Based on genome sequence analysis of thermophilic bacteriophage GBSV1, an open reading frame (ORF) [GenBank: EF079892] of GBSV1 shared homologies with phage replication proteins (Fig. 1), which contained a DnaD-like domain, suggesting that the protein encoded by this ORF had the DNA binding capacity. In order to identify its biological function, the ORF (termed as GBSV1-NSN gene) was expressed as GST fusion protein in E.coli. After induction with IPTG at 37°C, the induced and non-induced recombinant bacterium (containing GBSV1-NSN gene) and control bacterium (vector only) were analyzed by SDS-PAGE. A band (about 59.8 kDa) corresponding to the GST-GBSV1-NSN fusion protein was observed in the induced recombinant bacterium containing GBSV1-NSN gene (Fig. 2, lane 4), while no protein was found in the same positions in the induced and non-induced controls (vector only), showing that the GBSV1-NSN gene was expressed. After purification by affinity chromatography, a GST-GBSV1-NSN fusion protein was obtained (Fig. 2, lane 5). The fusion protein GST-GBSV1-NSN was efficiently cleaved by thrombin, yielding the purified GBSV1-NSN with a molecular mass of 33.8 kDa (Fig. 2, lane 6).

In an attempt to determine the DNA binding capacity of the recombinant GBSV1-NSN, the purified GBSV1-NSN protein was incubated with the genomic DNA of bacteriophage GBSV1. Surprisingly, the DNA was degraded by the GBSV1-NSN protein, suggesting it might be a nuclease. To evaluate its substrate specificity, the purified GBSV1-NSN protein was incubated with various nucleic acids including the circular pGEX-4T-2 plasmid dsDNA, the linear pGEX-4T-2 plasmid dsDNA, the PhiX174 virion ssDNA and the baker's yeast RNA. The results showed that all the nucleic acids could be degraded by the GBSV1-NSN protein (Fig. 3), indicating that it was a non-specific nuclease.

Homology analysis revealed that the GBSV1-NSN protein shared low identities with known nucleases. It had 10.0%, 13.3%, 13.5%, 10.9% and 12.1% identities with those of Staphylococcus aureus thermonuclease [GenBank: P00644], Serratia marcescens nuclease [GenBank: P13717], Anabaena sp. PCC 7120 nuclease [GenBank: YP_227663], Homo sapiens EndoG [GenBank: Q14249] and WSSV-NSN nuclease [GenBank: AAW88443], respectively. These data suggested that the GBSV1-NSN was a novel non-specific nuclease.

The nuclease activity estimated at different temperatures indicated that the optimum temperature for the recombinant GBSV1-NSN was 60°C (Fig. 4A). The GBSV1-NSN nuclease activity was decreased with higher temperatures above the optimum. Thermostability assays showed that the GBSV1-NSN nuclease was most stable at 60°C (pH 7.5), retaining more than 80% of its enzymatic activity for at least 2 hours (Fig. 4B).

Based on the effects of pH on enzymatic activity, the GBSV1-NSN nuclease exhibited optimal activity at pH 7.5 (Fig. 5A). The recombinant nuclease was stable in the neutral pH ranging from 7.0 to 8.0 (Fig. 5B). It presented higher stability after incubation at 60°C for 3 hours, showing 66, 74 and 71 % residual activity at pH 7.0, 7.5 and 8.0, respectively (Fig. 5B).

The effects of different metal ions on the GBSV1-NSN nuclease activity were evaluated as shown in Table 1. In the absence of metal ion, the GBSV1-NSN nuclease was active. The enzymatic activity was obviously stimulated in the presence of Mn²⁺ and Zn²⁺ at 10 mM concentration each. Metal ions Li⁺, Na⁺, K⁺, Cs⁺, Ca²⁺, Mg²⁺, Ni²⁺, Sr²⁺ and Ba²⁺ at different concentrations showed very slight inhibition of the GBSV1-NSN activity, retaining more than 80% of its initial activity. However, its activity was strongly inhibited by Cu²⁺ and Fe³⁺. With the increasing in Cu²⁺ or Fe³⁺ concentration, the nuclease activity decreased sharply.

The results indicated that the recombinant nuclease was active when some metal chelators, thiol reagents and detergents were used (Table 2). However, the enzymatic activity was reduced by some enzyme inhibitors or detergents, such as ethylene diamine tetraacetic acid (EDTA), citrate, dithiothreitol (DTT), β-mecaptoethanol (2-ME), guanidine hydrochloride, urea and SDS. Among them, 10 mM of DTT and 0.6 M of guanidine hydrochloride led to a significant reduction of enzyme activity by 34.1% and 51.0%, respectively. In contrast, the nuclease activity was enhanced by TritonX-100, Tween-20 or chaps to approximately 124.5 – 141.6%.

The kinetic parameters of the nuclease were obtained from Lineweaver-Burke plot of specific activities at 60°C with different substrates including the double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) and RNA. The results revealed that its K_m value was 75, 20, 30 μg/ml and V_max value was 2.5 × 10⁵, 5 × 10⁴, 5 × 10⁴ Kunitz units·mg^-1 for the dsDNA, ssDNA and RNA, respectively (Fig. 6). After calculations, the k_cat of GBSV1-NSN nuclease was estimated to be 1278, 241 and 300 s^-1 for the dsDNA, ssDNA and RNA substrates, respectively (Fig. 6).

During the culture of Geobacillus sp. 6K51, phage plaques were observed from a thermophilic bacterium isolated from an offshore hot spring in Xiamen of China. The thermophilic bacteriophage (termed as GBSV1) was purified from its host, which was grown at 60°C with shaking (150 rpm) in the medium consisting of 0.2% NaCl, 0.4% yeast extract and 0.8% tryptone (pH 7.0). The GBSV1 contained a double linear genomic DNA of 34683 bp, which had the capacity encoding 55 open reading frames (ORFs). The genome sequencing revealed that it was a novel bacteriophage.

The sequence analysis of GBSV1 genome revealed an ORF sharing high homologies with phage replication proteins. To characterize the protein encoded by this ORF (termed as GBSV1-NSN gene), it was expressed in Escherichia coli BL21 (DE3) as a fusion protein with glutathione S-transferase (GST). The GBSV1-NSN gene was amplified from the GBSV1 genomic DNA using primers 5'-CGGGATCCATGACGATATATCGCATCG-3' (BamHI site, italic) and 5'-GCGAATTCTTATAGAAGTCCATCGAGG-3' (EcoRI site, italic). The amplified fragment was cloned into pGEX-4T-2 downstream of GST. The recombinant bacteria were induced with 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) when OD₆₀₀ reached 0.6–0.8. After incubation for an additional 4 to 5 h at 37°C, cultures were harvested by centrifugation (4,000 × g, 10 min, 4°C) and resuspended in 20 ml of ice-cold phosphate-buffered saline (PBS, pH 7.4). Cells were lysed by sonication and the cell debris was removed by centrifugation at 10,000 × g for 15 min. The supernatants were mixed gently with 1 ml glutathione- sepharose beads (Sigma, America) for 2 h at 4°C. After rinsing with ice-cold PBS, the GST fusion protein was pooled with a reducing buffer of 50 mM Tris-HCl and 10 mM reduced glutathione\(pH 8.0). The purified fusion protein was incubated with thrombin (Sigma, America) in a reaction buffer of 50 mM Tris-HCl, 150 mM NaCl, 2.5 mM CaCl₂ and 0.1% β-mecaptoethanol\(pH 8.0) at 22°C for 8 h. Subsequently it was applied to glutathione-agarose beads (Sigma, USA) to remove GST. The purified protein was dialyzed in 50 mM Tris-HCl (pH8.0).

Homology searches were performed against the sequences in the GenBank database using the BLAST program. Alignments of deduced amino acid sequence were generated with DNAMAN program.

To determine the specificity of substrate for the recombinant GBSV1-NSN nuclease, 1 μg of the circular pGEX-4T-2 plasmid dsDNA, the linear pGEX-4T-2 plasmid dsDNA, the PhiX174 virion ssDNA (New England Biolabs, UK) or baker's yeast RNA (Sigma-Aldrich, USA) were respectively incubated with 1.5 μg of the purified GBSV1-NSN protein in 20 μl of a reaction buffer (20 mM Hepes, 50 mM KCl, 2 mM MgCl₂, 0.2 mM EDTA, 0.1 mg/ml BSA and 10% glycerol, pH 7.5) at 37°C. Six hours later, 10 μl of the reaction solution was analyzed by electrophoresis on a 1.0 % agarose gel. In the assays, 1.5 μg of commercial DNase I (TaKaRa, Japan) and RNase A (Sigma-Aldrich, USA) were included as controls. All assays were conducted in triplicate.

The spectrophotometric method was used to measure the nuclease activity according to the protocol as described previously 3. One Kunitz unit was defined as the amount of enzyme needed to cause an increase of absorbance of 0.001 ⊿A₂₆₀·ml^-1·min^-1·cm^-1 at 60°C. Each reaction mixture consisted of 100 μg/ml RNA or DNA, 20 mM Tris-HCl (pH 7.5) and the recombinant GBSV1-NSN nuclease. After incubation for 15 min at 60°C, the reactions were stopped by equal volume of 5% ice-cold perchloric acid. The solutions were held on ice for 10 min and then centrifuged for 5 min at 8000 × g. The supernatants were measured using a Thermo Spectronic UNICAM UV 300 spectrophotometer set at 260 nm.

The optimal temperature of the GBSV1-NSN nuclease was measured by performing the enzymatic activity assay at temperatures ranging from 20 to 80°C in the presence of herring sperm DNA (Sigma-Aldrich, USA) as substrate. The thermostability of nuclease was investigated at different temperatures 40, 50, 60 and 70°C after incubation of the enzyme solutions (295 μg/ml) in the absence of 100 μg/ml herring dsDNA (20 mM Tris-HCl, pH 7.5) for 15, 40, 60, 90 or 130 min, respectively. Residual activities were determined under nuclease activity assay conditions using 100 μg/ml of herring dsDNA as substrate. All assays were repeated for three times.

The effect of pH on nuclease activity was evaluated at the optimal temperature over a pH range of 3.0–10.0, using five different buffers (all at 20 mM concentrations): sodium formiate (pH 3.0, 3.5), sodium acetate (pH 3.5 to 6.0), imidazole (pH 6.0 to 7.0), Tris/HCl (pH 7.0 to 9.0) and ethanolamine (9.0 to 10.0), under nuclease activity assay conditions. Further study on the pH stability of nuclease was carried out at 60°C by pre-incubation of the enzyme solutions in Tris/HCl buffer systems (pH 5.0 to 10.0) in the absence of substrate for 15, 30, 60, 90, 120, 150 and 180 min, respectively. Then they were subjected to the nuclease activity assay. The assays were conducted in triplicate.

The effects of metal ions on nuclease activity were determined by measuring the residual activity of GBSV1-NSN after separate incubation of the enzyme solutions with various metal ions at 60°C and pH 7.5 for 10 min. The concentrations of metal ions in the incubation solutions with the nuclease and in the final nuclease reactions were 0.1, 1.0 or 10 mM and 0.05, 0.5 or 5 mM, respectively. The assays were carried out for three times.

To determine the effects of metal chelators, the nuclease was pre-incubated with ethylene diamine tetraacetic acid (EDTA) or citrate at 60°C for 10 min, followed by the nuclease activity assay. The effects of thiol reagents and detergents on the nuclease activity were also monitored by the same procedure as described above. The concentrations of EDTA, citrate, dithiothreitol (DTT) and β-mecaptoethanol (2-ME) were 1 mM or 10 mM in the incubation solution with the nuclease and 0.5 mM or 5 mM in the final nuclease reaction, respectively. The concentrations of SDS, TritonX-100, Tween-20 or 3- [(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (chaps) were 0.1% or 1% (w/v) in the incubation solution with the nuclease and 0.05% or 0.5% in the final nuclease reaction, respectively. The concentrations of guanidine hydrochloride and urea were 0.6 M and 1 M in the incubation solution with the nuclease and 0.3 M and 0.5 M in the final nuclease reaction, respectively. All assays were performed in triplicate.

The protein concentration of purified GBSV1-NSN was determined based on the dye-binding assay method of Bradford 26, with bovine serum albumin (Sigma, USA) as a standard.

Steady-state kinetic parameters for the GBSV1-NSN nuclease were determined using the Kunitz assay with herring sperm DNA (dsDNA), heat-denatured herring sperm DNA (ssDNA) and baker's yeast RNA. In order to determine K_m and V_max, the nucleic acid concentrations were varied in the range of 0–100 μg·ml^-1. All nuclease activity assays were conducted at 60°C in a Tris/HCl buffer (20 mM, pH 7.5). Typical Lineweaver-Burk plots were obtained when 1/[v] was plotted against 1/[S] 27. Kinetic parameters were estimated by linear regression from Lineweaver-Burk plots. All assays were conducted for three times.

To compare the K_m and k_cat of GBSV1-NSN with those of the known nucleases, the K_m values were converted from μg·ml^-1 to μM by using a mean molar mass of 330 g·mol ^-1·nt ^-128. The V_max values were converted to k_cat by assuming that the molar mass per nucleotide was 330 g·mol ^-1 and that a change in absorbance of 0.3 A₂₆₀ accompanied the degradation of 50 μg·ml^-1 DNA and 0.25 A₂₆₀ accompanied the degradation of 50 μg·ml^-1 RNA 24.

All the numerical data were analyzed by one-way ANOVA and expressed as mean ± standard deviation.