A Novel Halotolerant Thermoalkaliphilic Esterase from Marine Bacterium Erythrobacter seohaensis SW-135

Abstract

A novel esterase gene, e69, was cloned from Erythrobacter seohaensis SW-135, which was isolated from a tidal flat sediment of the Yellow Sea in Korea. This gene is 825 bp in length and codes for a 29.54 kDa protein containing 274 amino acids. Phylogenetic analysis showed that E69 is a new member of the bacterial lipolytic enzyme family IV. This enzyme exhibited the highest level of activity toward p-nitrophenyl (NP) butyrate but little or no activity toward the other p-NP esters tested. The optimum temperature and pH of the catalytic activity of E69 were 60°C and pH 10.5, respectively. The enzyme exhibited stable activity over a wide range of alkaline pH values (7.5-9.5). In addition, E69 was found to be a halotolerant esterase as it exhibited the highest hydrolytic activity in the presence of 0.5 M NaCl and was still active in the presence of 3 M NaCl. Moreover, it possessed some degree of tolerance to Triton X-100 and several organic solvents. Through homology modeling and comparison with other esterases, it was suggested that the absence of the cap domain and its narrow substrate-binding pocket might be responsible for its narrow substrate specificity. Sequence and structural analysis results suggested that its high ratio of negatively to positively charged residues, large hydrophobic surface area, and negative electrostatic potential on the surface may be responsible for its alkaline adaptation. The results of this study provide insight into marine alkaliphilic esterases, and the unique properties of E69 make it a promising candidate as a biocatalyst for industrial applications.

Keywords: alkaliphilic; esterase; family IV; halotolerant; homology modeling; marine.

FIGURE 1

Neighbor-joining phylogenetic tree based on amino acid sequences of E69 and related lipolytic enzymes. Sequence alignment was performed using ClustalX and the tree was constructed using the MEGA software. Bootstrap values are based on 1000 replicates and only values of >50% are shown. The scale bar indicates the number of amino acid substitutions per site.

FIGURE 2

Amino acid sequence alignment of E69 and related lipolytic enzymes. Accession numbers of the enzymes in the PDB or GenBank databases are given. Sequence alignment was performed using the ClustalX and ESPript programs. Identical and similar residues among groups are shown in white text on a red background and in red text on a white background, respectively. The triangles indicate the locations of the catalytic active site residues [serine (S), aspartate (D), and histidine (H)]. The red line indicates the sequences of the cap domain in E25 (PDB: 4Q05) and EstE5 (PDB: 3FAK).

FIGURE 3

Activity of E69 toward various substrates (A), and effects of pH (B) and temperature (C) on the activity and stability of E69. (A) Substrate specificity was determined using the p-NP esters. The highest activity was taken as 100%. ^∗p < 0.05, representing a significant difference from the 100% values (Student’s t-test). (B) Activities at various pH values (solid lines) were assayed at 40°C, with the activity obtained at pH 10.5 taken as 100%. The residual activities (dashed lines) after incubation at various pH values for 2 h were assayed at 40°C and pH 10.0, and the value obtained without treatment was taken as 100%. Assay was performed in different buffers: 100 mM citrate buffer (pH 4.0–6.0, filled diamonds), 100 mM phosphate buffer (pH 6.0–7.5, filled circles), 100 mM tricine buffer (pH 7.5–9.0, filled squares), and 50 mM CHES buffer (pH 9.0–10.5, filled triangles). (C) Activities at various temperatures (solid line with empty circles) were assayed at pH 10.0 and the value obtained at 60°C was taken as 100%. The residual activities after incubation at various temperatures for 1 h (dashed line with empty squares) or 2 h (dashed line with empty triangles) were assayed at 40°C and pH 10.0, and the value obtained without treatment was taken as 100%. Data are presented as mean ± SD (n = 3).

FIGURE 4

Effects of NaCl and metal ions on the activity of E69. (A) The effect of NaCl on the activity of E69 was determined using p-NP butyrate as the substrate. The value obtained without added NaCl was taken as 100%. (B) The effects of metal ions and EDTA on the activity of E69 were determined using p-NP butyrate as the substrate. All of the tests were performed at 40°C and pH 10.0. The value obtained without metal ions was taken as 100%. Data are presented as mean ± SD (n = 3). ^∗p < 0.05, representing a significant difference from the 100% values (Student’s t-test).

FIGURE 5

3D structure model of E69 and structural superimposition with other homologous esterases. (A) Cartoon representation of E69. The α helices and β strands are colored in blue and green, respectively. The catalytic triad residues are indicated as stick models colored in green. (B) The structural superposition of E69 (green), E25 (blue, PDB: 4Q05), MGS-MT1 (orange, PDB: 4Q3O), EstE5 (cyan, PDB: 3FAK), EstE7 (magenta, PDB: 3DNM), and E40 (yellow, PDB: 4XVC). The catalytic triad residues are indicated as stick models.

FIGURE 6

Structural comparison of E69 and its homologs, E25 (PDB: 4Q05) and EstE5 (PDB: 3FAK). (A) Electrostatic surface representations of E69 and its homologs, from -3.5 (red) to +3.5 kT/e (blue). Surface view of the structures showing the differences in (B) the cap domains and (C) the substrate-binding pockets. The cap domains and active sites are indicated in blue and red, respectively.