A Moderately Thermostable Alkaline Phosphatase from Geobacillus thermodenitrificans T2: Cloning, Expression and Biochemical Characterization
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- Zhang, Y., Ji, C., Zhang, X. et al. Appl Biochem Biotechnol (2008) 151: 81. doi:10.1007/s12010-008-8166-7
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A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (<35% identity), while it exhibits the conservation of the active site and structure element of Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 °C, respectively. The enzyme retained a high activity at 45–60 °C, while it could be quickly inactivated by a heat treatment at 80 °C for 15 min, exhibiting a half-life of 8 min at 70 °C. The Km and Vmax for pNPP were determined to be 31.5 μM and 430 μM/min at optimal conditions. A divalent cation is essential, with a combination of Mg2+ and Co2+ or Zn2+ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification.
Alkaline phosphatases (EC 22.214.171.124) (APs) catalyze the nonspecific hydrolysis of phosphomonoesters, optimally active at alkaline pH. These enzymes are ubiquitous in nature and play a vital role in phosphate metabolism and transportation . AP from Escherichia coli is the most extensively studied so far and has been served as a basis for comparison to enzymes with similar homology. E. coli AP is a homodimeric metalloenzyme and its catalytic site comprises a serine residue and two Zn2+ and one Mg2+ per monomer. Detailed catalytic mechanisms of APs have been deduced from structure of E. coli enzyme, which involves a double in-line displacement and the participation of all three metal ions [2–4].
APs have many biotechnological applications, such as molecular biology, immunoassay, and clinical diagnostics [5–7]. Previously, APs from diversified sources, with widely differing thermal profiles, have been isolated and characterized. In general, APs are very stable enzymes. For example, E. coli AP remains activity at 80 °C  and that from Pyrococcus abyssi even at 105 °C . However, these highly stable APs are difficult to be eliminated at the end of dephosphorylation reaction of DNA cloning and amplification. Although several thermal sensitive APs from psychrophilic or mesophilic organisms such as cold water shrimp, Antarctic bacterium TAB5 and calf intestinal are now available commercially and used in many molecular biology assays, their further applications are restricted due to low thermal resistance and shelf lives of these thermoliable APs. Thus moderately thermostable APs are attractive, since they are stable at room temperature and relatively more convenient, compared with highly stable APs, to be destroyed by heat treatment at the end of reactions. Furthermore, variants of APs from extremophilic organisms could serve as ideal model molecular for investigations of enzyme evolution, protein thermal adaptation mechanism as well as metal-dependent catalysis.
Geobacillus is a genus of moderately thermophilic bacilli with a high 16S rRNA sequence similarity (98.5–99.2%), which have attracted industrial interests for their potential application in biotechnology as an important source of thermostable enzymes . Up to now, several thermostable enzymes such as thermostable l-arabinose isomerase , lipase , α-amylase, and α-glucosidase  from Geobacillus spp. have been characterized, whereas no AP in them was investigated.
In this study, we describe the cloning of a gene coding for thermostable AP (named Gtd AP) from a thermophilic Gram-positive bacterium, Geobacillus thermodenitrificans T2. The recombinant Gtd AP was overexpressed and purified in E. coli and its biochemical properties were characterized. The recombinant enzyme was stable at temperature below 60 °C, while it could be quickly inactivated by a heat treatment at 80 °C for 15 min. As a moderately thermostable AP, Gtd AP might be of significant biotechnological interest and scientific research value.
Materials and Methods
Enzymes used in vector construction were from New England Biolabs. All the chemicals were purchased from Sigma (Sigma-Aldrich) unless otherwise specified.
Construction of Genomic Libraries and Screening for Thermostable Phosphatase Positive Clones
Genomic DNA from thermophilic bacterial was prepared with the phenol-chloroform extraction  and partially digested with Sau3A I. The DNA fragments >2 kb were recovered from agarose DNA gel and spliced into BamHI-digested, dephosphorylated pUC19 plasmid. E. coli DH 5α were transformed with the ligation mixture and plated on LB plates containing 100 μg/mL ampicillin and 0.1-mM isopropyl-d-thiogalactopyranoside (IPTG) to form about 103 colonies/10-cm dish. Then all colonies were lifted onto nitrocellulose filter papers and the filters were placed, colony side up, in a 10-cm plate and incubated with 3 ml buffer (0.1 M Tris–HCl, pH 8.5, 1% Triton X-100) at 70 °C for 5 min. Ten microliters of 0.6 M p-nitrophenyl phosphate (pNPP) (Merk, USA) was supplemented and incubated an additional 5 min at 70 °C. Colonies producing thermostable phosphatase would hydrolyze pNPP, releasing pNP that is yellow in color. By corresponding to the original plates, putative positive colonies were found and picked. Plasmids (termed pTP) from these colonies were prepared and sequenced using M13 forward/reverse sequencing primers by submission to DNA sequencing facility of Unit Gene, Inc (Shanghai, China).
Homology searches were performed with BLAST program at the NCBI web server (http://www.ncbi.nlm.nih.gov/BLAST). SignalP 3.0 Server was used to predict probably signal peptide and the cleavage sites (http://www.cbs.dtu.dk/services/SignalP). Multiple sequences alignments were performed by GeneDoc programmer (http://www.psc.edu/biomed/genedoc).
Recombinant Protein Expression
According to the sequencing result, three polymerase chain reaction (PCR) primers were designed to amplify the Gtd AP gene corresponding region from plasmid pTP (two forward primers: F-1, 5′-CTAGCTAGCTTCAAATCGAAACGCTGCGC-3′; F-2, 5′-CTAGCTAGCGCACCGTCCAAGCCCGCAA-3′, and a reverse primer: R, 5′-CCGCTCGAGAAATCCATGGCTTTCGTTGT-3′). The full-length coding sequence of Gtd AP was amplified using Pfu DNA polymerase with primers F-1 and R and Gtd AP without a deduced signal peptide sequence was amplified with primers F-2 and R, respectively. Each of the obtained fragments with expected size was gel-purified and cloned into the Nhe I and Xho I sites, respectively, of pET-28b (Novagen, USA). The fidelity of inserting fragments in pET vectors was confirmed by sequencing. E. coli Rosetta (DE3) pLyS harboring the constructed expression plasmids were grown in 1 L Luria-Bertani (LB) medium containing kanamycin (100 mg L−1) and chloramphenicol (34 mg L−1) until OD600 0.6–0.8. After induction for 10 h at 30 °C with 0.5-mM IPTG, cells were harvested by centrifugation.
Purification of Gtd AP Fusion Protein
The harvested cell pellet was suspended in a standard buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole pH 8.0) and ultrasonicated on ice. The lysate was centrifuged at 14,000×g for 20 min at 4 °C. The supernatant was used for purification procedure with Ni-NTA Superflow chromatography according to the manufacturer’s protocol (QIAGEN). Finally, the bound enzyme was completely eluted with an elution buffer (250 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, pH 8.0). Active fractions were pooled and phosphate buffer were replaced with 50 mM Tris–HCl buffer (pH 8.5) by ultrafiltration (Amicon-Ultra-15 column; Millipore, USA).
The protein samples were separated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined using bicinchoninic acid (BCA) protein assay (Pierce) with bovine serum albumin (BSA) as the protein standard. Protein purity was determined by high-performance liquid chromatography (HPLC) analysis using reverse phase column system (C-18 column, Agilent) and the chromatogram was analyzed by UNICORN V3.20 software.
Enzyme Activity Assay
A spectrophotometric assay was used to determine the AP activity . The standard assay was carried out in 0.5 ml of reaction mixture containing 0.1 M Tris–HCl buffer (pH 8.0), 2.4 mM pNPP, and 2.0 μg enzyme (enzymes used were preactivated by 5 mM Co2+ and Mg2+ ions, except as otherwise indicated). After incubation at 65 °C for 5 min, the reaction was terminated by adding equal volume of 2.0 M NaOH, and the released pNP (ɛ = 18,380 M−1 cm−1) was measured at 405 nm using a spectrophotometer (Hitachi Co., Japan). One unit of enzyme activity is defined as 1 μmol pNPP hydrolyzed per minute.
Temperature, pH, Thermostability Profiles, and Kinetic Parameters
The optimal pH of enzyme activity was investigated in 0.1 M Tri–HCl (pH 7.0–9.0) and 0.5 M diethanolamine (pH 9.0–13.0) buffer at 65 °C. All of the pH buffers were calibrated at high temperature. Temperature effect of activated Gtd AP was determined by standard assay from 25 to 100 °C. Thermostability of the enzyme was investigated by the standard assay after preincubating enzymes at indicated temperatures for various times. Kinetic parameters, Km and Vmax, were determined from data obtained by determining the initial rate of pNPP hydrolysis.
The Effect of Metal Ions and Inhibitors on Enzyme Activity
To examine the effects of metal ions and inhibitors, enzyme was preincubated in the absence or presence of various divalent ions (final concentration 5 mM) and inhibitors at 4 °C for 1 h, and then assayed the activity by standard procedure. All of the examined metal ions were in their chloride form.
Results and Discussion
Characterization of Thermophilic Strain T2
Thermophilic bacteria were isolated from a hot spring in the People’s Republic of China. Microscopic inspection revealed that strain T2 was a rod-shaped Bacillus, 0.4–0.8 μm wide, 4.0–8.0 μm long, which grow optimally at around 65 °C. And 16S rRNA gene of bacterium was amplified  and compared with known sequences from NCBI GenBank database. The partial sequence of 16S rRNA gene from strain T2 (GenBank Accession No. EF570295) exhibited a high level of homology (>99.2% sequence identity) with those of G. thermodenitrificans strains. On the basis of phenotype and 16S rRNA gene sequence analysis, the strain T2 was assigned to G. thermodenitrificans T2.
Cloning and Sequence Analysis of G. thermodenitrificans T-2 AP
Through activity-based screening of thermophilic strain T2 genomic library, one clone with apparent phosphatase activity to pNPP, a general phosphatase substrate, were detected from approximately 6,000 transformed colonies. According to the result of sequencing, the recombinant plasmid of this clone showed an insert of 1,778 bp. Meanwhile, it demonstrated a significant ORF of 1,275 bp encoding a polypeptide of 424 amino acids. This ORF was preceded at a spacing of 4 bp by a potential ribosome-binding sequence (5′-GGAG-3′), which was homologous to the consensus Shine–Dalgarno sequence . The E. coli promoter-like sequences, in the −35 and the −10 regions, were not found on the upstream region of the ORF. In the 3′-noncoding flanking region of the gene, there was no potential transcriptional termination sequence forming a stem-and-loop structure. BLASTP results, however, showed that the encoding protein from this ORF was homologous to other known alkaline phosphatases. Therefore, this ORF was suggested to be an alkaline phosphatase gene of G. thermodenitrificans T-2 (termed Gtd AP, GenBank Accession No. EU239359).
Most other bacterial APs are periplasmic proteins. E. coli AP and Bacillus subtilis AP III have 21 and 32 amino acids signal peptides, respectively [18, 19]. Thus, Gtd AP was subjected to the SignalP program designated for protein sequences from gram-positive bacteria for signal peptides prediction . The program located an N-terminal signal sequence consisting of 30 amino acids, which would be cleaved between Ala-30 and Ala-31. Accordingly, the mature Gtd AP protein was deduced to be composed of 394 amino acids, resulting in an estimated molecular mass of 42,149 Da and pI 6.04.
Expression and Purification of G. thermodenitrificans T-2 AP
To identify the deduced N-terminal signal sequence and further characterize Gtd AP, the encoding sequence of Gtd AP with and without the deduced N-terminal signal peptide (30 amino acids) were amplified by PCR and cloned into pET28b vectors.
Purification of recombinant Gtd AP from E. coli cells.
Activity of the G. thermodenitrificans T-2 AP
Values of kinetic constants were determined on the basis of the Lineweaver–Burk plots. Under the optimal conditions, the recombinant Gtd AP hydrolyzed an artificial substrate pNPP with a Km of 31.5 μM and Vmax of 430 μM/min. A similar Km was found in APs from mesophilic E. coli  and thermophilic Meiothermus ruber , which ranges from 21 to 55 μM.
Metal Ion Requirements for the Enzyme Activity
In contrast with typical Zn2+- and Mg2+-activated APs, the optimum complement to Gtd AP was a combination of Co2+ with Mg2+, which most significantly activated native enzyme compared with other added ions. Furthermore, the effect of Co2+ alone was slightly less than that of the addition of Co2+ in combination with Mg2+. Similar results are also found in other thermostable AP from Thermus yunnanensis  and Thermotoga maritime . For these known Co2+-activated APs, Zn2+ usually has a weak or inhibited function to enzyme activity. It is interesting that Gtd AP could be activated by both Co2+ and Zn2+ at a similar extent. This feature also distinguishes Gtd AP from others. A less activated effect was found for the addition of Ca2+, Mg2+ or Mn2+ in recombinant Gtd AP. No effect on the activity was observed when Ni2+ or Cd2+ was added.
Effect of various chemicals on recombinant Gtd AP activity. a
Relative activity (%)
5.4 ± 0.8
143.9 ± 6.3
75.2 ± 2.4
22.8 ± 1.12
83.2 ± 4.3
103.0 ± 6.0
32.8 ± 1.5
10.9 ± 1.2
111.8 ± 1.7
83.8 ± 2.8
87.3 ± 6.7
20.9 ± 2.1
1.2 ± 0.4
The present work reported cloning, sequencing, and biochemical characterization of a moderately thermostable AP from Geobacillus thermodenitrificans. The temperature activity and thermostability of Gtd AP may therefore provide a basis for thermostable enzyme utilization at moderate temperature such as 45–60 °C. On the other hand, the high yields of soluble recombinant Gtd AP in E. coli made the bulk production possible and easy, which meets the demands for reagent enzyme.
This work is supported by the National Basic Research Program of China (973 Program, 2007CB914304) and New Century Excellent Talents in University (NCET-06-0356).