Marine sediment sample S100 was collected from the South China Sea (13.5°N, 118°E) at a water depth of 3,939 m in September 2011. Temperature and salinity of bottom water in this area was 2.4°C and 3.46% (w/v), respectively. The sample was stored at -20°C until processing. Environmental genomic DNA was extracted from the sample by following the SDS-based extraction procedure described by Zhou et al. (1996).

The DNA extract was separated by pulsed-filed gel electrophoresis (PFGE), and DNA bands of ∼35 kbp in the gel were extracted by gelase enzymolysis and ethanol precipitation. A metagenomic DNA library was constructed using the CopyControl Fosmid Library Production Kit (Epicentre Biotechnologies, Madison, WI, USA) by following the manufacturer’s instructions. A total of 7,200 fosmid clones were obtained, which were spread onto Luria-Bertani (LB) agar plates supplemented with 1% (v/v) emulsified tributyrin to screen clones with lipolytic enzyme activity. Clones exhibiting a clear zone around the colony were selected to construct their respective subcloning libraries. Fosmid DNA was extracted from positive clones and partially digested by the restriction enzyme Sau3AI. The DNA fragments of 1.5–5 kbp were recovered from an agarose gel, end-repaired and ligated into the pUC19 vector that had been digested by BamHI and pretreated by bacterial alkaline phosphatase. The ligated products were transformed into E. coli TOP10 cells, and the transformants were spread onto LB agar plates supplemented with 100 μg/ml ampicillin and 1% (v/v) tributyrin. Transformants forming clear zones were sequenced. The open reading frames (ORFs) in the sequenced DNA fragments were predicted by the GeneMark program¹ and the genes encoding potential lipolytic enzymes were identified by using the blastx program against the NCBI non-redundant protein database (nr). Multiple sequence alignment was performed using MUSCLE (Edgar, 2004). Phylogenetic analysis was carried out with the neighbor-joining method using MEGA 6.0 (Tamura et al., 2013). The potential signal peptide sequence was predicted by SignalP 4.0 (Petersen et al., 2011).

The gene H8 encoding a lipolytic enzyme was amplified from the fosmid DNA using the primer pair of H8_F (5′-GGGAATTCCATATGCAGTCTGGCACGGTGAG-3′, NdeI digestion site was underlined) and H8_R (5′-CCGCTCGAGCGCCACCGCCGGTTGCGCC-3′, XhoI digestion site was underlined), and cloned into the expression vector pET-22b.

The constructed plasmid pET-22b-H8 was transformed into E. coli BL21 (DE3). Transformants were cultured at 37°C and 180 rpm in LB liquid medium containing 100 μg/mL ampicillin. When the OD₆₀₀ of cells reached approximately 0.6, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for the induction of protein expression. Then, the culture was incubated at 20°C and 110 rpm for 20 h. After incubation, the cells in the culture were harvested, resuspended in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0) and disrupted by pressure. The recombinant His-tagged protein in the extract was first purified by Ni affinity chromatography (Qiagen, USA), and further purified by gel filtration chromatography on a Superdex 200 column (GE healthcare, Sweden). Protein concentrations were determined by using the Pierce BCA Protein Assay Kit (Thermo Scientific, USA).

The esterase activity was measured by monitoring the hydrolysis of p-nitrophenyl (pNP) esters (Sigma, USA) using a spectrophotometric method (Shirai and Jackson, 1982). The reaction mixture contained 50 mM Tris-HCl buffer (pH 8.0), 0.02 ml of 10 mM substrate, and 0.02 ml enzyme with appropriate concentration in a final volume of 1 m1. After incubation at an indicated temperature for 5 min, the reaction was terminated by an addition of 0.1 ml 20% (w/v) SDS. The absorbance of the reaction mixture at 405 nm was measured to detect the amount of released p-nitrophenol (Li et al., 2014). The background hydrolysis of the substrate was determined by using a blank control with a composition identical with the reaction mixture except that the enzyme was replaced by buffer. One unit of enzyme (U) is defined as the amount of enzyme required to liberate 1 μmol p-nitrophenol per minute.

The substrate specificity of H8 was investigated using the substrates pNP acetate (C2), pNP butyrate (C4), pNP caproate (C6), pNP caprylate (C8), pNP decanoate (C10), pNP laurate (C12), pNP myristate (C14), and pNP palmitate (C16) (Sigma, USA). The optimum temperature for H8 activity was measured at temperatures ranging from 0 to 60°C at pH 8.0. For thermostability assay, the enzyme was incubated at temperatures ranging from 0 to 60°C for 1 h, and then the residual activity was measured at 35°C. The optimum pH of H8 was determined at 35°C in the Britton–Robinson buffers ranging from pH 4.0 to 13.0. For pH stability assay, the enzyme was incubated in buffers with a pH range of 4.0–13.0 at 25°C for 1 h, and then the residual activity was measured at pH 8.0 and 35°C. The effect of NaCl on H8 activity was determined at NaCl concentrations ranging from 0 to 4.8 M. For salt tolerance assay, the enzyme was incubated at 0°C for 1 h in buffers containing NaCl ranging from 0 to 4.6 M before the residual activity was measured at 35°C.

The effects of metal ions (K⁺, Li⁺, Ba²⁺, Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) and potential inhibitors (β-mercaptoethanol, DTT, Thiourea, Urea, EDTA, and PMSF) on H8 activity were examined at pH 8.0 and 35°C in a final concentration of 1 mM or 10 mM. The effects of organic solvents on H8 activity were examined using methanol, ethanol, isopropanol, acetone, acetonitrile, dimethylsulfoxide (DMSO), and dimethylformamide (DMF) at final concentrations of 10% and 20% (v/v). The effects of Tween 20, Tween 80, and Triton X-100 on H8 activity were examined at final concentrations of 0.001–0.1% (v/v). The effect of SDS on enzyme activity was measured at final concentrations of 0.001–0.1% (w/v).

Using plasmid pET-22b-H8 as the template, site-directed mutagenesis on H8 was performed with the QuikChange^® 146 mutagenesis kit II (Agilent technologies, USA) according to the method of QuikChange site-directed mutagenesis (Liu and Naismith, 2008). After verified by DNA sequencing, mutated plasmids were transformed into E. coli BL21 (DE3) for protein expression. The purification of H8 mutants was performed under the same conditions as those of the wild type (WT) H8.

Enzyme kinetic assays of H8 and its mutants were carried out at pH 7.5 (50 mM Tris-HCl) using pNPC6 at concentrations from 0.02 to 2.0 mM. Kinetic parameters were calculated by non-linear regression fit directly to the Michaelis–Menten equation using the Origin8.5 software.

Circular dichroism (CD) spectra of WT H8 and its mutants were recorded at 25°C on a J-810 spectropolarimeter (JASCO, Japan). All the spectra were collected from 200 to 250 nm at a scanning speed of 200 nm/min with a path length of 0.1 cm. Proteins for CD spectroscopy assays were at a concentration of 0.3 mg/ml in 50 mM Tris-HCl buffer (pH 8.0).

The nucleotide sequence of H8 has been deposited in the GenBank database under accession number KY273927.

A metagenomic library was constructed from a deep-sea sediment sample from the South China Sea, which contained a total of 7,200 fosmid clones. The metagenomic library represented approximately 252 Mbp environmental DNA assuming an average insert size of 35 kbp. By functional assays, 10 fosmid clones showing lipolytic enzyme activities were screened from this library. The sequences of putative lipolytic enzyme-encoding genes in the identified fosmids were determined by subcloning library construction and subsequent sequencing. Among these genes, the gene H8 containing 918 bp was predicted to encode a lipolytic enzyme and chosen for further analysis.

The gene H8 encodes a lipolytic enzyme of 305 amino acid residues with a predicted molecular weight of 32.8 kDa and a predicted pI of 9.09. Prediction by SignalP 4.0 suggested that H8 may lack an N-terminal signal sequence. Among the characterized lipolytic enzymes, H8 showed the highest sequence identity (46%) to a family V esterase (Est16) from a microbial consortium specialized for diesel oil degradation (Pereira et al., 2015). Phylogenetic tree also showed that H8 belongs to the family V of bacterial lipolytic enzymes (Figure 1). Based on sequence alignments with other proteins from family V, the catalytic triad of H8 was identified, which is composed of Ser120, Asp247, and His275 (Figure 2). The catalytic Ser120 is located in the conserved GASMGGMI motif, Asp247 in the conserved DPL motif, and His275 in the conserved MG/AHD motif.

H8 was over-expressed in E. coli BL21 (DE3), and the recombinant H8 protein was first purified by Ni affinity chromatography and then by gel filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the purified H8 displayed an apparent molecular weight of approximately 33 kDa, accordant to that predicted from its sequence (32.8 kDa) (Figure 3A). H8 could efficiently hydrolyze short-chain pNP esters (C4–C10), with the maximal activity toward pNPC6 (69.0 U/mg) (Figure 3B and Supplementary Figure 1). H8 showed a limited ability to degrade pNP esters longer than 10 carbon atoms, indicating that H8 is an esterase. H8 showed the highest activity at 35°C and retained 30% of its highest activity at 0°C (Figure 4A). H8 retained more than 80% of its highest activity after 1 h incubation at temperatures lower than 40°C, but lost all the activity after 1 h incubation at 50°C (Figure 4B). H8 had the highest activity at pH 10.0 (Figure 5A) and showed good tolerance in a range of pH 6.0–9.0, retaining over 80% of its highest activity after 1 h incubation in the buffers of pH 6.0–9.0 at 25°C (Figure 5B).

The effect of metal ions on the activity of H8 was also investigated (Table 1). H8 activity was almost unaffected by K⁺, Li⁺, Ca²⁺, Co²⁺ or Mg²⁺ at 1∼10 mM, but significantly inhibited by Zn²⁺ at 1∼10 mM, and Ba²⁺, Mn²⁺, and Ni²⁺ at 10 mM, and fully inhibited by Cu²⁺ and Fe²⁺ at 10 mM. EDTA had no effect on H8 activity, suggesting that the catalysis by H8 may not require metal ions. H8 activity was significantly inhibited by 10 mM PMSF, indicating that H8 is most likely a serine hydrolase. H8 activity was also severely inhibited by reductants DTT and β-mercaptoethanol. However, H8 showed high resistance to chaotropic agents urea and thiourea (Table 1). H8 activity was slightly increased by 0.001–0.1% (v/v) Tween 20, but fully inhibited by 0.01% (w/v) SDS (Table 2). Among all the tested organic solvents at 10% (v/v) concentration, methanol, ethanol, DMF, and DMSO slightly increased H8 activity, and other detergents slightly reduced H8 activity. At 20% (v/v) concentration, DMF and DMSO had nearly no effect on H8 activity, whereas other detergents significantly inhibited H8 activity (Table 3).

Because the H8 gene is isolated from a deep-sea sediment, we investigated the effect of NaCl of different concentrations on the activity and stability of H8. H8 still had full activity in NaCl with a concentration as high as 4.0 M (Figure 6A), indicating that H8 has high salt tolerance. Moreover, after 1 h incubation in 4.6 M NaCl, H8 still retained 80% activity (Figure 6B). These results show that H8 is a halotolerant enzyme.

Unlike most reported halophilic/halotolerant enzymes that have high acidic/basic residue ratios and relatively low pI values ranging from 4.3 to 6.8 (Ng et al., 2000; Kennedy et al., 2001; Dassarma et al., 2013), H8 contains more basic residues (10.49%) than acidic residues (9.18%) (Table 4), leading to a high predicted pI value of 9.09. By searching NCBI nr database using the H8 sequence as a query, more than 10 homologs of H8 are found to have more basic residues than acidic residues and high pI values (Table 4), suggesting that H8 and its homologs may represent an uncharacterized group of lipolytic enzymes rich in basic residues.

Until now, only one halophilic enzyme, the endonuclease VsEndA from V. salmonicida, is found to have an overwhelming number of basic residues distributed on the protein surface for its haloadaption (Altermark et al., 2008). To study the roles of the basic residues, especially the surface basic residues, in the salt tolerance of H8, we tried to obtain its crystal structure or modeled structure. Unfortunately, the crystal structure of H8 was unable to be solved due to the low resolution of the H8 crystals we obtained. In addition, due to the low sequence identity (lower than 28%) between H8 and reported proteins with resolved structures, no modeled structure of H8 could be constructed. Finally, according to multiple sequence alignment (Figure 2), we selected five basic residues (Arg195, Arg203, Arg216, Arg236, and Arg263) for site-directed mutation to acidic Glu to investigate their roles in H8 halotolerance. Residues Arg195 and Arg263 are partially conserved and residues Arg203, Arg216, and Arg236 are highly conserved in H8 homologs (Figure 2).

The effect of NaCl on the activities and stabilities of the mutants was measured and compared to WT H8 (Figure 7). Under their respective optimum temperatures, the effect of NaCl on the activities and stabilities of mutants R216E and R263E was similar to that of the WT (Figures 7A,B), suggesting that these two residues Arg216 and Arg263 may not be related to the salt tolerance of H8. In addition, mutants R216E and R263E had similar K_m and k_cat values and specific activities to the WT (Figure 8A and Table 5), indicating that these two mutations had little effect on the substrate binding and catalysis of H8 and that Arg216 and Arg263 are potentially surface residues.

Although the activity of mutant R203E was slightly stimulated by 1.2- to 1.3-fold in NaCl ranging from 2.0 to 4.0 M, its stability was significantly reduced in increased concentrations of NaCl. After incubated in 4.0 M NaCl for 1 h, H8 retained 80% activity, whereas R203E retained only 30% activity, indicating that mutant R203E is less tolerant than the WT under high salts. Mutation R203E had no impact on the K_m of H8, but significantly reduced its k_cat and specific activity (Figure 8A and Table 5). These data suggest that Arg203 might be directly or indirectly involved in the catalysis of H8.

The effect of NaCl on the stability of mutant R195E was similar to that of the WT, whereas its activity was significantly reduced by NaCl. Compared to WT H8, the activity of mutant R195E in 4 M NaCl was reduced by 51%, suggesting its potential role in the salt tolerance of H8. Mutation R195E had no impact on the specific activity of H8 and small impact on the K_m and k_cat values (Figure 8A and Table 5), indicating that Arg195 is potentially located on the surface of H8 protein.

For mutant R236E, both the activity and stability was significantly reduced in increased concentrations of NaCl, suggesting that Arg236 may play an important role in the salt tolerance of H8. Mutant R236E had only small effect on the substrate binding and catalysis of H8 (Table 5), suggesting that residue Arg236 is potentially a surface residue.

Circular dichroism spectral analysis showed that these mutations caused no visible changes in H8 structure (Figure 8B), indicating that the decrease in the enzymatic activity and stability of the mutants resulted from residue substitution rather than structural changes.