Tài liệu Báo cáo khoa học: The tandemly repeated domains of a b-propeller phytase act synergistically to increase catalytic efficiency doc

The tandemly repeated domains of a b-propeller phytase
act synergistically to increase catalytic efficiency
Zhongyuan Li*, Huoqing Huang*, Peilong Yang, Tiezheng Yuan, Pengjun Shi, Junqi Zhao,
Kun Meng and Bin Yao
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences,
Beijing, China
Keywords
dual domain; fusion protein; phytate;
synergistic catalysis; b-propeller phytase
Correspondence
B. Yao, Key Laboratory for Feed
Biotechnology of the Ministry of Agriculture,
Feed Research Institute, Chinese Academy
of Agricultural Sciences, No. 12
Zhongguancun South Street, Beijing
100081, China
Fax: +86 10 8210 6054
Tel: +86 10 8210 6053
E-mail: ;

*Z. Li and H. Huang contributed equally to
this paper
(Received 10 March 2011, revised 20 June
2011, accepted 23 June 2011)
doi:10.1111/j.1742-4658.2011.08223.x
b-Propeller phytases (BPPs) with tandemly repeated domains are abundant
in nature. Previous studies have shown that the intact domain is responsi-
ble for phytate hydrolysis, but the function of the other domain is rela-
tively unknown. In this study, a new dual-domain BPP (PhyH) from
Bacillus sp. HJB17 was identified to contain an incomplete N-terminal BPP
domain (PhyH-DI, residues 41–318) and a typical BPP domain (PhyH-DII,

residues 319–644) at the C-terminus. Purified recombinant PhyH and
PhyH-DII required Ca
2+
for phytase activity, showed activity at low tem-
peratures (0–35 °C) and pH 6.0–8.0, and remained active (at 37 °C) after
incubation at 60 °C and pH 6.0–12.0. Compared with PhyH-DII, PhyH is
catalytically more active against phytate (catalytic constant 27.72 versus
4.17 s
)1
), which indicates the importance of PhyH-DI in phytate degrada-
tion. PhyH-DI was found to hydrolyze phytate intermediate
D-Ins(1,4,5,6)
P
4
, and to act synergistically (a 1.2–2.5-fold increase in phosphate release)
with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid
phosphatase. Furthermore, fusion of PhyH-DI with PhyP or 168PhyA sig-
nificantly enhanced their catalytic efficiencies. This is the first report to elu-
cidate the substrate specificity of the incomplete domain and the functional
relationship of tandemly repeated domains in BPPs. We conjecture that
dual-domain BPPs have succeeded evolutionarily because they can increase
the amount of available phosphate by interacting together. Additionally,
fusing PhyH-DI to a single-domain phytase appears to be an efficient way
to improve the activity of the latter.
Introduction
Phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate, InsP
6
)
is the most abundant organic phosphorus compound
in nature [1,2]. Microbial mineralization of phytate by

phytase plays a significant role in the process of
phosphorus recycling. InsP
6
can be hydrolyzed com-
pletely to produce one inositol and six molecules
of inorganic phosphate, or partially to produce lower
inositol polyphosphate (IPP) isomers and inorganic
phosphates [3].
Among the four types of phytases that have been
identified, b-propeller phytase (BPP,
EC 3.1.3.8 or
EC 3.1.3.26) differs from the other three phytases (his-
tidine acid phosphatase (HAP), cysteine phytase and
purple acid phosphatase) by having a neutral (pH
7.0) rather than acidic pH optimum. Previous studies
have shown that BPP is the major class of phytate-
degrading enzyme in nature, which is widespread in
terrestrial and aquatic ecosystems [4,5]. Until now,
Abbreviations
BPP, b-propeller phytase; HAP, histidine acid phosphatase; InsP
6
, myo-inositol hexakisphosphate; IPP, inositol polyphosphate;
IPTG, isopropyl-b-
D-1-thiogalactopyranoside.
3032 FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS
only a small number of BPPs have been isolated and
studied, including Shewanella oneidensis MR-1 PhyS
[6], Bacillus subtilis PhyC [7], Bacillus sp. DS11 Phy
[8], B. subtilis 168 168PhyA [9], Bacillus licheniformis
PhyL [9], Pedobacter nyackensis MJ11 PhyP [10] and

Janthinobacterium sp. TN115 PhyA115 [11], all of
which are mesophilic or thermophilic (37–70 °C).
A typical BPP has a six-bladed propeller fold with
two phosphate-binding sites (cleavage site and affinity
site) and six calcium-binding sites, three of which are
high-affinity binding sites responsible for enzyme sta-
bility and three are low-affinity sites regulating the cat-
alytic activity of the enzyme [12,13]. BPP prefers the
hydrolysis of every second phosphate over adjacent
ones, and degrades InsP
6
gradually into InsP
5
, InsP
4
,
and the final product – Ins(2,4,6)P
3
and Ins(1,3,5)P
3
–
via two alternative pathways [14].
Bacterial BPPs containing two tandemly repeated
domains (dual domains) within a continuous sequence
have been found in S. oneidensis MR-1 PhyS [6] and
Janthinobacterium sp. TN115 [11]. However, few stud-
ies have been reported concerning the functions and
relationship of the dual domains. Here we describe the
physical properties that relate to the catalysis of a
newly isolated, dual-domain BPP (PhyH) from Bacillus

sp. HJB17. This enzyme is very active at neutral pH
(6.0–8.0) and at low temperatures (0–35 °C). We focus
on the function and relationship of the two single
domains. Additionally, the possibility that fusion of the
PhyH N-terminal domain to other single-domain BPPs
would improve the catalytic efficiency was assessed.
Results
Microorganism isolation
Using phytase screening and low phosphate media,
three strains with phytase activity were isolated from
the alpine tundra soil of China No. 1 Glacier in Xinji-
ang, China. Strain HJB17 exhibited the greatest phy-
tase activity, 0.33 ± 0.05 UÆmL
)1
, under optimal
growth conditions (pH 7.0 and 37 °C). According to
its 16S rDNA gene sequence (HQ610835), strain
HJB17 belongs to the genus Bacillus (99% 16S rDNA
gene sequence identity with that of Bacillus sp. SW41,
HM584798.1), and has been deposited in the Agricul-
tural Culture Collection of China under registration
number ACCC 05550.
Cloning and sequencing of BPP gene phyH
phyH (HM003046) was amplified using degenerate
PCR and thermal asymmetric interlaced PCR tech-
niques. The full-length gene contains 1932 base pairs
(643 amino acids). The deduced amino acid sequence
(PhyH) contains a putative signal peptide (40 amino
acids), an N-terminal domain (PhyH-DI, residues 41–
318) which shares 25% identity with that of Bacil-

lus amyloliquefaciens TS-Phy (YP090097), and a C-ter-
minal domain (PhyH-DII, residues 319–644) that has
49% identity with that of TS-Phy (YP090097) [15] and
41% with B. subtilis PhyC (CAM58513) [7]. Sequence
alignment of PhyH-DI and PhyH-DII with five homo-
logs identified six conserved residues (Pro30, Gly153,
Gln157, Asp188, Ala208 and Gly234). Some of the res-
idues involved in phosphate and calcium binding are
conserved in the PhyH-DII sequence, whereas only
one phosphate-binding residue is conserved in the
PhyH-DI sequence (Fig. S1). There are two pairs of
cysteine residues, Cys142 and Cys193 in PhyH-DII
and Cys445 and Cys495 in PhyH-DI. These residues
are at the same positions as Cys157, Cys206, Cys450
and Cys498 of PhyS [6], which form homologous disul-
fide bonds to stabilize BPPs [16].
The three-dimensional structures of PhyH-DI and
PhyH-DII were modeled using TS-Phy [15] as the tem-
plate (data not shown). PhyH-DI was predicted to
have a five-blade propeller structure and PhyH-DII a
six-blade b-propeller containing five four-stranded
sheets and one five-stranded sheet.
Expression and purification of PhyH, PhyH-DI and
PhyH-DII
PhyH, PhyH-DI and PhyH-DII were each expressed in
Escherichia coli. After isopropyl-b-d-1-thiogalactopyr-
anoside (IPTG) induction at 20 °C for 20 h, substan-
tial phytase activity was detected in all cultures, and
no activity was detected in cultures that harbored an
empty pET-22b(+). PhyH, PhyH-DI and PhyH-DII

were purified to homogeneity by Ni-affinity chroma-
tography and were found to have apparent molecular
weights of 67.0, 31.0 and 36.0 kDa (Fig. 1A), respec-
tively. Native gradient gel electrophoresis (Fig. 1B)
demonstrated that native PhyH might be a dimer. The
specific activities of PhyH and PhyH-DII against InsP
6
were 4.43 ± 0.55 and 1.82 ± 0.23 UÆmg
)1
, respec-
tively, at 35 °C. At the same temperature, PhyH-DI
had no activity against InsP
6
.
Biochemical properties of PhyH and PhyH-DII
Ca
2+
is required for BPP activity, and the optimal
concentration of Ca
2+
for the phytase activities of
PhyH and PhyH-DII was 1 mm (Fig. 2A). Both
enzymes exhibited optimal activities at pH 7.0, and
Z. Li et al. Catalysis of a dual-domain b-propeller phytase
FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS 3033
their apparent optimal temperatures were found to be
35 °C (Fig. 2B,C). At 0 °C, PhyH and PhyH-DII
retained 22% and 15.6% of their maximum activities,
respectively. PhyH appears to be the first BPP found
to be active at such a low temperature. PhyH was sta-

ble at neutral and alkaline pH; PhyH-DII was less sta-
ble under the same conditions (Fig. 2D).
PhyH was basically stable at 35 °C, and retained
60% of the initial activity at 45 °C for 90 min when
assayed at 35 °C (Fig. 3A,B). The presence of Ca
2+
increased the thermal stability of PhyH and PhyH-DII.
After incubation at 60 °C for 30 min, both enzymes
retained > 70% of their initial activity in the presence
of 10 mm Ca
2+
and < 30% of their activity without
Ca
2+
(Fig. 3C,D). PhyH at 35 °C showed a spectrum
(Fig. 4) with a weak minimum around 255 nm and a
large positive maximum around 298 nm. The structure
was intact after 12 h of incubation and failed to refold
after 5 min of boiling.
The kinetic values for PhyH and PhyH-DII towards
InsP
6
at 35 °C and 20 °C are given in Table 1. The higher
specific activity k
cat
and V
max
values and lower K
m
value

of PhyH suggests that PhyH is more catalytically efficient
and has a greater affinity for InsP
6
than PhyH-DII.
Substrate specificity of PhyH-DI
To understand the function of PhyH-DI, the substrate
specificities of PhyH-DI against several IPPs including
d-Ins(2)P
1
, d-Ins(1,4)P
2
, d-Ins(1,4,5)P
3
, d-Ins(1,4,5,6)P
4
and Ins(1,3,4,5,6)P
5
were determined. PhyH-DI has
a specific activity of 4.28 ± 0.56 UÆmg
)1
against
d-Ins(1,4,5,6)P
4
at 35 °C and cannot hydrolyze other
IPPs.
The function of PhyH-DI in InsP
6
degradation
When PhyH-DI was added into the reaction system of
PhyH-DII and InsP

6
, 1.63 fold phosphate was released
over that of PhyH-DII alone. More phosphate (1.17–
2.49 fold) was also released after subsequent addition
of PhyH-DI to a BPP (168PhyA or PhyP), or an HAP
(E. coli AppA) and InsP
6
reaction mixture (Table 2),
Fig. 1. Electrophoretic analysis of PhyH, PhyH-DI and PhyH-DII. (A)
SDS ⁄ PAGE analysis of purified PhyH, PhyH-DI and PhyH-DII. Lane
M, molecular weight markers; lane 1, culture supernatant of PhyH;
lane 2, culture supernatant of PhyH-DII; lane 3, culture supernatant
of PhyH-DI; lane 4, purified PhyH; lane 5, purified PhyH-DII; and
lane 6, purified PhyH-DI. (B) Non-denaturing gradient PAGE of
PhyH. Lane M, native high molecular weight markers; lane 1, native
PhyH stained with Coomassie Brilliant Blue R250.
0
20
40
60
80
100
120
3456789101112
pH of incubation
Relative activity (%)
PhyH PhyH-DII
0
20
40

60
80
100
120
012345
Concentration of Ca
2+
(m
M
)
Relative activity (%)
PhyH PhyH-DII
0
20
40
60
80
100
120
345678910
pH
Relative activity (%)
PhyH PhyH-DII
0
20
40
60
80
100
120

0 102030405060
Temperature (
嘙
C
)
Relative activity (%)
PhyH PhyH-DII
AB
CD
Fig. 2. Environmental factors that affect the catalytic activities of PhyH and PhyH-DII. (A) Effect of 0–5.0 mM Ca
2+
at 37 °C. (B) Effect of pH
at 37 °C. (C) Effect of temperature at pH 7.0. (D) pH stability. Residual activity was assayed under optimal conditions (1 m
M Ca
2+
,35°C, pH
7.0) after incubation in buffers at 37 °C for 1 h.
Catalysis of a dual-domain b-propeller phytase Z. Li et al.
3034 FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS
which suggests that PhyH-DI has the capacity to
increase the catalytic efficiency of PhyH-DII. Further-
more, the catalytic efficiency of PhyH was much greater
than that of PhyH-DI and PhyH-DII combined (5.73
fold) or twice the amount of PhyH-DII (5.45 fold).
This result reveals that the tandemly repeated BPP has
higher catalytic efficiency over single-domain phytase,
and the incomplete domain might play a key role.
Catalytic efficiencies of fusion BPP constructs
To verify the above conjecture, two fusion proteins,
PhyH-DI-168PhyA and PhyH-DI-PhyP, were con-

structed and expressed in E. coli. The catalytic con-
stants (k
cat
) of PhyH-DI-168PhyA and PhyH-DI-PhyP
towards InsP
6
are 12.28 s
)1
at 55 °C and 94.4 s
)1
at
37 °C, respectively, which are significantly greater than
those of the respective wild-type single-domain phyta-
ses (Table 1).
Discussion
In the present study, a BPP gene (phyH) was cloned
from Bacillus sp. HJB17, a strain isolated from the
alpine tundra soil of China No. 1 Glacier. It has been
reported that enzymes produced by psychrophilic
organisms are catalytically efficient at low tempera-
tures but are less stable at mesophilic temperatures
[17,18]. Bacillus sp. HJB17 has optimal growth at
37 °C and is not strictly psychrophilic. The apparent
optimal temperature of PhyH is 35 °C, similar to the
optimal temperature of strain HJB17, and the optimal
pH is 7.0. PhyH has some cold-adaptive properties,
retaining 20% of its maximal activity at 0 °C and
being thermolabile at 45 °C (Fig. 3). PhyH may be
0
20

40
60
80
100
120
Relative activity (%)
10 m
M
5 m
M
1 m
M
0 m
M
0
20
40
60
80
100
120
0 102030405060708090
Relative activity (%)
35 °C20°C
35 °C20°C45°C
45 °C
0
20
40
60

80
100
120
0 102030405060708090
Time at 35 °C (min)
Time at 60 °C (min) Time at 60 °C ( min)
Time at 45 °C (min)
Relative activity (%)
0
20
40
60
80
100
120
0 5 10 15 20 25 30
05
10
15 20 25 30
Relative activity (%)
10 m
M
5 m
M
1 m
M
0 m
M
AB
CD

Fig. 3. Thermostability of PhyH determined at 20, 35 and 45 °C after incubation at 35 °C (A) or 45 °C (B), and thermostability of PhyH (C)
and PhyH-DII (D) at 60 °C in the presence of 0–10 m
M Ca
2+
.
–1
–0.5
0
0.5
1
1.5
2
250 260 270 280 290 300 310 320
Wave le ngth (nm)
θ (mdeg)
Fig. 4. Near-UV CD spectra of PhyH incubated at 35 °C for 0 h
(black solid line) and 12 h (black dashed line) or boiled for 5 min
(gray solid line) and 2 h after boiling (gray dashed line).
Z. Li et al. Catalysis of a dual-domain b-propeller phytase
FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS 3035
used for aquaculture where the temperature is low
(28–30 °C) and the pH is neutral [19,20].
Sequence and structural analysis showed that PhyH
contains two domains – incomplete PhyH-DI and
complete PhyH-DII. Both PhyH and PhyH-DII
degrade InsP
6
efficiently, whereas PhyH-DI does not.
These observations have also been reported for PhyS
and its isolated domains, but the function of its N-ter-

minal domain was not characterized [6]. Given that
PhyH has a greater catalytic efficiency than does
PhyH-DII, we conjecture that PhyH-DI must be
involved in InsP
6
hydrolysis. To identify its role in the
catalysis process, we tested its substrate specificity and
action mode after incubation of InsP
6
with PhyH-DII
or other phytases. PhyH-DI can hydrolyze InsP
4
and
acts synergistically with other phytases to release 1.2–
2.5-fold phosphate. This is the first time the substrate
specificities and functions of the two domains have
been characterized in a tandemly repeated BPP. The
same phenomenon has been reported for an inositol
polyphosphatase PhyAmm, in which the complete D2
domain hydrolyzes the highly phosphorylated IPPs
and the incomplete D1 domain targets both IPPs
released by the D2 domain and unrelated di-, tri- and
tetra-phosphorylated IPPs present in the environment
[4,21]. Interestingly, the catalytic activity of PhyH is
much greater than the activity sum of PhyH-DI and
PhyH-DII and two times greater than that of PhyH-
DII. This large variance cannot be ascribed to the
function of PhyH-DI alone. The dual-domain phytase
was shown to be a dimer according to the native elec-
trophoresis. Thus, we presume the intact domain

(PhyH-DI) might mediate dimerization and further
enhance the catalytic efficiency of the dimer.
BPP is widespread in terrestrial and aquatic ecosys-
tems. Many putative nucleotide sequences homologous
to dual-domain BPP-like phytases are found in the
microbial genomes of the NCBI database, e.g. those of
Shewanella sp., Pseudomonas sp. and Idiomarina sp.
[5], suggesting the prevalence of dual-domain BPPs in
c-Proteobacteria. Notably, PhyH is the first Bacillus
dual-domain BPP identified, and its dual-domain struc-
ture is responsible for its higher catalytic efficiency.
This tandemly repeated structure probably evolves
from a single domain by gene duplication and persists
for better phosphate utilization.
Table 1. Kinetic parameters of BPPs and their fused counterparts with PhyH-DI.
Enzyme Specific activity (UÆmg
)1
) K
m
(lM) V
max
(lmolÆmin
)1
Æmg
)1
) k
cat
(s
)1
) k

cat
⁄ K
m
(lM
)1
Æs
)1
)
PhyH at 35 °C 4.43 ± 0.55 500 24.82 27.72 0.0540
PhyH at 20 °C 2.81 ± 0.28 1143 15.93 17.76 0.0155
PhyH-DII at 35 °C 1.82 ± 0.23 1432 5.56 4.17 0.0029
PhyH-DI-168PhyA at 55 °C 14.78 ± 0.84 191 10.24 12.28 0.6400
168PhyA at 55 °C 13.02 ± 0.56 240 8.45 5.92 0.2500
PhyH-DI-PhyP at 37 °C 29.82 ± 1.82 1086 78.13 94.40 0.0870
PhyP at 37 °C 21.61 ± 1.36 1280 71.90 44.94 0.0350
Table 2. Phytate hydrolysis by PhyH-DI with other BPPs and HAP.
Order of enzyme addition and reaction time
Amount of liberated inorganic
phosphate (lmol)
Folds of
increase
in activityFirst enzyme
Time
(min)
Second
enzyme
Time
(min) Observed yield
Expected
yield

PhyH-DI 120 – – 0.004 ± 0.003 – –
PhyH-DII 120 – – 0.302 ± 0.013 – –
PhyH-DII 120 PhyH-DI 120 0.500 ± 0.026 0.313 1.63
168PhyA 5 – – 0.536 ± 0.007 – –
168PhyA 5 PhyH-DI 120 1.345 ± 0.019 0.540 2.49
PhyP 5 – – 0.444 ± 0.008 – –
PhyP 5 PhyH-DI 120 0.523 ± 0.014 0.448 1.17
APPA 5 – – 1.978 ± 0.022 – –
APPA 5 PhyH-DI 120 2.310 ± 0.004 1.982 1.20
PhyH 120 – – 3.260 ± 0.170 – –
PhyH-DI ⁄ DII 120 – – 0.569 ± 0.015 – –
2 · PhyH-DII 120 – – 0.599 ± 0.017 – –
Catalysis of a dual-domain b-propeller phytase Z. Li et al.
3036 FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS
In the present study, we constructed two fusion
BPPs that showed greater catalytic constants towards
InsP
6
than the original enzymes. It appears that the
action of PhyH-DI is general, i.e. not limited to its
interaction with PhyH-DII, and can therefore enhance
the catalytic efficiencies of single-domain phytases.
Thus, PhyH-DI fused to another single-domain phy-
tase may improve the catalytic efficiency of the latter.
Materials and methods
Strains, plasmids and chemicals
E. coli Trans1-T1 (TransGen, Beijing, China) and pGEM-T
Easy (Promega, Madison, WI, USA) were used for gene
cloning and sequencing, respectively. E. coli BL21 (DE3)
(TaKaRa, Ostu, Japan) and pET-22b(+) (Novagen, San

Diego, CA, USA) were used for heterologous gene expres-
sion. T4 DNA ligase and restriction enzymes were supplied
by New England Biolabs (Hitchin, Herts, UK). LMW-SDS
marker kit and HMW native marker kit were purchased
from GE Healthcare (Uppsala, Sweden). Phytate (sodium
salt), d-Ins(2)P
1
, d-Ins(1,4)P
2
, d-Ins(1,4,5)P
3
, d-Ins(1,4,5,6)P
4
and Ins(1,3,4,5,6)P
5
were purchased from Sigma (St Louis,
MO, USA). Other chemicals were analytical grade and
commercially available.
Microorganism isolation from glacier soil
China No. 1 Glacier (43° 06.1183¢ N, 86° 50.1453¢ E) is
located in Xinjiang, China, where the average daily temper-
ature is below )20 °C. Samples of alpine tundra soil at an
elevation of 3525 m with an eastern exposure were collected
in September 2009 and stored at 4 °C. Bacteria were
screened for phytase activity at 4, 10, 20, and 37 °C using
two types of agar plates: one that contained phytase screen-
ing medium [10] and one that contained low phosphate
medium [22]. Phytase activity in the supernatants and cell
pellets of the retrieved strains was measured using the fer-
rous sulfate molybdenum blue method with a small modifi-

cation as described below [22,23]. Strain HJB17 with the
greatest activity against InsP
6
was identified on the basis of
its 16S rDNA gene sequence, and was subjected to further
experimentation.
Cloning, sequence and structure determination of
the phytase gene
Strain HJB17 was cultured in Luria–Bertani medium at
37 °C overnight and genomic DNA was extracted using the
TIANamp Bacteria DNA kit (Tiangen, Beijing, China).
The phytase gene was cloned from the genomic DNA of
strain HJB17 with the two-step PCR method according to
Huang et al. [10].
Nucleotide sequence assembly was performed using Vec-
tor NTI Advance 10.0 software (Invitrogen, Carlsbad, CA,
USA), and analyzed using the NCBI ORF Finder tool
( DNA and
protein sequence alignments used blastn and blastp
( respectively. The
SignalP 3.0 Server ( />was used for signal peptide analysis. A multiple protein
sequence alignment was performed using clustalw (http://
www.ebi.ac.uk/clustalW/) [24]. Protein structure was pre-
dicted using swiss-model (htt p://sw issmodel .expasy. org//
SWISS-MODEL.html) [25,26] and B. amyloliquefaciens TS-
Phy (1POO) [15] as the temp late.
Expression and purification of PhyH, PhyH-DI and
PhyH-DII in E. coli
Three genes phyH, phyH-DI and phyH-DII, the first two
lacking contiguous, upstream signal peptide sequences, were

each PCR amplified using the expression primers given in
Table S1 and were then cloned into the EcoRI–XhoI site of
a pET-22b(+) plasmid to construct the recombinant plas-
mids (pET-phyH, pET-phyH-DI and pET-phyH-DII;
Fig. 5). Each plasmid was transformed into E. coli BL21
(DE3) competent cells. Positive transformants were grown
in 25 mL Luria–Bertani medium (pH 7.0), 100 lgÆmL
)1
ampicillin at 37 °CtoanD
600
of 0.6. Protein expression
was induced by addition of IPTG (1 mm) and Ca
2+
(1 mm)
for 20 h at 20 °C. Culture supernatants and cell pellets
were assayed for phytase activity and separated by SDS ⁄
PAGE.
Purification was performed at 4 °C. The supernatants
were concentrated through a hollow fiber cartridge (cutoff
6 kDa; Motianmo, Tianjin, China) and the His-tagged
proteins in the supernatants were adsorbed onto a His-
Trap HP column (GE Healthcare) following Wang et al.
[27]. Purified PhyH was dialyzed against 20 mm Tris ⁄ HCl
(pH 7.0), 1 mm Ca
2+
, lyophilized, and dissolved in the
same buffer. Native electrophoresis was performed using a
non-denaturing 4%–15% (w ⁄ v) polyacrylamide gradient
gel at 15 mA at 4 °C. Gels were finally stained with Coo-
massie Brilliant Blue R250. Protein concentration was

determined using the Bradford assay with BSA as the
standard [28].
Phytase activity assay
Phytase activity was determined by measuring the amount
of phosphate released from InsP
6
using a modified ferrous
sulfate molybdenum blue method [23,29]. One unit (U) of
phytase activity was defined as the amount of enzyme
required to liberate 1 lmol phosphate per minute at
the corresponding temperature. All determinations were
performed in triplicate.
Z. Li et al. Catalysis of a dual-domain b-propeller phytase
FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS 3037
Characterization of purified recombinant PhyH,
PhyH-DI and PhyH-DII
To determine the effect of Ca
2+
, phytase activity was
assayed at 37 °C in solutions of 100 mm Tris ⁄ HCl (pH 7.0)
that contained 0–5.0 mm CaCl
2
. The optimal pH for
enzyme activity was determined in the presence of the opti-
mal Ca
2+
concentration at 37 °C for 30 min with the fol-
lowing buffers: 100 mm glycine ⁄ HCl (pH 1.0–3.5), 100 mm
sodium acetate ⁄ acetic acid (pH 3.5–6.0), 100 mm Tris ⁄ HCl
(pH 6.0–8.5) and 100 mm glycine ⁄ NaOH (pH 8.5–12.0).

Phytase activity was measured between 0 and 60 °Cto
determine the apparent optimal temperature for activity.
The effect of pH on enzyme stability was determined by
measuring the residual activity after incubating the enzymes
in buffered solutions with pH values of 3.0–10.0 at 37 °C
for 1 h.
The thermostability of PhyH at 35 and 45 °C was deter-
mined by measuring the residual activity at 20, 35 or 45 ° C
after incubation for various periods of time in 100 mm
Tris ⁄ HCl (pH 7.0). Additionally, the thermostabilities of
PhyH and PhyH-DII were also investigated at 60 °C in the
presence of 0–10 mm Ca
2+
. An enzyme solution without
treatment served as the control and was considered to have
100% activity.
Kinetic parameters for InsP
6
activity were determined in
100 mm Tris ⁄ HCl (pH 7.0) containing 1 mm Ca
2+
and
0.0125–2 mm InsP
6
. Reactions were run for 5 min at 35 °C
and 20 °C, respectively. The K
m
and V
max
values were

determined using Lineweaver–Burk plots [30] and the non-
linear regression computer program grafit. Three indepen-
dent experiments were averaged, and each experiment
included three replicates.
Circular dichroism spectroscopy
To verify the structural stability of PhyH at 35 °C or after
boiling, near-UV CD signals between 250 and 320 nm were
measured with a MOS-450 CD spectrometer (Bio-Logic,
Claix, France) equipped with a TCU-250 Peltier-type tem-
perature control system. PhyH at a concentration above
1mgÆmL
)1
in a 10-mm cell was subjected to signal mea-
surement within 8 min.
PhyH-DI substrate specificity
The substrate specificity of PhyH-DI was determined by
measuring its activity after incubation in 100 mm Tris ⁄ HCl
(pH 7.0) with 1 mmd-Ins(2)P
1
, d-Ins(1,4)P
2
, d-Ins(1,4,5)P
3
,
d-Ins(1,4,5,6)P
4
, Ins(1,3,4,5,6)P
5
or InsP
6

at 37 °C for
30 min. Each assay was replicated three times.
The function of PhyH-DI in InsP
6
hydrolysis
degradation
To determine the role of PhyH-DI in InsP
6
hydrolysis, its
effect on the enzymatic activities of PhyH-DII, 168PhyA
[9], PhyP [10] and E. coli AppA was characterized in a two-
step process. Each sample containing 1.5 mm sodium phy-
tate and 25 nmol of one of the phytases listed above, in
100 mm Tris ⁄ HCl (pH 6.0 or 7.0) for BPPs or in 100 mm
sodium acetate (pH 5.0) for AppA, was incubated at 37 °C
for 5 or 120 min and boiled for 5 min to inactivate the
enzyme present. Then the samples were adjusted to pH 7.0,
25 nmol PhyH-DI was or was not added, and the samples
were incubated at 37 °C for 120 min. The reactions were
terminated by addition of 1.5 mL trichloroacetic acid
PhyP-PhyDI-R
PhyDI-PhyP-F
PhyH-F
Phy168-R
PhyH-F
PhyH-R
PhyP-R
PhyDI-Phy168-F
phyH-DII
PhyH-F PhyDI-R

PhyDII-F
PhyH-R
PhyH-F
Phy168-PhyDI-R
phyH-DI
168phyA
phyH-DII
phyH-DI
phyH-DI
phyP
phyH-DI
pET-phyH
pET-phyH-DI
pET-phyH-DI-phyP
p
ET-phyH-DI-168phyA
pET-phyH-DI
Fig. 5. Expression plasmid construction. The arrows indicate the locations and directions of the PCR primers.
Catalysis of a dual-domain b-propeller phytase Z. Li et al.
3038 FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS
(10%, w ⁄ v) and subjected to the ferrous sulfate molybde-
num blue assay. Samples that contained only PhyH-DI or
one of the other phytases served as controls.
To understand how the two PhyH domains interact dur-
ing catalysis, samples that contained 25 nmol PhyH, a com-
bination of 25 nmol PhyH-DI and 25 nmol PhyH-DII, or
50 nmol PhyH-DII and 1.5 mm sodium phytate in 100 mm
Tris ⁄ HCl (pH 7.0) were incubated at 37 °C for 120 min,
terminated by the addition of 1.5 mL trichloroacetic acid,
and subjected to the ferrous sulfate molybdenum blue

assay.
Construction, expression and characterization of
fused BPPs
The fusion genes phyH-DI-phyP and phyH-DI-168phyA,
with phyH fused upstream, were constructed by overlapping
PCR [31] with the primers given in Table S1. EcoRI and
XhoI cleavage sites were introduced into the 5¢ end of
phyH-DI and the 3¢ end of phyP or 168phyA, respectively.
Each gene was restriction digested and inserted into pET-
22b(+). Expression, purification and characterization of
the two fusion proteins were conducted as described above.
Acknowledgements
This work was supported by the National Natural Sci-
ence Foundation of China (31001025), the Key Pro-
gram of Transgenic Plant Breeding (2008ZX08011-005)
and the earmarked fund for China Modern Agriculture
Research System (CARS-42). The authors declare
there is no conflict of interest in this paper.
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Supporting information
The following supplementary material is available:
Fig. S1. clustalw alignment of the PhyH-DI and
PhyH-DII amino acid sequences with those of the clo-

sely related BPPs, including Shewanella oneidensis MR-
1 PhyS-DI and PhyS-DII [6], Bacillus amyloliquefaciens
TS-Phy [15], Bacillus subtilis 168 168PhyA [9] and Pe-
dobacter nyackensis MJ11 PhyP [10].
Table S1. Primers used in this study.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
Catalysis of a dual-domain b-propeller phytase Z. Li et al.
3040 FEBS Journal 278 (2011) 3032–3040 ª 2011 The Authors Journal compilation ª 2011 FEBS