Purified recombinant PhyH and PhyH-DII required Ca2+for phytase activity, showed activity at low tem-peratures 0–35C and pH 6.0–8.0, and remained active at 37 C after incubation at 60C an
Trang 1act 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: yaobin@caas-bio.net.cn;
yaobin@mail.caas.net.cn
*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 Bacillussp 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 Ca2+for phytase activity, showed activity at low tem-peratures (0–35C) and pH 6.0–8.0, and remained active (at 37 C) after incubation at 60C 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)
P4, 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, InsP6)
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 InsP6 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.
Trang 2only 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 (37–70C)
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 InsP6 gradually into InsP5, InsP4,
and the final product – Ins(2,4,6)P3 and Ins(1,3,5)P3 –
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
Janthinobacteriumsp 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 (0–35C) 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 37C) 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 20C 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 InsP6 were 4.43 ± 0.55 and 1.82 ± 0.23 UÆmg)1, respec-tively, at 35C At the same temperature, PhyH-DI had no activity against InsP6
Biochemical properties of PhyH and PhyH-DII
Ca2+ is required for BPP activity, and the optimal concentration of Ca2+ for the phytase activities of PhyH and PhyH-DII was 1 mm (Fig 2A) Both enzymes exhibited optimal activities at pH 7.0, and
Trang 3their 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
ble at neutral and alkaline pH; PhyH-DII was less
sta-ble under the same conditions (Fig 2D)
PhyH was basically stable at 35C, and retained
60% of the initial activity at 45 C for 90 min when
assayed at 35C (Fig 3A,B) The presence of Ca2+
increased the thermal stability of PhyH and PhyH-DII
After incubation at 60C for 30 min, both enzymes
retained > 70% of their initial activity in the presence
of 10 mm Ca2+ and < 30% of their activity without
Ca2+ (Fig 3C,D) PhyH at 35C 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 InsP6at 35C and 20 C are given in Table 1 The higher specific activity kcatand Vmaxvalues and lower Kmvalue
of PhyH suggests that PhyH is more catalytically efficient and has a greater affinity for InsP6than 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)P1, d-Ins(1,4)P2, d-Ins(1,4,5)P3, d-Ins(1,4,5,6)P4
and Ins(1,3,4,5,6)P5 were determined PhyH-DI has
a specific activity of 4.28 ± 0.56 UÆmg)1 against
d-Ins(1,4,5,6)P4 at 35C and cannot hydrolyze other IPPs
The function of PhyH-DI in InsP6degradation When PhyH-DI was added into the reaction system of PhyH-DII and InsP6, 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 InsP6 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
3 4 5 6 7 8 9 10 11 12
pH of incubation
PhyH PhyH-DII
0 20 40 60 80 100 120
0 1 2 3 4 5
Concentration of Ca 2+ (m M )
0 20 40 60 80 100 120
3 4 5 6 7 8 9 10
pH
0 20 40 60 80 100 120
0 10 20 30 40 50 60
Temperature (嘙C )
PhyH PhyH-DII
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 mM Ca2+, 35 C, pH 7.0) after incubation in buffers at 37 C for 1 h.
Trang 4which 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 structed and expressed in E coli The catalytic con-stants (kcat) of PhyH-DI-168PhyA and PhyH-DI-PhyP towards InsP6 are 12.28 s)1 at 55C and 94.4 s)1 at
37C, 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 temperatempera-tures [17,18] Bacillus sp HJB17 has optimal growth at
37C and is not strictly psychrophilic The apparent optimal temperature of PhyH is35 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 0C and being thermolabile at 45C (Fig 3) PhyH may be
0 20 40 60 80 100 120
10 m M 5 m M 1 m M 0 m M
0 20 40 60 80 100 120
0 10 20 30 40 50 60 70 80 90
35 °C 20 °C 45 °C 35 °C 20 °C 45 °C
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70 80 90
Time at 35 °C (min)
Time at 45 °C (min)
0
20
40
60
80
100
120
10 m M 5 m M 1 m M 0 m M
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 mM Ca2+.
–1
–0.5
0
0.5
1
1.5
2
250 260 270 280 290 300 310 320
Wavelength (nm)
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).
Trang 5used for aquaculture where the temperature is low
(28–30C) 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 InsP6 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 InsP6 hydrolysis To identify its role in the
catalysis process, we tested its substrate specificity and
action mode after incubation of InsP6 with PhyH-DII
or other phytases PhyH-DI can hydrolyze InsP4 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 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)1Æmg)1) k cat (s)1) k cat ⁄ K m (lM)1Æs)1)
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 activity First enzyme
Time (min)
Second enzyme
Time
Expected yield
Trang 6In the present study, we constructed two fusion
BPPs that showed greater catalytic constants towards
InsP6 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 coliTrans1-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)P1, d-Ins(1,4)P2, d-Ins(1,4,5)P3, d-Ins(1,4,5,6)P4
and Ins(1,3,4,5,6)P5were 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 4C Bacteria were
screened for phytase activity at 4, 10, 20, and 37C 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 InsP6was 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
37C 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 (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) DNA and protein sequence alignments used blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/), respectively The SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/) 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 (http://swissmodel.expasy.org// SWISS-MODEL.html) [25,26] and B amyloliquefaciens TS-Phy (1POO) [15] as the template
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 37C to an D600 of 0.6 Protein expression was induced by addition of IPTG (1 mm) and Ca2+(1 mm) for 20 h at 20C Culture supernatants and cell pellets were assayed for phytase activity and separated by SDS⁄ PAGE
Purification was performed at 4C 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 Ca2+, 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 4C 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 InsP6 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
Trang 7Characterization of purified recombinant PhyH,
PhyH-DI and PhyH-DII
To determine the effect of Ca2+, phytase activity was
assayed at 37C in solutions of 100 mm Tris ⁄ HCl (pH 7.0)
that contained 0–5.0 mm CaCl2 The optimal pH for
enzyme activity was determined in the presence of the
opti-mal Ca2+ concentration at 37C 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 60C to
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 37C
for 1 h
The thermostability of PhyH at 35 and 45C was
deter-mined by measuring the residual activity at 20, 35 or 45C
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 60C in the
presence of 0–10 mm Ca2+ An enzyme solution without
treatment served as the control and was considered to have
100% activity
Kinetic parameters for InsP6activity were determined in
100 mm Tris⁄ HCl (pH 7.0) containing 1 mm Ca2+
and 0.0125–2 mm InsP6 Reactions were run for 5 min at 35C
and 20C, respectively The Km and Vmax 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 35C 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
1 mgÆmL)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 mm d-Ins(2)P1, d-Ins(1,4)P2, d-Ins(1,4,5)P3,
d-Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5 or InsP6 at 37C for
30 min Each assay was replicated three times
The function of PhyH-DI in InsP6hydrolysis degradation
To determine the role of PhyH-DI in InsP6 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 37C 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 37C 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
PhyP-R PhyDI-Phy168-F
phyH-DII
PhyH-F PhyDI-R
PhyDII-F PhyH-R
PhyH-F
Phy168-PhyDI-R
phyH-DII phyH-DI
phyH-DI
pET-phyH
pET-phyH-DI
pET-phyH-DI-phyP pET-phyH-DI-168phyA pET-phyH-DI
Fig 5 Expression plasmid construction The arrows indicate the locations and directions of the PCR primers.
Trang 8(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 nyackensisMJ11 PhyP [10]
Table S1 Primers used in this study
This supplementary material can be found in the online version of this article
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