far, phytases have been mainly if not solely used
as a feed supplement in diets for swine and poultry, and
to some extent for fish. The inclusion of phytases in
animal feed is attracting, increasing more attention
internationally. Laboratory experiments and field trials
have repeatedly demonstrated that 5001000 units of
phytase can replace c. 1 g of inorganic phosphorus
supplementation and reduce total phosphorus excretion
by 3050% (Yi et al. 1996; Kemme et al. 1997). Thus,
phytases perform a double duty, conserving expensive

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Journal of Applied Microbiology 112, 114 2011 The Society for Applied Microbiology

Engineering and application of phytase

HO
Z n 2+
O P
O
OH
O
O P
O
O

Starch

NH3

Ca2+
O
O P OH
O

O
O P O
O

O
O P O
OH

OH
OH

Phytase

H
H

H
HO

Protein
CH2

M.-Z. Yao et al.

O
O P O
OH
CH2OH
O
O
Starch

6 PO3

HO
OH
H

Ca2+
Protein
Zn2+

OH

Figure 1 The hydrolysis of phytate by phytase into inositol, phosphate and other divalent elements. The removal of phosphate groups by phytase
results in the release of metals, metal-binding enzymes and proteins.

and nonrenewable inorganic phosphorus resources by

reducing the need for their inclusion in animal feed,
while also protecting the environment from pollution
resulting from excessive manure phosphorus run-off. For
these reasons, phytases are increasingly used worldwide as
a phosphate-mobilizing feed supplement in the diets of
swine and poultry (Ketaren et al. 1993).
Although phytase was first shown to hydrolyse phytate
phosphorus in diets for chicks 40 years ago, its commercial application was not feasible for a long time because
of low yield, high costs and low enzymatic activity after
pelleting. Challenges in the above three areas have
prompted the rapid emergence of the field of phytase science and biotechnology. Phytase optimization by genetic
and protein engineering is actively being pursued as no
known phytase fulfils all properties for the ideal additive.
Here we highlight the current knowledge on phytases
with regard to their three-dimensional structure, their
enzyme mechanism, improvements in their bioavailability
to support the wider application of phytases in animal
and crop production.
Phytase nomenclature
Phytases (IP6 phosphohydrolase) are a class of phosphatase that sequentially hydrolyse phytate to lesser phosphomyo-inositol derivates and phosphate (Wyss et al. 1999).
Phytases are ubiquitously found in animals, plants and
micro-organisms. Examples include alkaline phytases
from Bacillus sp. that degrade plant metal-phytate complexes (Kerovuo et al. 2000a; Choi et al. 2001; Gulati
et al. 2007), phytate-degrading enzymes in calf, bird, reptile and fish blood (McCollum and Hart 1908; Rapoport
et al. 1941), and phytases in maize, barley, rice, wheat
and soybean (Hubel and Beck 1996; Dionisio et al. 2011;
Maugenest et al. 1999; Nagai and Funahashi 1962;
Hamada 1996).
Depending at which carbon in the myo-inositol ring of
phytate dephosphorylation is initiated, phytases can be
2

grouped into 3-phytases (myo-inositolhexakisphosphate3-phosphohydrolase, EC 3.1.3.8), 6-phytases (myo-inositol hexakisphosphate 6-phosphohydrolase, EC 3.1.3.26)
and 5-phytases (myo-inositol hexakisphosphate 5-phosphohydrolase, EC 3.1.3.72). 3-Phytases, in fact, include
fungal and bacterial representatives that dephosphorylate
phytates at either C1 or C3. (Sajidan et al. 2004). 6-Phytases are frequently found in grains and oil seeds of
higher plants, while 5-phytases have been isolated from
alfalfa, beans, peas and Selenomonas ruminantium (Chu
et al. 2004). Recently, a new protein tyrosine phosphatase
(PTP)-like inositol polyphosphatases (IPPases) phyAme
was identified that defines a new class of IPPase based on
its stereospecific reaction of cleaving InsP6 at positions
1D-3 or 1D4 (Puhl et al. 2009).
On the basis of their pH optima, phytases can be
broadly divided into two major classes: acid and alkaline
phytases. Acid phytases include those enzymes belonging
to the histidine acid phosphatases (HAPs), purple acid
phosphatases (PAPs) and PTP-like class of phosphatases
(the latter of which was identified more recently). To
date, the b-propeller phytases (BPPs) from Bacillus are
the only extensively characterized class of alkaline phytase
(Kerovuo et al. 1998; Tye et al. 2002). Phytases also exhibit variations in their catalytic mechanism and consequently have been categorized into HAPs, BPPs, PAPs or
cysteine phytases (Mullaney and Ullah 2003). Several fungal, bacterial and plant phytases belong to the HAPs class
of enzymes (Wyss et al. 1999). All of these phytases share
a conserved active site hepta-peptide motif RHGXRXP
and the catalytically active dipeptide HD, unique to this
class of enzymes (Etten et al. 1991). This group of
enzymes catalyses the phytic acid hydrolysis in a two-step
process: a nucleophilic attack on the phosphorous atom
by the histidine in the active site, followed by hydrolysis
of the resulting phospho-histidine intermediate (Vincent
et al. 1992). Phytases from Bacillus species constitute an
exception: These enzymes share a sequence identity of
9098% each other but are unrelated to HAPs and other

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Journal of Applied Microbiology 112, 114 2011 The Society for Applied Microbiology

M.-Z. Yao et al.

Engineering and application of phytase

phosphatases. Unlike other HAPs, they require Ca2+ for

activity and show a different pH optimum of 7080
(Kerovuo et al. 1998; Kim et al. 1998). Meanwhile, a phytase isolated from soybean was found to be unrelated to
previously characterized microbial or maize (Zea mays)
phytases, classified as HAPs. This soybean phytase is a
PAP, characterized by seven conserved residues (bold) in
the five conserved motifs DXG, GDXXY, GNH(D E),
VXXH and GHXH involved in the coordination of the
dimetal nuclear centre (Hegeman and Grabau 2001; Li
et al. 2002). In contrast, S. ruminantium phytase neither
contains the conserved RHGXRXP motif nor is affected
by divalent metal ions. The active site is located near a
conserved cysteine-containing (Cys241) P loop (Chu et al.
2004).
Phytase crystal structures
Crystal structure analyses of a number of phytases have
revealed a range of distinct folds for these enzymes and
have allowed their biophysical properties to be rationalized in terms of their structure. The crystal structure of
Aspergillus ficuum phytase at 25 A resolution revealed
three distinct domains, including a large a-helical domain
and b-sheet domain, and a small a-helical domain. The
large a-helical domain and small a-helical domain contain
five a-helixes and four a-helixes, respectively, and the bsheet domain contains eight b-sheets (Kostrewa et al.
1997). Crystal structure analysis of Escherichia coli phytase
with a resolution of 22 A also showed two domains. One
contains five a-helixes and two b-sheets, and the other
includes six a-helixes and nine b-sheets (Lim et al. 2000).
The crystal structure of phytases from Debaryomyces castellii (PhytDc) was determined at a resolution of 23 A.
The PhytDc structure is very similar to that of A. ficuum
phytases and can be divided into two parts: a large a-helical b-sheet domain with a six-stranded b-sheet, and a

(a)

small a-helical domain (Ragon et al. 2009). The crystal

structures of the phytases mentioned above belong to one
type of protein folding, which has an a-helical domain
and a conserved a-helical b-sheet domain, with two helices on each side of the seven-stranded sheet. The active
site is located at the interface between the two domains.
These structures closely resemble the overall folding in
other HAPs. A three-dimensional model of A. ficuum
phytase (1IHP) from the National Center for Biotechnology Informations (NCBI) website is shown in Fig. 2a.
The crystal structure of Bacillus amyloliquefaciens
phytase (TsPhy) at 21 A resolution revealed a six-bladed
b-propeller in which each blade consists of a four- or
five-stranded antiparallel b-sheet (Fig. 2b, PDB code
1H6L). The enzyme binds seven Ca2+: two near the
periphery, one in the central channel and four near the
top of the molecule. Unlike other b-propeller structures,
it does not show any conserved sequence repeats in the
b-sheet. The crystal structures of TsPhy at 21 A resolution in both the partially and the fully Ca2+-loaded states
were determined. And the dependence of thermostability
of TsPhy on Ca2+ was assessed by differential scanning
calorimetry. The binding of two Ca2+ to high-affinity
Ca2+-binding sites results in a dramatic increase in thermostability (with an increase of as much as c. 30C in the
melting temperature), because of the joining of loop segments remote in the amino acid sequence. Three Ca2+
bind to the active Ca2+-binding sites and create an ideal
conformation and charge distribution for the substrate.
Substrate binding to the active site would appear to be
followed by occupation of the fourth Ca2+ site to offset
the negative charge of the substrate phosphate group
already coordinated by lysine and arginine (Fu et al.
2008b).
Selenomonas ruminantium phosphatase (SrPhy) represents a third, dual-specificity phosphatase type with a
conserved cysteine (C241) in its so-called P loop. Two

(b)

(c)

Figure 2 Swiss-Pdb viewer-prepared molecular models from the National Center for Biotechnology Information (NCBI)s website (http://
www.ncbi.nlm.nih.gov), representing three types of phytases: (a) 1IHP, PhyA, a histidine acid phosphatase; (b) 1H6L Ts-Phy, a b propeller phytase;
(c) 1U26, SrPhy, a cysteine phytase.
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Journal of Applied Microbiology 112, 114 2011 The Society for Applied Microbiology

Engineering and application of phytase

M.-Z. Yao et al.

distinct crystal packing arrangements have been observed

of the complex of SrPhy with the inhibitor myo-inositol
hexasulfate. The inhibitor is bound to both standby and
inhibited conformations. In a pocket slightly away from
the conserved P loop Cys241 and at the substrate binding
site, the phosphate group to be hydrolysed is held close
to the -SH group of Cys241. Further, mutagenesis studies
verify that the P loop-containing phytase attracts and
hydrolyses the substrate (phytate) sequentially via a complicated mechanism (Chu et al. 2004). Figure 2c shows
1U26 to underscore the structural differences in these
three classes of enzyme.
Development of effective phytases
Identification of novel phytases
Phytases are produced in a wide range of plant, bacterial,
fungal and animal tissues. Ever since the discovery of
phytase in rice bran (Suzuki et al. 1907), a variety of phytases with different properties have been identified from
organisms (Table 1). Most scientific work has, however,
been performed on microbial phytases, particularly those
from filamentous fungi such as A. ficuum (Gibson 1987),
Aspergillus fumigatus (Pasamontes et al. 1997) or Mucor
piriformis (Howson and Davis 1983), Rhizopus oligosporus
(Casey and Walsh 2004) and Cladosporium species (Quan
et al. 2004). The search for phytases with higher thermostability resulted in the cloning of the phytase gene from
A. fumigatus (Pasamontes et al. 1997), the purified
enzyme of which retains 90% of its initial activity after
being maintained at 100C for 20 min. By contrast, the
phytase from Aspergillus niger remains only 30% active
after being heated to 70C for 20 min (Pasamontes et al.
1997; Wyss et al. 1998). The thermostability of A. fumigatus phytase, A. niger phytase and A. niger acid phosphatase was assessed by protein unfolding and refolding
experiments using circular dichroism spectroscopy and
protein fluorescence. Only A. fumigatus phytase could
refold into a fully active, native conformation even after
heat denaturation at 90C. In feed pelleting experiments
performed at 85C, the recovery of enzyme activity was
51% for A. fumigatus phytase but only 31% for A. niger
phytase and 14% for A. niger acid phosphatase (Wyss
et al. 1998). In contrast to most other phytate-degrading
enzymes, the enzyme from A. fumigatus has a broad pH
optimum; at least 80% of the maximal activity was
observed at pH values between 40 and 73 (Wyss et al.
1999).
Fungal phytases from Peniophora lycii, Agrocybe pediades, Ceriporia sp., and Trametes pubescens showed a high
degree of refolding after thermal unfolding, as evidenced
by differential scanning calorimetric studies (Lassen et al.
4

2001). The thermal stability of fungal phytases is often

attributed to highly reversible thermal unfolding, rather
than an intrinsic thermostability (Wyss et al. 1998). Fungal phytase have also been isolated from thermophilic
Rhiomucor pusillus and Thermocyces lanuginosus. The former has a temperature optimum of 70C, while the latter
retains its activity up to 75C, has a higher catalytic efficiency at 65C than other fungal phytases and has a comparably broad pH optimum as A. fumigatus phytase
(Chadha et al. 2004; Berka et al. 1998). Recently, a novel
phytase gene from A. niger N-3 was cloned and expressed
in Pichia pastoris. The purified enzyme of which retains
45% of its initial activity after being maintained at 90C
for 5 min. It showed a greater affinity for sodium phytate
than for p-nitrophenyl phosphate. Dual optimum pH values were obtained at 20 and 55. The activity at pH 20
was about 30% higher than that at pH 55, which is more
similar to conditions in the stomachs of monogastric animals (Shi et al. 2009). Two novel thermostable genes were
identified in Aspergillus japonicus BCC18313(TR86) and
BCC18081(TR170), respectively. The thermostable nature
of this phytases gives it valuable potential for applications
(Promdonkoy et al. 2009).
Apart from the phytase genes identified in fungi, others
have been cloned and identified in other microbes, motivated by their potential for applications. To find a phytase with high activity at low temperature and neutral
pH, two phytases have been isolated from Pedobacter nyackensis MJ11 CGMCC 2503 and Erwinia carotovora var.
carotovota ACCC 10276. The Pedobacter phytase belongs
to the BPP family and shares very low identity (approximately 285%) with Bacillus subtilis phytase. Compared
with the major commercial phytases and B. subtilis phytase, the purified recombinant enzyme from E. coli displayed higher activity and hydrolysed phytate from
soybean meal with better efficiency at neutral pH and
25C. These characteristics suggest that this phytase has a
great potential as an aquatic feed additive in the rapidly
developing aquaculture industry. The Erwinia phytase
contains a conserved active site hepta-peptide motif
RHGXRXP and the catalytically active dipeptide HD that
are typical of HAPs and shares a 50% amino acid identity
to the Klebsiella pneumoniae phytase (Huang et al.
2009a,b). And except for potential application in aquaculture, the latter is also attractive for food processing by
avoiding damage to the food in gradients at low temperatures (Greiner and Konietzny 2006). Moreover, owing to
its typical properties as a low-temperature-active enzyme,
it could be a good model protein to study the relationship between structure and function. The gene appA,
encoding a phytase from Yersinia kristeensenii, was cloned
and heterologously expressed in P. pastoris. The data
show that the Y. kristeensenii phytase is highly pH stable

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M.-Z. Yao et al.

Engineering and application of phytase

Table 1 Sources and properties of phytases, published during the last 10 years

Phytase source

Specific activity

Molecular
weight (kDa)

Temperature
optimum (C)

pH
optimum

Km
(lmol l)1)

Fungi
Aspergillus niger van Teighem
Aspergillus niger
Aspergillus ficuum
A. ficuum
A. ficuum

22 592 U mg)1

66
39
76
655
675816

5255
556
50
67
58

25
262, 505
20, 55
13
50

606
09
730
295
124

51 U mg)1
2 U ml)1
700 80 U mg)1
909 U mg)1
1080 110 U mg)1
3066 U mg)1

1210 30 U mg)1

88
74
59

72
625

62

55
50
5560
40
5055
55
70
50

55
5560
5560
35
4050
45
54
5055

114

152 31

370

Vats and Banerjee (2005)

Sariyska et al. (2005)
Zhang et al. (2004)
Zhang et al. (2010)
Ullah and Sethumadhavan
(2003)
Wang et al. (2007)
Uchida et al. (2006)
Lassen et al. (2001)
Quan et al. (2004)
Lassen et al. (2001)
Lee et al. (2007)
Chadha et al. (2004)
Lassen et al. (2001)

16 U mg)1
36 U mg)1

35 U mg)1
1269 U mg)1

769 U mg)1

99 U mg)1

310 U mg)1
2514 U mg)1
244 U mg)1
3960 U mg)1
2656 U mg)1

40
44
41
44
4146
47

453

69

45

38
45
48

6773
55
40
55
55
70
65
55
40
45
50
45
4045

45
55
55

67
70
6585
75
70
7080
70
45, 55
55
50
50
40
49

70
45
45

392
50

526

399

280

340

1280

Tran et al. (2010)

Rao et al. (2008)
Choi et al. (2001)
Farhat et al. (2008)
Tye et al. (2002)
Gulati et al.(2007)
Tye et al. (2002)
Gu et al. (2009)
Huang et al. (2009a)
Sajidan et al. (2004)
Palacios et al. (2005)
De Angelis et al. (2003)
Zinin et al. (2004)
Cho et al. (2005)
Huang et al. (2009b)
Huang et al. (2006)
Fu et al. (2008a)

165 U mg)1

330
80
51

40
50
65

46
45
5

30

Quan et al. (2002)

Watanabe et al. (2009)
Li et al. (2009b)

64
70

60
70

40
45

Vohra et al. (2010)

Hamada et al. (2005)

02 U mg)1

65
54
88
22
7072

45
65
55
55
58

60
45
80
50
4550

830
334 31
81

61

54

55
65

52
45

290
246 38

Bohn et al. (2007)

Dionisio et al. (2007)
Jog et al. (2005)
Gonnety et al. (2007)
Hegeman and Grabau
(2001)
Agostini and Ida (2006)
Dionisio et al. (2007)

Aspergillus fumigatus
Aspergillus oryzae
Ceriporia sp.
Cladosporium sp. FP-1
Peniophora lycii
Penicillium oxalicum PJ3
Rhizomucor pusillus
Trametes pubescens
Bacteria
Bacillus sp.
Bacillus sp.
Bacillus sp. KHU-10
Bacillus subtilis
B. subtilis
Bacillus laevolacticus
Bacillus licheniformes
Dickeya paradisiaca
Erwinia carotovora var. carotovota
Klebsiella sp. ASR1
Lactobacillus pentosus
Lactobacillus sanfranciscensis
Obesumbacterium proteus
Pseudomonas syringae MOK1
Pedobacter nyackensis
Yersinia intermedia
Yersinia kristeensenii
Yeasts
Candida krusei
Hansenula fabianii J640
Marine yeast Kodamaea
ohmeri BG3
Pichia anomala
Schwanniomyces occidentalis
Plants
Crude extract wheat
Hordeum vulgare
Lily pollen
Peanut
Soybean
Sunflower
Triticum aestivum L.

at pH 15110 and thermostable, providing significant

advantages for processing, transportation, storage and
application. Comparison of r-APPA with other wellknown phytases suggested that the Y. kristeensenii phytase

Reference

would be an attractive enzyme for feed industry use (Fu

et al. 2008a). A list of bacterial phytases with considerable
variations in biochemical properties is presented in
(Table 1).

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Engineering and application of phytase

M.-Z. Yao et al.

In addition, phytases from yeast have also been identified and characterized (motivated by their potential as a
feed additive for improving the phytate-phosphorus
digestibility in monogastric animals), such as the marine
yeast Kodamaea ohmeri BG3 (Li et al. 2008, 2009b), Pichia anomala (Kaur et al. 2010; Vohra et al. 2010) and
wastewater treatment yeast Hansenula fabianii J640 (Watanabe et al. 2009).

cally efficient, proteolysis-resistant, thermostable and

cheap (Lei and Stahl 2001). In reality, phytases possessing
all of these qualities may never be found or generated. To
obtain enzymes with modified and desired properties, two
different strategies are used: rational protein design and
directed (molecular) evolution, which are increasingly
applied in a synergistic manner to tailor-design the
enzyme for a given process (Chica et al. 2005; Bottcher
and Bornscheuer 2010) (see Fig. 3). One recent example
of the improvement of phytase activity was shown for the
phyI1s from A. niger 113, where two single mutant phyI1s
Q53R and K91D were obtained via a semi-rational sitedirected mutagenesis strategy. None of the single amino
acid residues in the two mutants was in a position

Protein engineering of phytases

Although properties of phytases vary, there is no single
wild-type enzyme that is perfect or ideal for field applications. Theoretically, an ideal phytase should be catalyti-

Develop effective phytases

Organism

Gene of phytase

Functional information

Semi-rational design

may be gained from small

Choice of method

library or point mutations

for phytase engineering

Whole-gene randomization
Yes

No

Yes

High-throughput
screening available?

No

Data about structure

Yes

and functional information

No

High-throughput
screening available?

available

partial

Semi-rational design

Directed Evolution

Rational design

Simultaneous
Random mutagenesis
ep PCR

Structral variation
Saturation
mutagenesis

and combination

Molecular modeling
and
Site-directed mutagenesis

Figure 3 Identification of novel phytases and selection of preferred experimental phytase engineering approaches, based on prior knowledge of
structure and function and the feasibility of a high-throughput screening system for screening. This figure is based partly on published articles
(Bottcher and Bornscheuer 2010; Chica et al. 2005), with slight modifications.

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Journal of Applied Microbiology 112, 114 2011 The Society for Applied Microbiology

M.-Z. Yao et al.

reported to be important for catalysis or substrate binding. Kinetic analysis of the phytase activity of the two
mutants (Q53R and K91D) showed a respective 22- and
15-fold increase in specific activity, and a 147- and 116fold increased affinity for sodium phytate. In addition,
the overall catalytic efficiency (kcat Km) of the two
mutants was improved 408- and 284-fold compared with
that of the wild type (Tian et al. 2010). A semi-rational
protein engineering strategy based on 3D structure and
sequence alignment was used to take advantage of the
desirable characteristics of A. niger NRRL 3135 phytase
(Anp) and A. fumigatus ATCC 13073 phytase (Afp); these
were chosen because they are quite different but possess
many mutually complementary properties (Bei et al.
2009). Another example for engineering of the thermostability of phytases is given by A. niger PhyA phytase; crystal structure comparisons with its close homolog, the
thermostable A. fumigatus phytase (Afp), suggested thermostability associations with several key residues (E35,
S42, R168 and R248) that formed a hydrogen bond network in the E35-to-S42 region, and ionic interactions
between R168 and D161, and R248 and D244. Finally,
four mutants showed improved thermostability; the best
response came from the quadruple mutant (A58E P65S
Q191R T271R), which retained 20% greater (P < 005)
activity after being heated at 80C for 10 min and had a
7C higher melting temperature than that of wild-type
PhyA (Zhang et al. 2007).
The E. coli phytase appA gene product was chosen as a
candidate for increasing thermal tolerance to promote the
survival of the enzyme during pelleting, because of its high
specific activity and specificity and its favourable pH profile
for gastric activity. To create an optimized phytase gene, site
saturation mutagenesis (GSSM) technology was employed
to identify mutations that increased enzymatic performance.
The technology, which generates a library of all possible single-site mutations in an enzyme, can target individual
aspects of a phenotype in association with an appropriate
high-throughput screening assay. In this case, the dual goals
of increased thermal tolerance and maintenance of high
turnover were established. Combining GSSM technology
with an assay that challenged all of the GSSM constructs
with a heat step revealed mutations that increased thermal
tolerance. Combining these mutations led to a phytase
Phy9X with an optimal phenotype for economic use as an
animal feed supplement (Garrett et al. 2004).
Another E. coli phytase (AppA2) was evolved to a thermostable phytase via epPCR. After a mutant library of
AppA2 was generated via error-prone polymerase
chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in P. pastoris
for characterization of thermostability. Compared with
the wild-type enzyme, two variants (K46E and

Engineering and application of phytase

K65E K97M S209G) showed an improvement of over

20% in thermostability (80C for 10 min) and 67C
increases in melting temperatures (Tm) (Kim and Lei
2008). To highlight the rapidly growing number of successful phytase engineering studies using rational protein
design and directed (molecular) evolution, previously
reported examples are summarized in Table 2.
Potential biotechnological applications of
phytases
Because of the potential value of phytases for improving
the efficiency of phosphorus use, biotechnology has led the
rapid development of the field to its current stage. With
the development of heterologous gene expression, large
amounts enzymes could be produced at relatively low cost.
Microbial expression systems
A transgenic approach has proved to be a powerful tool for
expressing specific phytase genes on a large scale in yeast
strains, which have been verified to be an elite expression
system for heterologous genes. Several studies have already
investigated the use of various yeast expression systems as
an alternative to the current phytase production method
using overexpression in filamentous fungi. It was found that
phytases used in ecotopic expression could be derived from
a host of micro-organism sources (Xiong et al. 2006; Lei
et al. 2007). They created a P. pastoris that expressed the
modified phytase gene (phy-pl-sh), with the MF4I
sequence producing 122 g of phytase per litre of fluid culture and a phytase activity of 10540 U ml)1. Using the
phytase gene (phytDc) from D. castellii and an alphaamylase gene (AMY) from Debaryomyces occidentalis as the
target genes, Lim et al. (2008) developed an industrial
strain of S. cerevisiae. Phytase has also been expressed in
Stretptomyces lividans (Stahl et al. 2003) and Lactobacillus
plantarum (Kerovuo et al. 2000b). The latter expression
system offers the possibility of combining phytase with
beneficial probiotic lactic acid bacteria. Recently, a great
research effort has been made towards the use of transgenic
rhizobacteria overexpressing citrobacter braakii appA on
phytate-P availability to mung bean plants. This is the first
report of the overexpression of phytase in rhizobacterial
strains and its exploitation for plant growth enhancement
(Patel et al. 2010).
Transgenic plants and animals
Transgenic plants might contain sufficient phytase activity
that they could replace additional supplementation of
feed and food with microbial phytases, thereby reducing
the downstream processing and formulation costs

2011 The Authors

Journal of Applied Microbiology 112, 114 2011 The Society for Applied Microbiology

N A, not applicable.

3D structure and
PCR overlap assembly
sequence alignment

Two fungal
phytases

Combination of desirable
properties

Structure-guided
Whole-gene synthesis
consensus approach

Mn2+-dNTP random
mutation method

Thermos b-propeller Broaden pH profiles

phytase

High thermal stability,

NA
low optimal temperature
and pH

Gene site saturation

mutagenesis (GSSM),
high-throughput
screening

NA

Protease-resistant
phytase from
Penicillium sp.

Error-prone PCR,
high-throughput
screening

NA

Enhance the thermal

tolerance and gastric
performance

Improve thermostability

Escherichia
coli (AppA)

Site-directed
mutagenesis
strategy

Sequence alignment

E. coli (AppA)

Improve activity

A. niger 113
(PhyI1s)

3D structure and
DNA shuffling
sequence alignment

Improve thermostability

Site-directed
mutagenesis

Crystal structure

Improve thermostability

Conclusions comments

Hydrogen bond network

and ionic interactions
play an important role
Replacing one a-helix phytase in
Sequence alignment in
A. terreus with a A. niger phytase
structure-based hybrid
resulted in an enzyme with higher
enzymes has potential as
thermostability
an additional strategy to
improve defined enzyme
characteristics
22- and 15-fold increase in specific
Amino acid residues near
activity, and 147- and 116-fold
the catalytic active centre
increase in affinity for sodium phytate or substrate specificity
site as well as some
residues far from this
site can effect phytase
characteristics
The thermostability of AppA phytase
The mutant I408L could be
I408L showed a 233% increase
used for the large-scale
compared with WT
commercial production
of phytases
No loss of activity after heating
GSSM technology is
at 62C for 1 h, and 27% of its
particularly useful, providing
initial activity after heating for
comprehensive codon
variation to chart an optimal
10 min at 85C; a 35-fold
mutational path, to selectively
enhancement in gastric stability
and rapidly target aspects of
an enzymes phenotype
Two mutants with improved thermal
Facilitating the interaction
stability and optimal temperature
between the substrate and
and pH retained their high
the catalytic centre,
resistance to pepsin
weakening the bonding with
the side chain of D353
Showed activity over a pH range
P257, D336 likely play an
of 259; showed new properties
important role, form a larger
at pH 55 and 75
number of hydrogen bonds
Phytases with desirable properties
The region-shuffling scheme
were obtained by systematic
described could be adopted
evaluation of substitutions
for other phytases with great
comparability and compatibility

Mutant (A58E P65S Q191R T271R)

showed a higher thermostability

Experimental strategies Results

Aspergillus
niger phytase
(PhyA)
Aspergillus
terreus phytase

Design

Project goal

Target

Table 2 Previously reported examples of phytase engineering

Bei et al. (2009))

Viader-Salvado
et al. (2010)

Zhao et al. (2010)

Garrett et al. (2004)

Zhu et al. (2010)

Tian et al. (2010)

Jermutus et al. (2001)

Zhang et al. (2007)

References

Engineering and application of phytase

M.-Z. Yao et al.

2011 The Authors

Journal of Applied Microbiology 112, 114 2011 The Society for Applied Microbiology

M.-Z. Yao et al.

Engineering and application of phytase

involved in the commercial production of phytases. Alternatively, transgenic plants could be used as bioreactors
for the production of phytase as a supplement. Meanwhile, transgenic animals producing endogenous phytase
along with other digestive enzymes are known to increase
the bioavailability of plant phytate, which in turn leads to
reduced phosphorus output in manure.
Phytases have been expressed in several dicotyledonous
plants like tobacco (Pen et al. 1993), Arabidopsis thaliana
(Richardson et al. 2001; Xiao et al. 2005), alfalfa (Ullah
et al. 2002), canola (Ponstein et al. 2002), soybean (Chiera
et al. 2004) and so on. Aspergillus phytase expressed in
maize seeds exhibited an increase in iron bioavailability, as
evidenced by in vitro digestion and Caco-2 cell model studies (Drakakaki et al. 2005). Aspergillus fumigatus phytase
expressed in tobacco exhibited high thermostability and
retained 287% of the initial activity, even after incubation
at 90C for 15 min (Wang et al. 2007). The expression of
B. subtilis phytase in tobacco indicated that the Bacillus
phytase transgene could only improve the phytate-phosphorus uptake by transgenic plants under sterilized conditions, and its effectiveness might be limited under natural
conditions because of microbial decomposition and mineral fixation. The microbial community in the rhizosphere
appears to be resistant to the impact of single-gene changes
in plants designed to alter rhizosphere biochemistry and
nutrient cycling (Kong et al. 2005; George et al. 2009).
Overexpression of heterologous phytases in transgenic
potato not only offers an ideal feed additive for improving
phytate-P digestibility in monogastric animals, but also
improves tuber yield, enhances P acquisition from organic
fertilizers and has the potential for phytoremediation
(Hong et al. 2008). Transgenic soybean plants expressing

Arabidopsis (Arabidopsis thaliana) PAPs (AtPAP15) exhibited enhanced bioavailability of phosphorus (Wang et al.
2009). In plants, the phosphate-absorption sites are in the
root system, especially the root hairs. It is therefore better
for the phytases ectopically expressed in transgenic plants
to be secreted in the rhizosphere where the phytate and its
derivatives are degraded by the biochemical reactions
involved in the encoded phytases. Therefore, in transgenic
soybean plants in which the A. ficuum phytase (AfPhyA)
was integrated, a promoter from the Arabidopsis Pky10
gene and the carrot extensin signal peptide were used to
drive the root-specific and secretory expression of the AfPhyA gene. The phytase activity and inorganic phosphate
levels in transgenic soybean root secretions were
47 U mg)1 protein and 439 lmol l)1, respectively, compared with 08 U mg)1 protein and 120 lmol l)1 in control soybeans, suggesting that the transgenic techniques
could be of great value for the generation of crop varieties
with high P-use efficiency in the future (Li et al. 2009a).
Expression levels of heterologous phytases in diverse transgenic plants are presented in Table 3.
Transgenic mice and pigs have been generated by overexpressing phytase in their salivary glands (Golovan et al.
2001a,b). A transgenic mouse model has been developed
with an appA phytase gene from E. coli, driven by the
upstream promoter of a pig parotid secretory protein
gene. Expression of salivary phytase reduced faecal phytates by 85 and 125% in two transgenic mouse lines
(Yin et al. 2006). The transgenic Enviropig, expressing
E. coli appA phytase, could secrete active phytase into its
saliva and showed a substantial reduction (60%) in the
excretion of phosphorus compared with nontransgenic
animals (Forsberg et al. 2003).

Table 3 Expression of phytase in transgenic plants

Transgenic plant
Alfalfa
Canola
Maize
Medicago truncatula
Potato
Potato
Rice
Soybean
Soybean
Soybean
Soybean
Tobacco
Tobacco
Tobacco
Tobacco
Trifolium subterraneum L.

Source of phytase
Aspergillus ficuum
Aspergillus niger
A. niger
A. niger
A. ficuum
Escherichia coli
Schwanniomyces occidentalis
A. niger
Aspergillus awamori
A. ficuum
Arabidopsis thaliana
A. niger
A. ficuum
Aspergillus fumigatus
A. niger
A. niger

Tissues expressed
Leaves
Seeds
Seeds
Cell suspension culture
Leaves
Plant
Leaves
Cell suspension culture
Seeds
Roots
Roots
Leaves
Leaves
Cell suspension culture
Seeds
Shoots

2011 The Authors

Journal of Applied Microbiology 112, 114 2011 The Society for Applied Microbiology

Enzyme activity
)1

3893 nKat g per FW

41 FTU per g

2979 nKat mg)1 protein

106 U g)1 per FW

920 pKat lg)1 per protein
125 FTU per kg
47 U mg)1 protein
2400 ng mg)1 per DW
3280 nKat mg)1

15 FTU per g
305 nKat g)1 per FW

References
Ullah et al. (2002)
Peng et al. (2006)
Chen et al. (2008)
Pires et al. (2008)
Ullah et al. (2003)
Hong et al. (2008)
Hamada et al. (2005b)
Li et al. (1997)
Gao et al. (2007)
Li et al. (2009a)
Wang et al. (2009)
Verwoerd et al. (1995)
Ullah et al. (1999)
Wang et al. (2007)
Pen et al. (1993)
George et al. (2004)

Engineering and application of phytase

M.-Z. Yao et al.

Conclusions and future perspectives

Along with the motivation provided by increasing concerns relating to phosphorus pollution in the areas of
intensive livestock, developments in the phytase field are
driven by their considerable potential in commercial and
environmental applications. However, a limited number
of phytases have been reported and studied, and our scientific knowledge of phytases has yet to yield a solution
to meet the nutritional and environmental requirements
that a real-world solution demands. Further research into
identifying new phytases, engineering better phytases and
developing more cost-effective expression systems should
be continued. With the collaborative efforts of scientists
from different fields, effective solutions for the biotechnological development of phytases for mineral nutrition and
environmental protection should be available in the near
future.
Acknowledgements
This project was supported by grants from the National
Natural Science Foundation of China (no. 31071924), the
Natural Science Foundation of Shanxi Province
(2010011040-1) and the Shanxi Scholarship Council of
China.
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