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: [email protected];
[email protected]
*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,

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

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-

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-

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

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

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

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

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

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

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-

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-

(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

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

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