Identification and Characterization of a Novel Salt-Tolerant Esterase from the Deep-Sea Sediment of the South China Sea

Abstract

Marine esterases play an important role in marine organic carbon degradation and cycling. Halotolerant esterases from the sea may have good potentials in industrial processes requiring high salts. Although a large number of marine esterases have been characterized, reports on halotolerant esterases are only a few. Here, a fosmid library containing 7,200 clones was constructed from a deep-sea sediment sample from the South China Sea. A gene H8 encoding an esterase was identified from this library by functional screening and expressed in Escherichia coli. Phylogenetic analysis showed that H8 is a new member of family V of bacterial lipolytic enzymes. H8 could effectively hydrolyze short-chain monoesters (C4-C10), with the highest activity toward p-nitrophenyl hexanoate. The optimal temperature and pH for H8 activity were 35°C and pH 10.0, respectively. H8 had high salt tolerance, remaining stable in 4.5 M NaCl, which suggests that H8 is well adapted to the marine saline environment and that H8 may have industrial potentials. Unlike reported halophilic/halotolerant enzymes with high acidic/basic residue ratios and low pI values, H8 contains a large number of basic residues, leading to its high basic/acidic residue ratio and high predicted pI (9.09). Moreover, more than 10 homologous sequences with similar basic/acidic residue ratios and predicted pI values were found in database, suggesting that H8 and its homologs represent a new group of halotolerant esterases. We also investigated the role of basic residues in H8 halotolerance by site-directed mutation. Mutation of Arg195, Arg203 or Arg236 to acidic Glu significantly decreased the activity and/or stability of H8 under high salts, suggesting that these basic residues play a role in the salt tolerance of H8. These results shed light on marine bacterial esterases and halotolerant enzymes.

Keywords: basic residues; deep-sea sediment; esterase; metagenomics; salt-tolerance.

FIGURE 1

Phylogenetic tree of esterase H8 and representative lipolytic enzyme sequences from family V. The tree was built by Neighbor-Joining method with JTT-matrix based model using 218 amino acid positions. Bootstrap analysis of 1000 replicates was executed and values above 50% are shown. The scale for branch length is shown below the tree. Lipases from family I were used as outgroups.

FIGURE 2

Multiple sequence alignment of H8 and its homologs. Identical and similar amino acids are shaded in black and gray, respectively. Stars indicate amino acid residues belonging to the catalytic triad, and circles indicate basic amino acid residues (Arg195, Arg203, Arg216, Arg236, and Arg263) selected for site-directed mutation. Sequence analysis suggested that residues Arg203, Arg216, and Arg236 are highly conserved, and residues Arg195 and Arg263 are partially conserved.

FIGURE 3

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified esterase H8 and the substrate specificity of H8. (A) SDS-PAGE analysis of purified H8. Lane 1, purified H8; lane M, protein mass markers. (B) Substrate specificity of H8 evaluated with pNP esters. The graph shows data from triplicate experiments (mean ± SD).

FIGURE 4

Effect of temperature on the activity and stability of H8. (A) Effect of temperature on H8 activity. The highest activity of H8 at 35°C (69.2 U/mg) was taken as 100%. (B) Effect of temperature on the stability of H8. The enzyme was incubated at 0–60°C for 1 h. The remaining activity was measured under optimal conditions. The activity at 0°C (66.4 U/mg) was taken as 100%. The graphs show data from triplicate experiments (mean ± SD).

FIGURE 5

Effect of pH on the activity and stability of H8. (A) Effect of pH on the activity of H8. The activity was measured at 35°C in Britton–Robinson buffers ranging from pH 4.0 to 13.0. The highest activity at pH 10.0 (70.2 U/mg) was taken as 100%. (B) Effect of pH on the stability of H8. The enzyme was incubated in buffers ranging from pH 4.0 to 13.0 at 25°C for 1 h. The remaining activity was measured under optimal conditions. The highest activity at pH 8.0 (54.4 U/mg) was taken as 100%. The graphs show data from triplicate experiments (mean ± SD).

FIGURE 6

Effect of NaCl on the activity and stability of H8. (A) Effect of NaCl on the activity of H8. The activity was measured at 35°C in 50 mM Tris-HCl buffer with different concentrations of NaCl. The activity in 0 M NaCl (70.9 U/mg) was taken as 100%. (B) Effect of NaCl on the stability of H8. The enzyme was incubated at 0°C for 1 h in buffers containing NaCl ranging from 0 to 4.6 M. The remaining activity was measured under optimal conditions. The activity in 0 M NaCl (68.6 U/mg) was taken as 100%. The graphs show data from triplicate experiments (mean ± SD).

FIGURE 7

Effect of NaCl on the activity and stability of the mutants of H8. (A) Effect of NaCl on the activity of WT H8 and its mutants. The activities of H8 and its mutants were determined at different NaCl concentrations at their respective optimum temperatures. The activities of WT H8 (70.9 U/mg), R195E (65.6 U/mg), R203E (7.5 U/mg), R216E (82.6 U/mg), R236E (53.0 U/mg), and R263E (62.0 U/mg) in 0 M NaCl were taken as 100%, respectively. (B) Effect of NaCl on the stability of WT H8 and its mutants. The enzymes were incubated in buffers containing different NaCl concentrations at 0°C for 1 h, and the residual activity was measured at their optimum temperatures, respectively. The activities of WT H8 (68.6 U/mg), R195E (64.5 U/mg), R203E (6.7 U/mg), R216E (72.8 U/mg), R236E (48.8 U/mg), and R263E (61.0 U/mg) in 0 M NaCl were taken as 100%, respectively. The graphs show data from triplicate experiments (mean ± SD).

FIGURE 8

Relative specific activities and circular dichroism (CD) spectra of WT H8 and its mutants. (A) Relative specific activities of WT H8 and its mutants. The specific activity of WT H8 (69.7 U/mg) was defined as 100%. (B) CD spectra of WT H8 and its mutants. 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. The graphs show data from triplicate experiments (mean ± SD).