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Acta Biochim Biophys
Sin 2007, 39: 844850
doi:10.1111/j.1745-7270.2007.00347.x
Expression and
Characterization of Recombinant Thermostable Alkaline Phosphatase from a Novel
Thermophilic Bacterium Thermus thermophilus XM
Jianbo LI1, Limei
XU2,
and Feng YANG2*
1 School of Life Sciences, Xiamen
University, Xiamen 361005, China;
2
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography,
State Oceanic Administration, Xiamen 361005, China
Received: April 10,
2007
Accepted: June 20,
2007
This work was
supported by a grant from the China Ocean Mineral Resources R & D
Association
*Corresponding
author: Tel, 86-592-2195276; Fax, 86-592-2085376; E-mail,
Abstract A gene (tap) encoding a
thermostable alkaline phosphatase from the thermophilic bacterium Thermus
thermophilus XM was cloned and sequenced. It is 1506 bp long and encodes a protein
of 501 amino acid residues with a calculated molecular mass of 54.7 kDa.
Comparison of the deduced amino acid sequence with other alkaline phosphatases
showed that the regions in the vicinity of the phosphorylation site and metal
binding sites are highly conserved. The recombinant thermostable
alkaline phosphatase was expressed as a His6-tagged
fusion protein in Escherichia coli and its enzymatic properties were
characterized after purification. The pH and temperature optima for the
recombinant thermostable alkaline phosphatases activity were pH 12 and 75 ?C.
As expected, the enzyme displayed high thermostability, retaining more than 50%
activity after incubating for 6 h at 80 ?C. Its catalytic function was
accelerated in the presence of 0.1 mM Co2+, Fe2+, Mg2+, or Mn2+ but was
strongly inhibited by 2.0 mM Fe2+. Under optimal conditions, the Michaelis
constant (Km) for cleavage of p-nitrophenyl-phosphate
was 0.034 mM. Although it has much in common with other alkaline phosphatases,
the recombinant thermostable alkaline phosphatase possesses some unique
features, such as high optimal pH and good thermostability.
Keywords gene expression; Thermus thermophilus XM; thermostable
alkaline phosphatase
Alkaline phosphatases (APases; EC 3.1.3.1) exist widely in nature
from microorganisms to mammals, and play an important role in fundamental
biochemical processes, especially phosphate transportation and metabolism [1–3]. They can be
applied extensively in diagnostics, immunology, and molecular biology as
sensitive biological markers in processes such as enzyme-linked immunosorbent
assay, Western blotting analysis, nucleic acid hybridization, and in situ
hybridization [4–6]. APase from Escherichia coli has been extensively studied
in terms of biosynthesis, structure, and catalytic mechanism [7–13]. E. coli
APase is located in the periplasmic space as a homodimer and each monomer comprises
449 amino acid residues and contains two Zn2+ and one
Mg2+. At present, E. coli APase and calf intestine APase are the
most commonly used, but their inherently low thermal resistance and shelf-lives
have restricted their further applications under some special circumstances,
for example, high temperature and high pH. Compared with common APase,
thermostable APase (TAPase) has many beneficial qualities, such as high
thermostability, high reaction rates, and excellent resistance to denaturation
or microbial contamination [14]. Due to these advantages, there has been
increasing attention to TAPase from thermophilic bacteria. So far, a number of
TAPases have been isolated and their corresponding genes have been cloned and
characterized from various sources, such as Thermus species, Thermotoga
neapolitana, Meiothermus ruber, Bacillus stearothermophilusi,
and Pyrococcus abyssi [15–25]. In this study, we cloned and sequenced a tap gene from T.
thermophilus XM, discussed the characteristics of the deduced primary
structure of the enzyme, expressed it in E. coli and provided a
preliminary characterization of the recombinant TAPase (rTAPase).
Materials and Methods
Identification of strain
A thermophilic bacterium, whose optimal growth temperature is in the
range of 70–75 ?C in Luria Bertani (LB) medium, was isolated from a hot spring
at Dongfu beach (Xiamen, China). After incubation in LB medium for 24 h at 75
?C with shaking at 150 rpm, the bacterial cells were harvested and the genomic
DNA was purified using the Wizard genomic DNA purification kit (Promega,
Madison, USA). To identify the isolate, the 16S ribosomal RNA (rRNA) gene was
amplified by polymerase chain reaction (PCR) using universal primers, 27F (5‘-AGAGTTTGATCCTGGCTCAG-3‘)
and 1492R (5‘-GGTTACCTTGTTACGACTT-3‘). The PCR product was
cloned into pMD-18T vector (TaKaRa, Dalian, China) and sequenced. The
nucleotide sequence homology was analyzed using the blast program (http://www.ncbi.nlm.nih.gov/blast).
Cloning of the tap gene
Based on the complete coding region of the TAPase genes from the
genus Thermus, the following specific primers were synthesized: tapN (5‘-GGGGGATCCAAGCGAAGGGACATCCTG-3‘,
sense primer, BamHI restriction site underlined); and tapC (5‘-GGGAAGCTTTTAGGCCCAGACGTCCTC-3‘,
antisense primer, HindIII restriction site underlined). The amplified
fragment of 1506 bp was obtained by PCR with genomic DNA of T. thermophilus
XM as the template, cloned into pQE30 vector (Qiagen, Hilden, Germany), and
further confirmed by sequencing. The 25 ml PCR mixture consisted of
100 ng genomic DNA of T. thermophilus XM, 0.4 mM each primer, 200 mM each dNTP, 2.5
mM Mg2+, 1.25 U LA-Taq DNA polymerase (TaKaRa), and the buffer
supplied by the manufacturer. The reaction was carried out for 25 cycles as
follows: denaturation at 94 ?C for 30 s; annealing at 58 ?C for 30 s; and
extension at 72 ?C for 2 min.
Sequence homology searches were carried out using the blast program. Sequence analysis and
alignment were carried out with dnaman
software (version 5.1; Lynnon BioSoft, Vaudreuil, Canada).
Expression and purification of
rTAPase
After incubation at 37 ?C overnight, E. coli XL-Blue
containing the recombinant plasmid pQE30-tap and the control (vector only) were
inoculated into new media at a ratio of 1:100. When the A600 was 0.6, the cultures were induced with 0.5 mM isopropyl-b–D-thiogalactoside
for an additional 6 h at 37 ?C. Cells were harvested by centrifugation, then
suspended in 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. After
sonication, the insoluble debris was removed by centrifugation at 18,000 g
for 20 min and the supernatant was heated for 20 min at 70 ?C. The resulting
precipitate was again removed by centrifugation. Subsequently, the His6-tagged rTAPase in the supernatant fraction was purified using
Ni-NTA metal-affinity chromatography according to the instructions of the
QIAexpressionist system (Qiagen, Valencia, USA). The purified rTAPase was
dialyzed against the buffer containing 10 mM Tris-HCl, pH 8.0, 50% glycerol,
and the protein concentration was determined using the CB protein assay kit
(Calbiochem, La Jolla, USA).
Gel electrophoresis and
Western blot analysis
Protein samples were separated by 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis [26] then transferred onto a
polyvinylidene fluoride membrane (GE Healthcare, Princeton, USA) by semi-dry
blotting at a constant current of 0.5 mA/cm2 for 1.5
h. The membrane was immersed in blocking buffer (20 mM Tris-HCl, pH 7.5, 150
mM NaCl, 3% bovine serum albumin, and 0.05% Tween-20) at room temperature for 1
h, followed by incubation with mouse anti-His antibody (GE Healthcare; diluted
1:3000) in blocking buffer at 4 ?C overnight. Subsequently, a secondary
antibody, alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G
(Promega) was added at a dilution of 1:7500 in blocking buffer at room temperature
for 1 h, then the immunoblot signals were detected using NBT-BCIP substrate
(Roche, Mannheim, Germany) in detection solution (50 mM Tris-HCl, 100 mM NaCl,
and 5 mM MgCl2, pH 9.5).
Measurement of rTAPase
activity
The enzymatic reaction was carried out at 75 ?C for 10 min in 1.0 ml
appropriate buffer containing 6 mM p-nitrophenyl-phosphate disodium salt
hexahydrate (pNPP; Amresco, Solon, USA) and 1.5 mg of rTAPase, and
terminated on ice. The activity of rTAPase was measured spectrophotometrically
at 410 nm. To correct the rate of non-enzymatic hydrolysis of pNPP, the
reaction mixture without enzyme was used as a reagent blank. One unit of
activity was defined as the amount of enzyme required to release 1 mmol of p-nitrophenol
from pNPP in 1 min under the above assay conditions.
Characterization of rTAPase
The rTAPase was characterized by optimum pH, optimum temperature,
thermostability, and the effect of metal ions on its activity. Unless otherwise
indicated, the following experiments were carried out using the reaction
conditions described in the above section and the data were presented as the
mean values of at least three independent experiments.
To assess the effect of pH, rTAPase activity was measured at various
pH values ranging from 7.5 to 13.0. Tris-HCl buffer (100 mM) was used in the pH
range 7.5–9.0, and 100 mM diethanolamine buffer was used in the range 8.5–13.0. The optimum temperature was measured by carrying out the rTAPase
activity assay at temperatures ranging from 40 ?C to 95 ?C at intervals of 5 ?C
and at pH 12.0. To determine the thermostability, the enzyme was incubated for
6 h at different temperatures (50 ?C, 65 ?C, 80 ?C, and 95 ?C). Samples were
taken out every 30 min and the residual activity was then measured as described
above. To test the effect of metal ions, rTAPase activity was determined by
measuring the residual activity after incubation of the enzyme solutions with
various metal ions such as CaCl2, CoCl2, CuSO4, FeSO4, MgCl2, MnSO4, and ZnSO4 at 0.1 mM or 2.0 mM concentration.
Michaelis constant (Km) of
rTAPase
The Km value of rTAPase was
determined under optimum conditions using different concentrations of pNPP
from 0 to 6 mM. A typical plot was obtained when v was plotted against v/[S]
(v, initial rate; [S], substrate concentration), according to the
method of Eadie-Hofstee and the Km value
was estimated by linear regression from it.
Results
Cloning and sequencing
analysis of the tap gene
At present, analysis based on the 16S rRNA gene sequence is
considered to be a standard in bacterial identification. For the thermophilic
bacterium from Xiamen, its 16S rRNA gene was amplified and sequenced (GenBank
accession No. DQ647385). Blast
analysis revealed that the strain had 99% similarity with T. thermophilus
HB8 (GenBank accession No. AP008226). Based on sequence homology, the strain
was considered to be a T. thermophilus sp., so it was named T.
thermophilus XM.A DNA fragment (approximately 1.5 kb) was amplified by PCR using tap
gene primers and sequenced. Sequence analysis revealed that the gene consisted
of 1506 bp (GenBank accession No. DQ645419) and was predicted to code a
polypeptide of 501 amino acids with a calculated molecular mass of 54.7 kDa and
isoelectric point of 8.7. The result from the DNA blast search showed that the tap gene shared over 93%
sequence identity with other tap genes from the genus Thermus. In
particular, it had the highest identity with T. thermophilus HB8 (99%).
Comparison of amino acid sequences of TAPase and other bacterial
APase
When the deduced amino acid sequence of the gene was compared with
other APases from thermophilic, mesophilic, and psychrophilic bacteria, TAPase
had the highest identity with that from genus Thermus, such as T.
thermophilus HB27 (99.6%) and T. caldophilus (98.4%), followed by M.
ruber APase (64.3%). Identity was lower with Thermotoga neapolitana APase
(27.3%), Bacillus subtilis APase (24.2%), Antarctic psychrophilic
bacterium TAB5 APase (20.6%), and E. coli APase (18.2%). Although the
whole sequence homology is not high between different APases, the alignment
analysis showed that their active site regions involved in metal ion
coordination, phosphorylation, and phosphate binding are highly conserved (Fig.
1), except that the residues D153 and K328 (E. coli numbering) are
replaced by histidine (compared with E. coli APase).
Expression and purification of
rTAPase
The tap gene was cloned into a His6-tagged expression
vector pQE30, then the recombinant plasmid pQE30-tap and empty vector pQE30
were transformed into XL-Blue. After induction with isopropyl-b–D-thiogalactoside,
total cellular proteins were separated on 10% sodium dodecyl
sulfate-polyacrylamide gel and visualized by staining with Coomassie Brilliant
Blue. A protein an with apparent molecular mass of approximately 55 kDa,
consistent with the predicted 54.7 kDa molecular mass of TAPase, was observed
in XL-Blue cells transformed with pQE30-tap [Fig. 2(A), lane 1] but
absent from empty vector pQE30 [Fig. 2(A), lane 2]. The His6-tagged protein was purified by Ni-agarose chromatography and its
concentration was determined to be 0.15 mg/ml. The purified product contained
two bands of molecular masses 55 and 43 kDa, the slower migrating band being
more abundant than the faster migrating band [Fig. 2(A), lane 3].
Western blot analysis revealed that the 55 kDa protein was recognized
specifically by anti-His antibody, indicating that it was indeed rTAPase [Fig.
2(B)].
Properties of rTAPase
Generally, the pH value of reaction solution can significantly
affect enzyme activity. Like other APases, the rTAPase was activated under
alkaline conditions. Fig. 3 shows the pH profile of purified rTAPase activity.
The optimal pH range of the enzyme was found to be 11.5–12.5, and maximal activity
occurred at pH 12.0. It was nearly inactive below pH 7.5. The purified rTAPase had its maximal enzymatic activity at 75–80 ?C, and
approximately 50% of the activity was retained at temperatures above 40 ?C and
below 85 ?C, indicating that the enzyme is capable of functioning over a broad
temperature range (Fig. 4). In order to better estimate the effect of
temperature on enzyme activity, thermostability was determined by incubating
the rTAPase at temperatures ranging from 50 ?C to 95 ?C for different time
points. As shown in Fig. 5, the rTAPase can be characterized as a
moderately thermostable enzyme: over 50% of the activity was retained even
after 6 h at 80 ?C. However, the activity was sharply decreased after treatment
for 1.5 h at 95 ?C.As APases are metalloenzymes [2], metal ions as co-factors might
play important roles in enzymatic reactions. The effects of some divalent metal
ions on the rTAPase activity are shown in Table 1. Mg2+ is the most optimal for enzymatic reaction activity, but Zn2+, Ca2+, and Cu2+ show an
inhibitory effect. Co2+, Fe2+, and Mn2+ (0.1 mM) can also significantly enhance the activity of rTAPase,
especially Co2+, however, there is a strong inhibition in the
presence of 2 mM Fe2+. As expected, the enzymatic activity of
rTAPase was strongly influenced in the presence of the chelating agent EDTA
(data not shown).The Km value of the rTAPase for pNPP
was estimated to be 0.0340.009 mM (Fig. 6), similar to those previously
reported in other bacteria, such as T. caldophilus APase (0.036 mM)
[20], M. ruber APase (0.055 mM) [23], and E. coli
APase (ranging from 0.021 mM to 0.040 mM) [28,29], suggesting that the initial
velocity of the reaction catalyzed by APases from both thermophilic and
mesophilic bacteria is close.
Discussion
APases from thermophilic microorganisms are becoming more and more
interesting in diagnostics, immunology and molecular biology such as non-radioactive
labeling, due to their inherent advantages which are more resistant to thermal
denaturation during preparation or labelling procedures, easier to transport
and store, and longer half-life of enzyme activity.In this study, a
tap gene was cloned from
T. thermophilus XM. Although the sequence homology is very low between
different APases, the amino acid residues in the active site were highly
conserved (Fig. 1). All ligands to Zn1, Zn2 and Mg, S102 and R166 are
absolutely conserved in all APases. In E. coli APase, Zn1 is coordinated
by two imidazole bases of H331 and H412, as well as one carboxyl of D327. Zn2
is bound by two carboxyls of D51 and D369 along with one imidazole base of
H370. Mg is bound by residues D51, T155, and E322 [12]. However, the secondary
ligands of the Mg ion, that is, D153 and K328, are replaced by histidine in T.
thermophilus XM APase (Fig. 1). D153 is a part of the Mg ion binding
site, and K328 is important for phosphate binding in E. coli APase. The
same substitutions were found in T. thermophilus HB27, T. caldophilus,
M. ruber, and mammalian APases. Previous studies showed that the
replacement of D153 resulted in an enhancement of enzymatic activity when the
Zn2+ bound in this site is replaced by Mg2+ [27].The characterization studies showed that rTAPase is one of the most
characterized alkaline APases so far identified. By analogy with the mammalian
APase, which have higher pH optima than the E. coli APase, we can
speculate that the substitution of amino acid residues corresponding to D153
and K328 in rTAPase by histidine is responsible for the more alkaline activity
profile of the rTAPase [30]. However, the reason for alkaline phosphatase’s
resistance to extreme pH remains unclear. In fact, we are trying to reveal the
inherent mechanism of this resistance to extreme alkaline in another
experiment.APase is a metalloenzyme, and different metal ions could have
different influences on rTAPase activity. Many studies showed that the same
metal ion has different effects (enhancer or inhibitor) on the activity of
APases from different sources, for example, Zn2+ could
be an inhibitor for M. ruber APase but be an enhancer for Thermotoga
neapolitana APase; Co2+ could enhance the activity of T.
thermophilus XM APase but inhibit the activity of M. ruber APase [22–25]. We
hypothesize that the phenomenon might be the result of the differences between
the active sites conformation of different APases. As shown in Fig. 1,
although almost all metal coordinating amino acid residues in the active sites
are conserved, the residues close to metal binding sites are variable from
different sources. It suggested that these varieties might result in some
conformational changes to affect the binding affinity for different metal ions.
In
summary, a tap gene from T. thermophilus XM was successfully
cloned and expressed in E. coli. The recombinant enzyme shared common
characteristics of alkaline phosphatase and displayed good thermostability. In
addition, it also revealed a unique property in that it showing a strong
competence for high alkaline conditions compared with other APases. These
characteristics suggest that the enzyme has potential value in experimental
science. For instance, it could be applied in nucleic acid hybridization and in
situ hybridization under high temperature and alkaline environments instead
of APases from other sources.
References
1 Coleman
JE, Gettins P. Alkaline phosphatase, solution structure, and mechanism. Adv Enzymol
Relat Areas Mol Biol 1983, 55: 381–452
2 Posen
S. Alkaline phosphatase. Ann Intern Med 1967, 67: 183–203
3 Trowsdale
J, Martin D, Bicknell D, Campbell I. Alkaline phosphatases. Biochem Soc Trans
1990, 18: 178–180
4 Blake
MS, Johnston KH, Russell-Jones GJ, Gotschlich EC. A rapid, sensitive method for
detection of alkaline phosphatase-conjugated anti-antibody on Western blots.
Anal Biochem 1984, 136: 175–179
5 Jablonski
E, Moomaw EW, Tullis RH, Ruth JL. Preparation of oligodeoxynucleotide-alkaline
phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res
1986, 14: 6115–6128
6 Augood
SJ, McGowan EM, Finsen BR, Heppelmann B, Emson PC. Non-radioactive in situ
hybridization using alkaline phosphatase-labelled oligonucleotides. Int Rev
Neurobiol 2002, 47: 173–201
7 Derman
AI, Beckwith J. Escherichia coli alkaline phosphatase fails to acquire
disulfide bonds when retained in the cytoplasm. J Bacteriol 1991, 173: 7719–7722
8 Karamyshev
AL, Karamysheva ZN, Kajava AV, Ksenzenko VN, Nesmeyanova MA. Processing of Escherichia
coli alkaline phosphatase: Role of the primary structure of the signal
peptide cleavage region. J Mol Biol 1998, 277: 859–870
9 Bradshaw
RA, Cancedda F, Ericsson LH, Neumann PA, Piccoli SP, Schlesinger MJ, Shriefer K
et al. Amino acid sequence of Escherichia coli alkaline
phosphatase. Proc Nat Acad Sci USA 1981, 78: 3473–3477
10 Chang
CN, Kuang WJ, Chen EY. Nucleotide sequence of the alkaline phosphatase gene of Escherichia
coli. Gene 1986, 44: 121–125
11 Coleman
JE. Structure and mechanism of alkaline phosphatase. Annu Rev Biophys Biomol
Struct 1992, 21: 441–483
12 Kim EE,
Wyckoff HW. Structure of alkaline phosphatases. Clin Chim Acta 1990, 186: 175–187
13 Kim EE,
Wyckoff HW. Reaction mechanism of alkaline phosphatase based on crystal
structures. Two-metal ion catalysis. J Mol Biol 1991, 218: 449–464
14 Sterner
R, Liebl W. Thermophilic adaptation of proteins. Crit Rev Biochem Mol Biol
2001, 36: 39–106
15 Angelini
S, Moreno R, Gouffi K, Santini C, Yamagishi A, Berenguer J, Wu L. Export of Thermus
thermophilus alkaline phosphatase via the twin-arginine translocation
pathway in Escherichia coli. FEBS Lett 2001, 506: 103–107
16 Gong N,
Chen C, Xie L, Chen H, Lin X, Zhang R. Characterization of a thermostable
alkaline phosphatase from a novel species Thermus yunnanensis sp. nov.
and investigation of its cobalt activation at high temperature. Biochim Biophys
Acta 2005, 1750: 103–111
17 Ji CN,
Jiang T, Chen MQ, Sheng XY, Mao YM. Purification, crystallization and
preliminary X-ray studies of thermostable alkaline phosphatase from Thermus
sp. 3041. Acta Crystallogr D Biol Crystallogr 2001, 57: 614–615
18 Kim YJ,
Park TS, Kim HK, Kwon ST. Purification and characterization of a thermostable
alkaline phosphatase produced by Thermus caldophilus GK24. J Biochem Mol
Biol 1997, 30: 262–268
19 Park T,
Lee JH, Kim HK, Hoe HS, Kwon ST. Nucleotide sequence of the gene for alkaline
phosphatase of Thermus caldophilus GK24 and characteristics of the deduced
primary structure of the enzyme. FEMS Microbiol Lett 1999, 180: 133–139
20 Pantazaki
AA, Karagiorgas AA, Liakopoulou-Kyriakides M, Kyriakidis DA. Hyperalkaline and
thermostable phosphatase in Thermus thermophilus. Appl Biochem Biotechnol
1998, 75: 249–259
21 Yeh MF,
Trela JM. Purification and characterization of a repressible alkaline
phosphatase from Thermus aquaticus. J Biol Chem 1976, 251: 3134–3139
22 Dong G,
Zeikus JG. Purification and characterization of alkaline phosphatase from Thermotoga
neapolitana. Enzyme Microb Technol 1997, 21: 335
23 Yurchenko
JV, Budilov AV, Deyev SM, Khromov IS, Sobolev AY. Cloning of an alkaline
phosphatase gene from the moderately thermophilic bacterium Meiothermus
ruber and characterization of the recombinant enzyme. Mol Genet Genomics
2003, 270: 87–93
24 Mori S,
Okamoto M, Nishibori M, Ichimura M, Sakiyama J, Endo H. Purification and
characterization of alkaline phosphatase from Bacillus stearothermophilus. Biotechnol
Appl Biochem 1999, 29: 235–239
25 Zappa
S, Rolland JL, Flament D, Gueguen Y, Boudrant J, Dietrich J. Characterization
of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus
abyssi. Appl Environ Microbiol 2001, 67: 4504–4511
26 Laemmli
UK. Cleavage of structural proteins during the assembly of the head of the
bacteriophage T4. Nature 1970, 227: 680–685
27 Murphy
JE, Xu X, Kantrowitz ER. Conversion of a magnesium binding site into a zinc
binding site by a single amino acid substitution in Escherichia coli
alkaline phosphatase. J Biol Chem 1993, 268: 21497–21500
28 Janeway
CM, Xu X, Murphy JE, Chaidaroglou A, Kantrowitz ER. Magnesium in the active
site of Escherichia coli alkaline phosphatase is important for both
structural stabilization and catalysis. Biochemistry 1993, 32: 1601–1609
29 Krupianko
VI, Kolot MN, Nesmeianova MA. Comparative study of properties of periplasmic
and membrane-bound alkaline phosphatase of E. coli. Biokhimiia 1981, 46:
1249–1257
30 Murphy
JE, Tibbitts TT, Kantrowitz ER. Mutations at positions 153 and 328 in Escherichia
coli alkaline phosphatase provide insight towards the structure and
function of mammalian and yeast alkaline phosphatases. J Mol Biol 1995, 253:
604–617